000X9969/92$5.00+ 0.00 Copyright 0 1992Pergamon Press Ltd
Archs oral Bid. Vol. 37, No. 5, pp. 355-361, 1992 Printed in Great Britain. All rights reserved
LEVELS OF SALIVARY CYSTATINS IN PERIODONTALLY HEALTHY AND DISEASED OLDER ADULTS A.
AGUIRRE, L. A. TESTA-WEINTRAUB, J. A. BANDERAS, R. DUNFORD
Department
and M. J.
LEVINE
of Oral Biology and Dental Research Institute, 109 Foster Hall, School of Dental Medicine, State University of New York at Buffalo, Buffalo, NY 14214, U.S.A. (Accepted 13 November 1991)
Summary-Cystatins are cysteine protease inhibitors present in a variety of tissues and body fluids, including saliva. One possible function of these molecules may be to modulate tissue destruction in periodontal diseases. To investigate the potential role of salivary cystatins in these events, the levels of cystatins in saliva from periodontally healthy and diseased individuals were measured by enzyme-linked immunosorbent assay. Flow rates and total protein content were determined in all the samples collected, while cysteine protease inhibitory activity was assessed in submandibular-sublingual secretions. Statistical analysis showed no significant differences in the levels and activity of salivary cystatins in periodontally healthy and diseased individuals. These findings suggest that comparing the levels of cystatins in glandular salivas may not be a suitable indicator of periodontal disease status. Key words: cystatins. saliva, cysteine protease inhibitor, adults, periodontitis, ageing.
INTRODUCTION
The cystatin superfamily comprises a diverse group of cysteine protease inhibitors widely distributed in mammalian tissues and plasma. They protect the organism against the uncontrolled action of endogenous and/or exogenous cysteine proteinases (Lindhal et al., 1988). Originally, members of the cystatin superfamily were grouped into three families; family I or stefins, family II or cystatins (including salivary cystatins) and family III or kininogens (Barrett et al., 1986a, b). However, additional families were suggested by Rawlings and Barrett (1990) to accommodate those proteins that cannot be included in the three established families. Human salivary cystatin genes are part of a multigene family composed of seven members segregated on chromosome 20 (Saitoh et al., 1989). These were first recognized as cysteine-containing phosphoproteins (Shomers et cd, 1982a), but it was not until recently that they were identified as cysteine proteinase inhibitors (Isemura, Saitoh and Sanada, 1984a, 1986, 1987; Isemura et al., 1984b). Salivary cystatins S (SAP-l), SN (SA-I) and SA contain 121 amino acids and have -90% sequence homology. In addition, salivary cystatins have a 54% sequence homology with serum cystatin C, which is present in the gingival crevicular fluid and consequently in the oral milieu. Impaired regulation of proteolysis is an important biochemical aspect in the aetiology of periodontal disease (Sandholm, 1986). Recent studies have shown increased levels of cysteine proteases (cathepsins D, B and L) with increasing severity of inflammation in immunosorbent Abbreviations : ELISA, enzyme-linked assay; SDS-PAGE, sodium dodecyl sulphate-polyacrylamide gel electrophoresis.
partially purified gingival homogenates (Lah et al., 1985) and crevicular fluid (Eisenhauer et al., 1983; Lah et al., 1986). Likewise, gingival homogenates obtained from periodontally diseased sites show a decrease in levels of cystatin C that is inversely correlated with the degree of periodontal disease (Skaleric et al., 1989). However, it has not been determined if levels of glandular salivary cystatins are also associated with periodontal disease status. Such an association might serve as an indicator of disease activity. Accordingly, our goal was to see if there is a correlation between the levels of glandular salivary cystatins and their cysteine protease inhibitory activity, and periodontal status. MATERIALS AND METHODS
Materials
Lashley cups were from H & I Instrumentation (Teaneck, NJ, U.S.A.). Block-Brotman universal collectors were from Flory Dental Prosthetics (Star City, WV, U.S.A.). Citric acid (anhydrous), ammonium sulphate, sodium hydroxide and Btij 35 were from Fisher Scientific Company (Fair Lawn, NJ, U.S.A.). Vinyl polysiloxane impression material (Express, light body-fast set), dispenser and disposable mixing tips were from 3 M (St Paul, MN, U.S.A.). Papain (P-3125), disodium p-nitrophenyl phosphate, N-abenzoyl-L-arginine-7-amido-4-methyl-coumarin (B7260), alkaline phosphatase from bovine intestinal mucosa (P-6774) and bovine serum albumin (A-7906) were from Sigma (St Louis, MO, U.S.A.). Nitrocellulose membranes (0.45 pm), horseradish peroxidaseconjugated goat anti-rabbit serum (IgG H + L chains) and 4-chloro-1-naphthol were from Bio-Rad (Richmond, CA, U.S.A.). Freund’s complete and incomplete adjuvants were from GIBCO (Grand Island, NY, U.S.A.). ELISA plates (96 wells, 355
356
A. AGU~RRE el al.
modified flat bottom, 25805-96) were from Corning Glass Works (Corning, NY, U.S.A.). Patient population
Clinically healthy individuals of both sexes (age 55-74) were recruited from the general population of Buffalo, NY, U.S.A. Some of these individuals were taking prescription medication, in particular antihypertensive drugs. Patients selected had at least 16 natural teeth (two of which were molars) with no fixed prosthetic devices obscuring the cementumenamel junction and no previous history of periodontal surgical treatment. The criterion (Grossi et al., 1990) used to categorize the subject’s periodontal status was as follows. Group A consisted of individuals without periodontitis (all teeth had interproximal attachment loss of 3 mm or less); group B was composed of individuals with moderate to severe periodontitis (periodontal attachment loss of 6 mm or more in the interproximal sites of at least two different teeth). All participants were screened by an examiner who had undergone suitable training for calibration (Miller et af., 1987). A total of 52 subjects were selected, 22 individuals in group A and 30 in group B. Saliva collection
Subjects refrained from eating, drinking, smoking and oral hygiene for 2 h for saliva collection. To collect submandibular-sublingual saliva a customized Block-Brotman device was used (Block and Brotman, 1962); parotid saliva was collected with a Lashley cup (Lashley, 1916). Salivas were stimulated by the application of 2% citric acid to the sides of the tongue at 30-s intervals and the first ml of saliva was discarded (Baum, 1981). Saliva was collected into chilled preweighed polypropylene tubes whereby flow rates were determined gravimetrically and expressed as ml/min/gland. The salivas were then divided into IOO-~1samples and frozen at -20°C. Before analysis, samples were slowly thawed in an ice bath and Na,EDTA was added to give a final concentration of 5 mM for submandibular-sublingual and 1 mM for parotid saliva (Fox et al., 1986). Protein content
Total protein content of salivary samples was determined from the absorbance at 215 nm as described by Arneberg (1971). Purification ofcystatin SN and preparation of antibog),
Salivary cystatin SN was purified (Al-Hashimi, Dickinson and Levine, 1988; Ramasubbu et al.. 1991) for use as an immunogen to raise polyclonal anti-
bodies and as a standard for ELISA and cysteine protease inhibition assays. Protein content of cystatin preparations was determined by amino acid analysis (Al-Hashimi et al., 1988). To obtain monospecific polyclonal antisera, 6-week-old female New Zealand white rabbits were immunized as described by Aguirre et al. (1987). In brief, subcutaneous dorsal injections of cystatin SN (200 p g protein/ml 0.154 M NaCl) emulsified with Freund’s complete adjuvant (1: 1, v/v) were used to prime the animals. Subcutaneous booster injections were given at &week intervals with 100 pg protein/ml of 0.154M NaCl emulsified with Freund’s incomplete adjuvant. After 10 days, titres were monitored by SDS-PAGE (Laemmli, 1970) and anionic-PAGE (Ornstein, 1964) followed by immunoblotting (Towbin, Staehlin and Gordon, 1979). This demonstrated that the rabbit antiserum (dilution: l/60,000) was specific for salivary cystatins, as shown by the presence of single bands in both salivas with mobility comparable to purified cystatin SN [Plate Fig. 1 (B)]. Furthermore, anionic-PAGE/immunoblotting revealed that this antiserum recognized several members of the salivary cystatin family and was not reactive with amylase [Plate Fig. 2 (B)]. For ELISA, concentrated IgG fractions were obtained by ammonium sulphate precipitation (Beutner et al., 1987) and subsequently conjugated to alkaline phosphatase (Voller, Bidwell and Bartlett, 1976). immunoquantitation of salivary cystatins
Salivary cystatins were quantitated by an ELISA, double-antibody sandwich technique in 96-well plates. In brief, plates were first coated with IgG fractions (1: 500 in 0.05 M sodium carbonate buffer, pH 9.6) and incubated at 37°C for 2 h. After washing (0.154 M NaCI, 0.05% Tween-20), saliva samples were assayed at three different dilutions, each in triplicate. Standard curves were generated using known quantities of cystatin SN (0.062.1 ng protein, each in triplicate). The plates were then incubated at 37’C for 1 h. After a second washing, IgG conjugated to alkaline phosphatase was added and incubated overnight at 25’C. After another wash, disodium p-nitrophenyl phosphate (1 mg/ml of 0.05 M sodium carbonate buffer, pH 9.8 with 1 mM MgCl,) was added for colour development and incubated for 60 min at 25’C. Plates were read at 405 nm in a 2550 ELISA Reader (Bio-Rad, Richmond, CA, U.S.A.) interfaced to a Macintosh computer. Salivary cystatin values were obtained from a regression line derived from the standards (Mac ReaderTM 2.0 Program) and the results expressed as /*g/ml saliva, pg/ml/min
Plate I Fig. I. Panel A shows a 10% SDS-PAGE gel stained with Coomassie blue and the corresponding immunoblot in panel B. Lane I, low molecular-weight standards; lane 2, submandibular-sublingual saliva (25 pg); lane 3, cystatin SN (2.5 pg): and lane 4 parotid saliva (25 pg). Fig. 2. Panel A shows a 7.5% anionic-PAGE gel stamed with Coomassie blue and the corresponding immunoblot in panel B. Lane 1, submandibular-sublingual saliva (IO pg); lane 2, salivary a-amylase [Scannapieco et al. (1989) 3 rg]; lanes 3-5, enriched fractions of cystatin SN (0.5 pg), cystatin SA (2.5 pg) and cystatin S (1 .O pg), respectively (prepared as described by Al-Hashimi et al., 1988; Ramasubbu et al., 1991). No bands were seen when the primary antibody was substituted with rabbit non-immune sera.
Salivary cystatins in older adults
(I 1
A
B
_,
1234
(2)
234
A
12
351
B
1
345 Plate
I
2
3
4
5
A.
358 Table
I. Salivary
flow rates and protein
AGUIRRE
et al.
concentration individuals
in periodontally
Sialometry (ml/mitt/gland f SEM) Submandibular sublingual
Group Healthy (A) Range Diseased Range
(B)
Total Values are the means
0.31 + 0.03 0.0770.60 (n = 22) 0.31 f 0.03 0.10-0.71 (n = 28) 0.31 (50) for the number
or pg/mg of total salivary protein. day-to-day reproducibility of assays, stimulated submandibular-sublingual salivas from four clinically healthy pooled, subdivided and frozen at samples were used as controls for protein and ELISA assays.
Submandibular sublingual
0.38 f 0.07 0.03-l .65 (n = 22) 0.32 + 0.03 0.03GO.77 (n = 30) 0.34
Cysteine protease inhibitory activity The cysteine protease inhibitory activity of salivas was assessed by a modification of the procedure of Barrett and Kirschke (1981). The volume of submandibular-sublingual saliva used was based upon the concentration of cystatin previously determined immunologically. As the immunological data indicated that large volumes of parotid saliva would be required, only submandibular-sublingual (n = 44) was assayed. For each sample, five different cystatin concentrations (0.62; 1.25; 2.5; 5.0 and 10 kg, each in duplicate) were mixed with 200 ~1 of 0.4 M sodium potassium phosphate, pH 6.0, containing 8 mM dithiothreitol and 4 mM EDTA. Papain (0.4 units in 0.1% Brij 35) was added and the solutions incubated for 10 min at 40°C. Subsequently, 250 ~1 of a freshly prepared solution containing 20 PM N-a-benzoylL-arginine-7-amido-4-methylcoumarin (in distilled water) were added and incubated for 10 min at 4OC. The enzymatic reaction was ended by the addition of 0.1 M sodium monochloroacetate in 0.1 M sodium acetate, pH 4.0. Release of the N-a-benzoyl-targinine-7-amido-4-methylcoumarin was determined by fluorescence spectroscopy (excitation wavelength = 345 nm; emission wavelength = 438 nm) in a 65040 fluorescence spectrophotometer (Perkin-Elmer, Norwalk, CT. U.S.A.).
Parotid
1.72&0.10 0.87-2.77 (It = 22) 1.85 & 0.09 1.143.49 (n = 28) 1.79
(52)
To monitor the 2% citric acidand parotid subjects were -20°C. These all subsequent
and diseased
Total protein (mg/ml f SEM)
Parotid
of subjects
healthy
2.87 k 0.25 0.96606 (n = 22) 3.1 I + 0.23 1.15-6.83 (n = 30) 3.01 (52)
(50) indicated
in the parentheses.
were done, a significance level of 1% was chosen reduce the risk of a statistical type I error.
to
RESULTS
Sialometry The average salivary flow rates (ml/min/gland) for periodontally healthy and diseased subjects are shown in Table 1. No significant differences were found in the flow rates of parotid (p = 0.7248) and submandibular-sublingual saliva (p = 0.8679) between periodontally healthy and diseased subjects. Salivary protein content Protein concentrations by the Arneberg method are shown in Table 1. No statistically significant differences were observed between periodontally healthy and diseased subjects (submandibular-sublingual saliva: p = 0.3951; parotid saliva: p = 0.3083). Quantitation of salivary cystatins Immunochemical quantitation of cystatins in submandibular-sublingual saliva revealed a mean value Salivary Cystatin Concentration 10
500 0
8
0 0
Statistical analysis The non-parametric Mann-Whitney U-test was used to analyse the data. The variables evaluated were salivary flow rate (ml/min/gland), protein concentration (mg/ml), salivary cystatin concentration @g/ml), salivary cystatin secretion rate (pg/ml/min) and cystatin content per total salivary protein @g/ml protein). These variables were determined for both salivas. In addition, an examination of the cysteine protease inhibitory activity for submandibularsublingual saliva was done. Because several statistical tests for variables involving salivary cystatin levels
Fig. 3. The left-hand side of the diagram shows the cystatin concentration (O-500 pg/ml) in submandibular-sublingual saliva (HSMSL) of 22 subjects without periodontal disease (group A) and 28 individuals with periodontal disease (group B). The right-hand side of the diagram shows the cystatin values (O--l0 pg/ml) for parotid saliva (HPS). Each data point represents a single individual. The mean and SEM are represented by the corresponding bars.
359
Salivary cystatins in older adults Table 2. Rate of cystatin secretion and cystatin content in periodontally healthy and diseased
individuals Cystatin content
Cystatin secretion (pg/min/gland k SEM) Submandibularr sublingual
Group Healthy (A) Range Diseased (B) Range Total
Parotid
40.62 + 7.59 1.19-145.50 28.30 _+4.42 3.85-108.57 34.46 k 6.00
0.51 +0.16 o.Ot-3.10 0.34 + 0.92 0.00-2.03 0.45 & 0.12
of 129.7 f 100.9 pg/ml for subjects without periodontal disease (group A) and 92.0 f 58.1 pg/ml for subjects with periodontal disease (group B). The cystatin concentration in parotid saliva had a mean value of 1.6 f 2.0 pg/ml for group A (n = 22) and 1.2 + 1.8 pg/ml for group B (n = 30), almost lOO-fold less than in submandibular-sublingual saliva (Text Fig. 3). The SD for both secretions were rather large, indicating a wide individual variation in cystatin concentrations (Text Fig. 3). However, no statistically significant differences were observed between groups A and B (submandibular-sublingual saliva: p = 0.1537; parotid saliva: p = 0.5784). The antibody used for these studies cross-reacts with cystatin S, SA, and SN [Plate Fig. 2 (B)]. Therefore the relative amounts of these different cystatins in salivas cannot be distinguished. Assessment of the rate of cystatin secretion (pg/ml/min) for both salivas was also made (Table 2). Again, no significant differences were observed between groups A and B (submandibular-sublingual saliva: p = 0.1537; parotid saliva; p = 0.5784). Similarly, no statistically significant differences were observed between groups A and B when salivary cystatin content was expressed on the basis of total salivary protein (submandibular-sublingual saliva: p = 0.0820; parotid saliva: p = 0.3544; Table 2). Cysteine protease
inhibition activity
Next, studies were done to determine munochemical quantitation of cystatins
if the imcorrelated
HSMSL Cysteine Protease Inhibition
450.
00
400 350 300 ;
250 200 150
0
0
0%
00;
i
100’ i
50 O-
? A
f
(p g/mg protein + SEM)
i +P w B
Fig. 4. Cysteine protease inhibitor activity of submandibular-sublingual from 18 subjects without periodontal disease (group A) and 26 individuals with periodontal disease (group B). Each data point represents the volume of saliva from a single individual that was needed to achieve 100% inhibition of papain activity. The mean and SEM are represented by the corresponding bars.
Submandibularsublingual 70.04 k 8.95 4.64191.17 50.49 + 5.30 5.81-120.76 60.26 + 7.12
Parotid 0.52+0.12 0.00-l .85 0.38kO.ll 0.00-2.4 I 0.45*0.11
with the biological activity of these proteins in saliva. Under our assay conditions, _ 1Opg of purified salivary cystatin SN were required to give 100% inhibition of papain activity. Therefore for each submandibular-sublingual sample, portions containing varying amounts of cystatin (0.62-10 pg) were tested. Preliminary screening of the data showed that complete inhibition of papain activity by submandibular-sublingual salivas was directly correlated to salivary cystatin concentration. As shown in Text Fig. 4, each data point represents the volume of that saliva from a single individual which was needed to achieve 100% inhibition of papain activity. Wide individual variation in cysteine protease inhibition activity was observed in periodontally healthy (group A) and diseased (group B) subjects. However, there were no statistically significant differences between the two groups for the amounts of cystatin required to give 100% inhibition of papain activity. DISCUSSION
Our data for the flow rate of stimulated parotid saliva (0.34 ml/min/gland; n = 52) are approximately the same as those obtained in several earlier studies (Ben-Aryeh et al., 1986; Wolf et al., 1990; Ship, Fox and Baum, 1991) but are lower than those reported by others (Benedek-Spat, 1973; Baum, 1981; Heft and Baum, 1984). Likewise, the average flow rate for stimulated submandibular-sublingual saliva (2 = 0.31 ml/min/gland; n = 50) was similar to that reported by several groups (Tylenda et al., 1988; Wolf et al., 1990; Ship et al., 1991). In contrast, our data were higher than those reported by Pedersen et al. (1985) but lower than those reported by Ericson, Hedin and Wiberg (1972). Differences among these various cross-sectional studies may be the result of several factors including the collection technique/device or the type and frequency of gustatory stimulation. Another factor to be considered is the consumption of prescription and non-prescription medications, many of which have been associated with salivary gland hypofunction (Sreebny and Schwartz, 1986). Interestingly, the use of prescription medication by some of our subjects had no apparent effect on salivary flow rates, as demonstrated by segregation and comparison of data between medicated and non-medicated groups (data not shown). For the analysis of total protein concentration in human salivas, both the Lowry and Arneberg methods have been used. The Lowry method is dependent on the presence of aromatic amino acids
360
A. AGURREet al.
(Lowry et al., 1951) while the Arneberg method relies on peptide-bond absorbance at 215 nm (Arneberg, 1971). Because the Lowry assay tends to underestimate the total protein content of saliva samples, we chose the Arneberg method, which has been adapted for use with small amounts of salivary fluids (Johnson and Cortez, 1988). Our data for the total protein concentration in parotid saliva (n = 3.01 mg/ml; n = 52) were similar to those (a = 2.87 mg/ml, n = 50) reported by Baum (1981) in which a population with an age range of 60-88 yr of age was screened. We both used the Arneberg method, suggesting that it provides a reliable and reproducible technique for the assessment of total salivary protein concentration. The majority of cystatins (S, SA and SN) in whole saliva arise from the salivary glands while the small amount of cystatin C is probably derived from the crevicular fluid (Skaleric et al., 1989). Our findings are similar to those obtained by Shomers et al. (1982b), who used rocket immunoelectrophoresis to determine salivary cystatin levels. In that study, the concentration of cystatin in submandibular-sublingual saliva was 163 f 137 pg/ml (n = 30) and in parotid saliva, 5.0 + 3.3 pg/ml (n = 15). The acinar parenchyma of healthy parotid glands is composed exclusively of serous cells while the submandibular and sublingual glands contain both serous and mucous cells. Immunocytochemical studies have shown that salivary cystatins are produced in the serous cells of parotid and submandibular glands (Isemura et al., 1984b; Bobek, Aguirre and Levine, 1991). Our data and those reported by Shomers et al. that human submandibular(1982b) indicate sublingual saliva contains approx. 100 times as much cystatin as parotid saliva. These findings are supported by the work of Rathman et al. (1990), who found that the cysteine protease inhibitory activity of submandibular saliva was much higher than that of parotid fluids. In situ hybridization studies have shown a higher content of cystatin transcripts in the human submandibular gland than in the parotid (Bobek, Aguirre and Levine, 1991). These observations are somewhat perplexing because the parotid contains a higher abundance of serous acinar cells. The molecular events responsible for this difference remain to be elucidated. A study by Ship et al. (1991) suggests that longitudinal studies of salivary secretions that examine individual changes over time may prove a more appropriate strategy for assessing the impact of saliva on oral health. Their observations were based upon the large individual variations obtained in cross-sectional sialochemical studies. Thus, monitoring an individual’s salivary cystatin levels over time may provide a better indicator of oral health status than comparisons made from cross-sectional studies. Still, such longitudinal measurements may not provide a subtle enough indicator of periodontal disease status. It may be necessary to monitor crevicular fluid for serum cystatins. For example, a role of serum cystatin in the modulation of periodontal disease was suggested by Skaleric et al. (1989), who found an inverse correlation between the levels of cystatin C in gingival homogenates and the degree of gingivitis and
periodontitis. Further studies explore these possibilities.
are
underway
to
Acknowledgements-This
study was supported in part by USPHS Grants DE52559, DE08240 and DE07585 We thank Dr Sara Grossi for evaluating the periodontal status of the subjects involved in this study. We also thank Drs Pamela Jones and Frank Scannapieco for their critical review of this manuscript. REFERENCES
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Sandholm L. (1986) Proteases and their inhibitors in chronic inflammatory periodontal disease. J. clin. Periodonr. 13, 19-25.
Scannapieco F. A., Bergey E. J., Reddy M. S. and Levine M. J. (1989) Characterization of salivary alpha-amylase binding to Streptococcus sanguis. Infect. Immun. 57, 2853-2863.
Ship J. A., Fox P. C. and Baum B. J. (1991) How much saliva is enough? JADA 122, 63369. Shomers J. P., Tabak L. A., Levine M. J., Mandel I. D. and Ellison S. A. (1982a) Characterization of cysteinecontaining phosphoproteins from human submandibularsublingual saliva. J. denf. Res. 6, 764-767. Shomers J. P., Tabak L. A., Levine M. J., Mandel I. D. and Hay D. I. (1982b) Properties of cysteine-containing phosphoproteins from human submandibular-sublingual saliva. J. dent. Res. 61, 397-399. Skaleric U., Babnik J., Curin V., Lah T. and Turk V. (1989) Immunochemical quantitation of cysteine proteinase inhibitory cystatin C in inflamed human gingiva. Archs oral Biol. 4, 301-305.
Sreebny L. M. and Schwartz S. (1986) Reference guide to drugs and dry mouth. Gerodontologia 5, 75-99. Towbin H., Staehlin T. and Gordon J. (1979) Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc. nam. Acad. Sri., U.S.A. 76, 43504354.
Tylenda C. A., Ship J. A., Fox P. C. and Baum B. J. (1988) Evaluation of submandibular salivary flow rate in different age groups. J. dent. Res. 67, 122551228. Voller A., Bidwell D. E. and Bartlett A. (1976) Enzyme immunoassays in diagnostic medicine: Theory and practice. Bull. Wld Hlth Org. 53, 5565. Wolf A., Fox P. C., Ship J. A., Atkinson J. C., Macynski A. A. and Baum B. J. (1990) Oral mucosal status and major salivary gland function. Oral Surg. 70, 49-54.
Archs oral Bid. Vol. 37, No. 5, pp. 355-361, 1992 Printed in Great Britain. All rights reserved
LEVELS OF SALIVARY CYSTATINS IN PERIODONTALLY HEALTHY AND DISEASED OLDER ADULTS A.
AGUIRRE, L. A. TESTA-WEINTRAUB, J. A. BANDERAS, R. DUNFORD
Department
and M. J.
LEVINE
of Oral Biology and Dental Research Institute, 109 Foster Hall, School of Dental Medicine, State University of New York at Buffalo, Buffalo, NY 14214, U.S.A. (Accepted 13 November 1991)
Summary-Cystatins are cysteine protease inhibitors present in a variety of tissues and body fluids, including saliva. One possible function of these molecules may be to modulate tissue destruction in periodontal diseases. To investigate the potential role of salivary cystatins in these events, the levels of cystatins in saliva from periodontally healthy and diseased individuals were measured by enzyme-linked immunosorbent assay. Flow rates and total protein content were determined in all the samples collected, while cysteine protease inhibitory activity was assessed in submandibular-sublingual secretions. Statistical analysis showed no significant differences in the levels and activity of salivary cystatins in periodontally healthy and diseased individuals. These findings suggest that comparing the levels of cystatins in glandular salivas may not be a suitable indicator of periodontal disease status. Key words: cystatins. saliva, cysteine protease inhibitor, adults, periodontitis, ageing.
INTRODUCTION
The cystatin superfamily comprises a diverse group of cysteine protease inhibitors widely distributed in mammalian tissues and plasma. They protect the organism against the uncontrolled action of endogenous and/or exogenous cysteine proteinases (Lindhal et al., 1988). Originally, members of the cystatin superfamily were grouped into three families; family I or stefins, family II or cystatins (including salivary cystatins) and family III or kininogens (Barrett et al., 1986a, b). However, additional families were suggested by Rawlings and Barrett (1990) to accommodate those proteins that cannot be included in the three established families. Human salivary cystatin genes are part of a multigene family composed of seven members segregated on chromosome 20 (Saitoh et al., 1989). These were first recognized as cysteine-containing phosphoproteins (Shomers et cd, 1982a), but it was not until recently that they were identified as cysteine proteinase inhibitors (Isemura, Saitoh and Sanada, 1984a, 1986, 1987; Isemura et al., 1984b). Salivary cystatins S (SAP-l), SN (SA-I) and SA contain 121 amino acids and have -90% sequence homology. In addition, salivary cystatins have a 54% sequence homology with serum cystatin C, which is present in the gingival crevicular fluid and consequently in the oral milieu. Impaired regulation of proteolysis is an important biochemical aspect in the aetiology of periodontal disease (Sandholm, 1986). Recent studies have shown increased levels of cysteine proteases (cathepsins D, B and L) with increasing severity of inflammation in immunosorbent Abbreviations : ELISA, enzyme-linked assay; SDS-PAGE, sodium dodecyl sulphate-polyacrylamide gel electrophoresis.
partially purified gingival homogenates (Lah et al., 1985) and crevicular fluid (Eisenhauer et al., 1983; Lah et al., 1986). Likewise, gingival homogenates obtained from periodontally diseased sites show a decrease in levels of cystatin C that is inversely correlated with the degree of periodontal disease (Skaleric et al., 1989). However, it has not been determined if levels of glandular salivary cystatins are also associated with periodontal disease status. Such an association might serve as an indicator of disease activity. Accordingly, our goal was to see if there is a correlation between the levels of glandular salivary cystatins and their cysteine protease inhibitory activity, and periodontal status. MATERIALS AND METHODS
Materials
Lashley cups were from H & I Instrumentation (Teaneck, NJ, U.S.A.). Block-Brotman universal collectors were from Flory Dental Prosthetics (Star City, WV, U.S.A.). Citric acid (anhydrous), ammonium sulphate, sodium hydroxide and Btij 35 were from Fisher Scientific Company (Fair Lawn, NJ, U.S.A.). Vinyl polysiloxane impression material (Express, light body-fast set), dispenser and disposable mixing tips were from 3 M (St Paul, MN, U.S.A.). Papain (P-3125), disodium p-nitrophenyl phosphate, N-abenzoyl-L-arginine-7-amido-4-methyl-coumarin (B7260), alkaline phosphatase from bovine intestinal mucosa (P-6774) and bovine serum albumin (A-7906) were from Sigma (St Louis, MO, U.S.A.). Nitrocellulose membranes (0.45 pm), horseradish peroxidaseconjugated goat anti-rabbit serum (IgG H + L chains) and 4-chloro-1-naphthol were from Bio-Rad (Richmond, CA, U.S.A.). Freund’s complete and incomplete adjuvants were from GIBCO (Grand Island, NY, U.S.A.). ELISA plates (96 wells, 355
356
A. AGU~RRE el al.
modified flat bottom, 25805-96) were from Corning Glass Works (Corning, NY, U.S.A.). Patient population
Clinically healthy individuals of both sexes (age 55-74) were recruited from the general population of Buffalo, NY, U.S.A. Some of these individuals were taking prescription medication, in particular antihypertensive drugs. Patients selected had at least 16 natural teeth (two of which were molars) with no fixed prosthetic devices obscuring the cementumenamel junction and no previous history of periodontal surgical treatment. The criterion (Grossi et al., 1990) used to categorize the subject’s periodontal status was as follows. Group A consisted of individuals without periodontitis (all teeth had interproximal attachment loss of 3 mm or less); group B was composed of individuals with moderate to severe periodontitis (periodontal attachment loss of 6 mm or more in the interproximal sites of at least two different teeth). All participants were screened by an examiner who had undergone suitable training for calibration (Miller et af., 1987). A total of 52 subjects were selected, 22 individuals in group A and 30 in group B. Saliva collection
Subjects refrained from eating, drinking, smoking and oral hygiene for 2 h for saliva collection. To collect submandibular-sublingual saliva a customized Block-Brotman device was used (Block and Brotman, 1962); parotid saliva was collected with a Lashley cup (Lashley, 1916). Salivas were stimulated by the application of 2% citric acid to the sides of the tongue at 30-s intervals and the first ml of saliva was discarded (Baum, 1981). Saliva was collected into chilled preweighed polypropylene tubes whereby flow rates were determined gravimetrically and expressed as ml/min/gland. The salivas were then divided into IOO-~1samples and frozen at -20°C. Before analysis, samples were slowly thawed in an ice bath and Na,EDTA was added to give a final concentration of 5 mM for submandibular-sublingual and 1 mM for parotid saliva (Fox et al., 1986). Protein content
Total protein content of salivary samples was determined from the absorbance at 215 nm as described by Arneberg (1971). Purification ofcystatin SN and preparation of antibog),
Salivary cystatin SN was purified (Al-Hashimi, Dickinson and Levine, 1988; Ramasubbu et al.. 1991) for use as an immunogen to raise polyclonal anti-
bodies and as a standard for ELISA and cysteine protease inhibition assays. Protein content of cystatin preparations was determined by amino acid analysis (Al-Hashimi et al., 1988). To obtain monospecific polyclonal antisera, 6-week-old female New Zealand white rabbits were immunized as described by Aguirre et al. (1987). In brief, subcutaneous dorsal injections of cystatin SN (200 p g protein/ml 0.154 M NaCl) emulsified with Freund’s complete adjuvant (1: 1, v/v) were used to prime the animals. Subcutaneous booster injections were given at &week intervals with 100 pg protein/ml of 0.154M NaCl emulsified with Freund’s incomplete adjuvant. After 10 days, titres were monitored by SDS-PAGE (Laemmli, 1970) and anionic-PAGE (Ornstein, 1964) followed by immunoblotting (Towbin, Staehlin and Gordon, 1979). This demonstrated that the rabbit antiserum (dilution: l/60,000) was specific for salivary cystatins, as shown by the presence of single bands in both salivas with mobility comparable to purified cystatin SN [Plate Fig. 1 (B)]. Furthermore, anionic-PAGE/immunoblotting revealed that this antiserum recognized several members of the salivary cystatin family and was not reactive with amylase [Plate Fig. 2 (B)]. For ELISA, concentrated IgG fractions were obtained by ammonium sulphate precipitation (Beutner et al., 1987) and subsequently conjugated to alkaline phosphatase (Voller, Bidwell and Bartlett, 1976). immunoquantitation of salivary cystatins
Salivary cystatins were quantitated by an ELISA, double-antibody sandwich technique in 96-well plates. In brief, plates were first coated with IgG fractions (1: 500 in 0.05 M sodium carbonate buffer, pH 9.6) and incubated at 37°C for 2 h. After washing (0.154 M NaCI, 0.05% Tween-20), saliva samples were assayed at three different dilutions, each in triplicate. Standard curves were generated using known quantities of cystatin SN (0.062.1 ng protein, each in triplicate). The plates were then incubated at 37’C for 1 h. After a second washing, IgG conjugated to alkaline phosphatase was added and incubated overnight at 25’C. After another wash, disodium p-nitrophenyl phosphate (1 mg/ml of 0.05 M sodium carbonate buffer, pH 9.8 with 1 mM MgCl,) was added for colour development and incubated for 60 min at 25’C. Plates were read at 405 nm in a 2550 ELISA Reader (Bio-Rad, Richmond, CA, U.S.A.) interfaced to a Macintosh computer. Salivary cystatin values were obtained from a regression line derived from the standards (Mac ReaderTM 2.0 Program) and the results expressed as /*g/ml saliva, pg/ml/min
Plate I Fig. I. Panel A shows a 10% SDS-PAGE gel stained with Coomassie blue and the corresponding immunoblot in panel B. Lane I, low molecular-weight standards; lane 2, submandibular-sublingual saliva (25 pg); lane 3, cystatin SN (2.5 pg): and lane 4 parotid saliva (25 pg). Fig. 2. Panel A shows a 7.5% anionic-PAGE gel stamed with Coomassie blue and the corresponding immunoblot in panel B. Lane 1, submandibular-sublingual saliva (IO pg); lane 2, salivary a-amylase [Scannapieco et al. (1989) 3 rg]; lanes 3-5, enriched fractions of cystatin SN (0.5 pg), cystatin SA (2.5 pg) and cystatin S (1 .O pg), respectively (prepared as described by Al-Hashimi et al., 1988; Ramasubbu et al., 1991). No bands were seen when the primary antibody was substituted with rabbit non-immune sera.
Salivary cystatins in older adults
(I 1
A
B
_,
1234
(2)
234
A
12
351
B
1
345 Plate
I
2
3
4
5
A.
358 Table
I. Salivary
flow rates and protein
AGUIRRE
et al.
concentration individuals
in periodontally
Sialometry (ml/mitt/gland f SEM) Submandibular sublingual
Group Healthy (A) Range Diseased Range
(B)
Total Values are the means
0.31 + 0.03 0.0770.60 (n = 22) 0.31 f 0.03 0.10-0.71 (n = 28) 0.31 (50) for the number
or pg/mg of total salivary protein. day-to-day reproducibility of assays, stimulated submandibular-sublingual salivas from four clinically healthy pooled, subdivided and frozen at samples were used as controls for protein and ELISA assays.
Submandibular sublingual
0.38 f 0.07 0.03-l .65 (n = 22) 0.32 + 0.03 0.03GO.77 (n = 30) 0.34
Cysteine protease inhibitory activity The cysteine protease inhibitory activity of salivas was assessed by a modification of the procedure of Barrett and Kirschke (1981). The volume of submandibular-sublingual saliva used was based upon the concentration of cystatin previously determined immunologically. As the immunological data indicated that large volumes of parotid saliva would be required, only submandibular-sublingual (n = 44) was assayed. For each sample, five different cystatin concentrations (0.62; 1.25; 2.5; 5.0 and 10 kg, each in duplicate) were mixed with 200 ~1 of 0.4 M sodium potassium phosphate, pH 6.0, containing 8 mM dithiothreitol and 4 mM EDTA. Papain (0.4 units in 0.1% Brij 35) was added and the solutions incubated for 10 min at 40°C. Subsequently, 250 ~1 of a freshly prepared solution containing 20 PM N-a-benzoylL-arginine-7-amido-4-methylcoumarin (in distilled water) were added and incubated for 10 min at 4OC. The enzymatic reaction was ended by the addition of 0.1 M sodium monochloroacetate in 0.1 M sodium acetate, pH 4.0. Release of the N-a-benzoyl-targinine-7-amido-4-methylcoumarin was determined by fluorescence spectroscopy (excitation wavelength = 345 nm; emission wavelength = 438 nm) in a 65040 fluorescence spectrophotometer (Perkin-Elmer, Norwalk, CT. U.S.A.).
Parotid
1.72&0.10 0.87-2.77 (It = 22) 1.85 & 0.09 1.143.49 (n = 28) 1.79
(52)
To monitor the 2% citric acidand parotid subjects were -20°C. These all subsequent
and diseased
Total protein (mg/ml f SEM)
Parotid
of subjects
healthy
2.87 k 0.25 0.96606 (n = 22) 3.1 I + 0.23 1.15-6.83 (n = 30) 3.01 (52)
(50) indicated
in the parentheses.
were done, a significance level of 1% was chosen reduce the risk of a statistical type I error.
to
RESULTS
Sialometry The average salivary flow rates (ml/min/gland) for periodontally healthy and diseased subjects are shown in Table 1. No significant differences were found in the flow rates of parotid (p = 0.7248) and submandibular-sublingual saliva (p = 0.8679) between periodontally healthy and diseased subjects. Salivary protein content Protein concentrations by the Arneberg method are shown in Table 1. No statistically significant differences were observed between periodontally healthy and diseased subjects (submandibular-sublingual saliva: p = 0.3951; parotid saliva: p = 0.3083). Quantitation of salivary cystatins Immunochemical quantitation of cystatins in submandibular-sublingual saliva revealed a mean value Salivary Cystatin Concentration 10
500 0
8
0 0
Statistical analysis The non-parametric Mann-Whitney U-test was used to analyse the data. The variables evaluated were salivary flow rate (ml/min/gland), protein concentration (mg/ml), salivary cystatin concentration @g/ml), salivary cystatin secretion rate (pg/ml/min) and cystatin content per total salivary protein @g/ml protein). These variables were determined for both salivas. In addition, an examination of the cysteine protease inhibitory activity for submandibularsublingual saliva was done. Because several statistical tests for variables involving salivary cystatin levels
Fig. 3. The left-hand side of the diagram shows the cystatin concentration (O-500 pg/ml) in submandibular-sublingual saliva (HSMSL) of 22 subjects without periodontal disease (group A) and 28 individuals with periodontal disease (group B). The right-hand side of the diagram shows the cystatin values (O--l0 pg/ml) for parotid saliva (HPS). Each data point represents a single individual. The mean and SEM are represented by the corresponding bars.
359
Salivary cystatins in older adults Table 2. Rate of cystatin secretion and cystatin content in periodontally healthy and diseased
individuals Cystatin content
Cystatin secretion (pg/min/gland k SEM) Submandibularr sublingual
Group Healthy (A) Range Diseased (B) Range Total
Parotid
40.62 + 7.59 1.19-145.50 28.30 _+4.42 3.85-108.57 34.46 k 6.00
0.51 +0.16 o.Ot-3.10 0.34 + 0.92 0.00-2.03 0.45 & 0.12
of 129.7 f 100.9 pg/ml for subjects without periodontal disease (group A) and 92.0 f 58.1 pg/ml for subjects with periodontal disease (group B). The cystatin concentration in parotid saliva had a mean value of 1.6 f 2.0 pg/ml for group A (n = 22) and 1.2 + 1.8 pg/ml for group B (n = 30), almost lOO-fold less than in submandibular-sublingual saliva (Text Fig. 3). The SD for both secretions were rather large, indicating a wide individual variation in cystatin concentrations (Text Fig. 3). However, no statistically significant differences were observed between groups A and B (submandibular-sublingual saliva: p = 0.1537; parotid saliva: p = 0.5784). The antibody used for these studies cross-reacts with cystatin S, SA, and SN [Plate Fig. 2 (B)]. Therefore the relative amounts of these different cystatins in salivas cannot be distinguished. Assessment of the rate of cystatin secretion (pg/ml/min) for both salivas was also made (Table 2). Again, no significant differences were observed between groups A and B (submandibular-sublingual saliva: p = 0.1537; parotid saliva; p = 0.5784). Similarly, no statistically significant differences were observed between groups A and B when salivary cystatin content was expressed on the basis of total salivary protein (submandibular-sublingual saliva: p = 0.0820; parotid saliva: p = 0.3544; Table 2). Cysteine protease
inhibition activity
Next, studies were done to determine munochemical quantitation of cystatins
if the imcorrelated
HSMSL Cysteine Protease Inhibition
450.
00
400 350 300 ;
250 200 150
0
0
0%
00;
i
100’ i
50 O-
? A
f
(p g/mg protein + SEM)
i +P w B
Fig. 4. Cysteine protease inhibitor activity of submandibular-sublingual from 18 subjects without periodontal disease (group A) and 26 individuals with periodontal disease (group B). Each data point represents the volume of saliva from a single individual that was needed to achieve 100% inhibition of papain activity. The mean and SEM are represented by the corresponding bars.
Submandibularsublingual 70.04 k 8.95 4.64191.17 50.49 + 5.30 5.81-120.76 60.26 + 7.12
Parotid 0.52+0.12 0.00-l .85 0.38kO.ll 0.00-2.4 I 0.45*0.11
with the biological activity of these proteins in saliva. Under our assay conditions, _ 1Opg of purified salivary cystatin SN were required to give 100% inhibition of papain activity. Therefore for each submandibular-sublingual sample, portions containing varying amounts of cystatin (0.62-10 pg) were tested. Preliminary screening of the data showed that complete inhibition of papain activity by submandibular-sublingual salivas was directly correlated to salivary cystatin concentration. As shown in Text Fig. 4, each data point represents the volume of that saliva from a single individual which was needed to achieve 100% inhibition of papain activity. Wide individual variation in cysteine protease inhibition activity was observed in periodontally healthy (group A) and diseased (group B) subjects. However, there were no statistically significant differences between the two groups for the amounts of cystatin required to give 100% inhibition of papain activity. DISCUSSION
Our data for the flow rate of stimulated parotid saliva (0.34 ml/min/gland; n = 52) are approximately the same as those obtained in several earlier studies (Ben-Aryeh et al., 1986; Wolf et al., 1990; Ship, Fox and Baum, 1991) but are lower than those reported by others (Benedek-Spat, 1973; Baum, 1981; Heft and Baum, 1984). Likewise, the average flow rate for stimulated submandibular-sublingual saliva (2 = 0.31 ml/min/gland; n = 50) was similar to that reported by several groups (Tylenda et al., 1988; Wolf et al., 1990; Ship et al., 1991). In contrast, our data were higher than those reported by Pedersen et al. (1985) but lower than those reported by Ericson, Hedin and Wiberg (1972). Differences among these various cross-sectional studies may be the result of several factors including the collection technique/device or the type and frequency of gustatory stimulation. Another factor to be considered is the consumption of prescription and non-prescription medications, many of which have been associated with salivary gland hypofunction (Sreebny and Schwartz, 1986). Interestingly, the use of prescription medication by some of our subjects had no apparent effect on salivary flow rates, as demonstrated by segregation and comparison of data between medicated and non-medicated groups (data not shown). For the analysis of total protein concentration in human salivas, both the Lowry and Arneberg methods have been used. The Lowry method is dependent on the presence of aromatic amino acids
360
A. AGURREet al.
(Lowry et al., 1951) while the Arneberg method relies on peptide-bond absorbance at 215 nm (Arneberg, 1971). Because the Lowry assay tends to underestimate the total protein content of saliva samples, we chose the Arneberg method, which has been adapted for use with small amounts of salivary fluids (Johnson and Cortez, 1988). Our data for the total protein concentration in parotid saliva (n = 3.01 mg/ml; n = 52) were similar to those (a = 2.87 mg/ml, n = 50) reported by Baum (1981) in which a population with an age range of 60-88 yr of age was screened. We both used the Arneberg method, suggesting that it provides a reliable and reproducible technique for the assessment of total salivary protein concentration. The majority of cystatins (S, SA and SN) in whole saliva arise from the salivary glands while the small amount of cystatin C is probably derived from the crevicular fluid (Skaleric et al., 1989). Our findings are similar to those obtained by Shomers et al. (1982b), who used rocket immunoelectrophoresis to determine salivary cystatin levels. In that study, the concentration of cystatin in submandibular-sublingual saliva was 163 f 137 pg/ml (n = 30) and in parotid saliva, 5.0 + 3.3 pg/ml (n = 15). The acinar parenchyma of healthy parotid glands is composed exclusively of serous cells while the submandibular and sublingual glands contain both serous and mucous cells. Immunocytochemical studies have shown that salivary cystatins are produced in the serous cells of parotid and submandibular glands (Isemura et al., 1984b; Bobek, Aguirre and Levine, 1991). Our data and those reported by Shomers et al. that human submandibular(1982b) indicate sublingual saliva contains approx. 100 times as much cystatin as parotid saliva. These findings are supported by the work of Rathman et al. (1990), who found that the cysteine protease inhibitory activity of submandibular saliva was much higher than that of parotid fluids. In situ hybridization studies have shown a higher content of cystatin transcripts in the human submandibular gland than in the parotid (Bobek, Aguirre and Levine, 1991). These observations are somewhat perplexing because the parotid contains a higher abundance of serous acinar cells. The molecular events responsible for this difference remain to be elucidated. A study by Ship et al. (1991) suggests that longitudinal studies of salivary secretions that examine individual changes over time may prove a more appropriate strategy for assessing the impact of saliva on oral health. Their observations were based upon the large individual variations obtained in cross-sectional sialochemical studies. Thus, monitoring an individual’s salivary cystatin levels over time may provide a better indicator of oral health status than comparisons made from cross-sectional studies. Still, such longitudinal measurements may not provide a subtle enough indicator of periodontal disease status. It may be necessary to monitor crevicular fluid for serum cystatins. For example, a role of serum cystatin in the modulation of periodontal disease was suggested by Skaleric et al. (1989), who found an inverse correlation between the levels of cystatin C in gingival homogenates and the degree of gingivitis and
periodontitis. Further studies explore these possibilities.
are
underway
to
Acknowledgements-This
study was supported in part by USPHS Grants DE52559, DE08240 and DE07585 We thank Dr Sara Grossi for evaluating the periodontal status of the subjects involved in this study. We also thank Drs Pamela Jones and Frank Scannapieco for their critical review of this manuscript. REFERENCES
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Eisenhauer D. A., Hutchinson R., Javed T. and McDonald J. K. (1983) Identification of a cathepsin B-like protease in creiicular fluid. J. dent. Res. 62, $17-921. _ Ericson S., Hedin M. and ‘Wiberg A. (1972) Variability of the submandibular flow rate in man with special reference to the size of the gland. Odonl. Reuy 23, 41 l-420. Fox P. C.. van der Ven P. F.. Baum B. J. and MandelI. D. (1986) hlocarpine for the treatment of xerostomia associated with salivary gland dysfunction. Oral Surg. 61, 243-248. Grossi S. G., Dunford R. G., Reynolds H. S., Zambon J. J. and Christersson L. (1990) Clinical and microbiological parameters in the diagnosis of severe periodontal disease in older adults. J. dent. Res. 69, Abstract No. 2021. Heft M. W. and Baum B. J. (1984) Unstimulated and stimulated parotid salivary flow rate in individuals of different ages. J. dent. Res. 63, 1182-I 185.
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