Increase of extracellular cathepsin D activity in gingival washings during experimental gingivitis in man

Archs oral Bid.

Vol.22,pp. 375lo 378.PergamonPress1977.Printed

in Great Britain.

INCREASE OF EXTRACELLULAR CATHEPSIN D ACTIVITY. IN GINGIVAL WASHINGS DURING EXPERIMENTAL GINGIVITIS IN MAN A. TZAMOURANIS,J. MATTHYS, I. ISHIKAWA and G. CIMAWNI Division of Physiopathology

and Periodontology, Dental School, Medical Faculty, University of Geneva, Switzerland

Summary--The free and total activities of cathepsin D were measured at regular intervals in the ging:lval washings, collected with individual appliances, in five subjects, during a period of experim’mtal gingivitis. Counts of intact neutrophils and epithelial cells were made. The concentration of both free and total enzyme followed the changes in the clinical signs of gingivitis. The estimated amount of intracellular cathepsin D activity decreased in polymorphonuclear cells during the time of increasing inflammation. The specific activities of enzyme related to the number of neutrophils tended to decrease during the same period.

IINTRODUCTION Lysosomal hydrolases may be involved in the tissue damage associated with gingivitis and periodontitis (Bang, Cimasoni and Held, 1970; Ishikawa, Cimasoni and Ahmed-Zadeh.. 1972; Attstriim, 1975). Latency has been demonstrated for acid phosphatase, fi-glucuronidase and cathepsin D in homogenates of human gingiva (Hasegawa and Cimasoni, 1975) and the concentration or the free portion of these enzymes increased significantly in gingivitis (Hasegawa, Cimasoni and Vuagnat, 1975). In gingival washings collected during experimental gingivitis in man, the concentration of various bacterial and lysosomal hydrolases increased, with maximum values appearing at the time of maximum inflammation (Baehni et al., 1975). However no precise data are available on the process of liberation of lysosomal enzymes either from leucocytes, after their migration into the gingival crevice, or from desquamating epithelial cells. As cathepsin D has a lysosoma1 distribution (Barrett, 1969), a study was undertaken of both the free and total concentrations of cathepsin D at the gingival margin.

MATERIALS AND METHODS Gingiual washings

For the collection of the gingival washings, individual appliances were constructed, allowing the rinsing of the upper crevicular region in the absence of any salivary contaminat Ion (Oppenheim, 1970; Cimasoni, 1974). The appliance consisted of a hard acrylic plate covering the total maxillary area, with soft acrylic borders and a groove along the buccal and palatal marginal region, connected to a peristaltic pump through four plastic tubings. The washings were made by circulating 5 ml of sterile physiological saline for 5 min. Experimental

gingivitis

According to the procedure described by Liie. Theilade and Jensen (19b5), five male subjects 21-24 years

old were given intensive prophylaxis for a period of two weeks until the average gingival index of inflammation approached as close as possible to zero (Ltie, 1967). They were instructed to stop brushing their upper teeth for 21 days and then were allowed to resume their usual dental hygiene. The gingival index of inflammation (L6e, 1967), plaque index (Lije, 1967) and intensity of crevicular fluid flow (Valazza et al., 1972) were evaluated at regular intervals before, during and after the period of experimental gingivitis. The gingival and plaque indices were measured on six points of each tooth and the average calculated for each patient. The flow of crevicular fluid was measured at the right canine tooth and the two central incisors by inserting four filter-paper strips around each of these teeth and weighing the amount of fluid collected per unit of time (Valazza et al., 1972). Gingival washings were performed 2 days before the no-brushing period (day -2), at the start of that period (day 0) and at days +7, + 14, +21, f26 and +34. Determination

of Cathepsin D

Cathepsin D (EC 3.4.4.23) was determined by a modification of the method of Anson (1938), using haemoglobin from beef blood (Sigma Chemical Co., St. Louis, U.S.A.) dissolved at a concentration of 1.5 per cent in 0.1 M Mcllvaine (citric acid-sodium phosphate) buffer, pH 3.5 as substrate. A sample of 0.5 ml of washing was mixed with 0.5ml of substrate solution and incubated for 15 h at 37°C. To terminate the reaction, 1 ml of cold 0.3 M-trichloroacetic acid (T.C.A.) was added and the mixture was filtered. The aromatic degradation products in a 0.5 ml aliquot of the filtrate were measured spectrophotometrically by the method of Lowry et al. (1951) using tyrosine (Sigma Chemical Co, St. Louis, USA) in 0.15 M T.C.A. as a standard. Blank assays were performed on substrate solution incubated in the absence of gingival washing and to which, following termination of the incubation with T.C.A., 0.5 ml of washing was added. The solution was filtered and the concentration of the degradation products determined. The 315

A. Tzamouranis,

376

J. Matthys,

values of the blank determinations were subtracted from those of the test analysis. For the analysis of the total enzymic activity, 2 ml of washing was mixed with 0.1 ml of a 2 per cent solution of Triton X-100 (B.D.H. Chemicals Ltd, Poole, England), resulting in a final detergent concentration of 0.1 per cent. The free portion of enzyme activity was determined after centrifuging the washing solution at 1809 and 4°C for 1Omin.

I. Ishikawa

and G. Cimasoni

3 63”-

Counting of cells Polymorphonuclear leucocytes and epithelial cells with no evident signs of lysis were counted in the washings by using a modification of the method routinely used for cell counting in urine. One millilitre of washing was centrifuged for 5 min at 1000 g in thin microcentrifugation tubes. A sample of 0.95 ml of clear supernatant was then discarded and the counting of the two types of cells was performed under the microscope after transferring the concentrated residue to a Neubauer haematocytometer (American Optical Corp., Buffalo, N.Y., U.S.A.). The original concentration of cells was calculated by dividing the readings by a factor of 20. RESULTS

Gingivitis Figure 1 shows that the average gingival inflammation (GI) and the plaque index (PI), after reaching their lowest value around day 0, started to increase during the no-brushing period and reached a peak at day 21. This was followed by a rapid decline when the hygiene procedures were resumed. The amount of crevicular fluid also increased throughout the nohygiene period and followed, in general, the same pattern of changes as the plaque and gingival indices. Cell counts and actioity of cathepsin D The cell counting showed a rapid increase in the number of polymorphonuclear leucocytes during the no-brushing period; there were 4 to 5 times more

Fig. 2. Concentration (mean f standard error of the mean) of free (0) and total (m) cathepsin D activities in the washings of five subjects during experimental gingivitis. The enzyme activity is expressed as a difference in extinction between experimental and blank at 750nm in 1 ml of washing.

polymorphonuclear leucocytes at day 21 when compared with the number of cells in the washings on days 0 and 28. The number of epithelial cells started to increase only in the final phase of the no-brushing period and reached at day 21 a value which was twice that on day 0. Both the free and the total concentrations of cathepsin D showed a rapid and significant increase during the period of experimental gingivitis (Fig. 2). In general, their pattern of change closely followed that of the clinical indices (Fig. 1). The index obtained by dividing the difference between free and total enzyme activities by the number of polymorphonuclear leucocytes in a given volume of washing gives an estimate of the intracellular cathepsin D activity. This ratio showed a significant decrease from day 0 to day 21 and tended to increase therafter (Fig. 3). A similar pattern was found

Fig. 1. The pattern of variation of the gingival index GI: m---H), the plaque index (PI: t-+) and the intensity of gingival fluid flow (GF: A--.--.-A) before, during and after withdrawal oral hygiene. Each point represents the average of five subjects.

of

Cathepsin D in gingival washings

Fig. 3. Variation of the intracellular concentration of cathepsin D in neutrophils, estimated by the difference between total and free enzymes activity divided by the number of cells in 1 ml of gingival washing. when using the number of epithelial cells instead of leucocytes in the same type of ratio, i.e. total minus free enzyme activity/number of epithelial cells in 1 ml of washing. The variations in the total and free cathepsin D activities, each divided by the numbers of polymorphonuclear leucocytes in 1 ml of washing, showed a rapid decrease from day 0 to day 21 and tended to increase during the period of recovery (Fig. 4). DISCUSSION

Polymorphonuclear leucocytes are probably the main source of the increasing cathepsin D during the time of plaque accumulation, the number of epithelial cells being comparatively low. Human poiymorphonuclear leucocytes have been shown to contain cathepsin D (Ishikawa and Cimasoni, 1977) and these cells are known to predominate in the crevicular area, mononuclear cells accounting for less than 5 per cent (Attstriim, 1975) a’r 8 per cent (Skapski and Lehner, 1975) of the total. Furthermore, as confirmed in our study, the number of neutrophils increases considerably during a period of plaque accumulation and

Fig. 4. Specific activities of free (0) and total (B) cathepsin D. The enzyme activities in 1 ml of washing were divided by the number of polymorphonuclear leucocytes counted in the same volume.

311

gingivitis (Schiott and Liie, 1970; Page and Schroeder, 1976). Polymorphonuclear leucocytes possess a life span of only a few days and cannot synthesize proteins (Ross. 1969). They liberate lysosomal enzymes during phagocytosis or at the moment of degeneration (Attstrom, 1975). Little is known, however, about the viability of polymorphonuclear cells migrating through the gingival crevice (Cimasoni, 1975). Skapski and Lehner (1975), using the trypan blue exclusion test, found that more than 80 per cent of the polymorphonuclear cells collected at the gingival margin were still viable, and evidence of phagocytosis has been observed in gingival fluid neutrophils (Frank and Cimasoni, 1972). It was, however, not determined whether the cells are capable of phagocytosis of bacteria once they have migrated into the crevice. According to Raeste (1972), only 0.6 per cent of the neutrophils collected in mouth rinses have the appearance of intact blood cells; 50 per cent show initial signs of injury with others in more advanced stages of degeneration. Although the proportion of degenerated cells and the degree of lysis were not determined in our investigation, the difference between free and total cathepsin D activity (Fig. 2) suggests that a significant proportion of lysosomal enzyme is within the cells, at least when little or no inflammation is present. This confirms the histochemica1 observations of Lange (1967) that polymorphonuclear leucocytes harvested at the gingival crevice react normally for various intracellular enzymes. Furthermore parallel to the increasing degree of inflammation, less lysosomal enzyme seems to be present within crevice neutrophils (Fig. 3) as could happen with increasingly active phagocytosis. Although the concentrations of both free and total enzyme increased significantly during the inflammatory phase (Fig. 2) their specific activities, referred to the numbers of neutrophils in a volume of washing, did not remain constant and even decreased steadily (Fig. 4). Free lysosomal bodies are known to occur in the gingival crevice (Attstrom, 1975) but their relative contribution to the cathepsin D activity would be difficult to determine. Analysis of the results (Figs. 3 and 4) suggests that the state of polymorphonuclear leucocytes is probably not the same during the various stages of gingivitis. The cells migrating at the time of maximum inflammation contain the lowest level of cathepsin D (Fig. 3) and liberate a minimal amount of enzyme (Fig. 4). This suggests that the number of dying cells, or the intensity of degeneration is proportional to the severity of inflammatory reaction. A similar observation has been made concerning desquamating epitheha1 cells (Cornaz, Cimasoni and Pataki, 1974); the epithelial cells collected at the gingival margin in gingivitis contained less acid phosphatase when compared to cases of normal gingiva. The increasing concentration of extracellular cathepsin D at the gingival margin gives further support to the hypothesis of active participation of lysosomal enzymes in the pathogenesis of periodontitis. Cathepsin D is one of the chief protein-digesting enzymes in lysosomes, present at high concentrations in inflamed tissues. Its action is not hindered by serum inhibitors.

378

A. Tzamouranis,

J. Matthys,

The pattern of liberation of cathepsin D is probably not different from that of other lysosomal enzymes in the migrating leucocytes or in the desquamating epithelial cells at the gingival margin. As pointed out

by Page and Schroeder (1976), an uncontrolled production of lymphokines, prostaglandins and hydrolytic enzymes could be one mechanism of tissue destruction in periodontitis and might account for the difference in susceptibility to bacterial plaque of different individuals.

Acknowledgements--This research was aided by grant 3 604-0.75 of the Swiss national Fund for scientific research. We would like to thank Mrs I. Condacci and Miss E. Andersen for the very diligent technical assistance.

REFERENCES

Anson M. L. 1938. The estimation of pepsin, trypsin papain and cathepsin with haemoglobin. J. gen. Physiol. 22, 79-89. AttstrGm R. 1975. The roles of gingival epithelium and phagocytosing leucocytes in gingival defence. J. c/in. Periodont. 2, 25-32. Baehni P., Sudan J. M., Steube W. and Cimasoni G. 1975. Enzymes lysosomiales dans les produits de rinGage de la rkgion parodontale marginale au tours de la gingivite expbrimentale chez I’homme. J. Biol. buccnle 3, 143-156. Bang J., Cimasoni G. and Held A. J. 1970. Beta-glucuronidase correlated with inflammation in the exudate from human gingiva. Archs oral Biol. 15, 445451. Barrett A. J. 1969. Cathepsin D. In: Lysosomes in Biology and Pathology (Edited by Dingle J. T. and Fell H. B.), Vol. 2, pp. 288-292. North-Holland, Amsterdam. Cimasoni G. 1974. The crevicular fluid. In: Monographs in Oral Science. (Edited by Myers H. M.) Vol. 3 Chap. VI, p. 45. Karger Basel. Cornaz A., Cimasoni G. and Pataki A. 1974. Decrease of acid phosphatase activity in the epithelial cells from inflamed gingivae. Experientia 30, 143-144.

I. Ishikawa

and G. Cimasoni

Frank R. and Cimasoni G. 1972. Electron microscopy of acid phosphatase in the exudate of inflamed gingivae. J. periodont. Res. I, 213-225. Hasegawa K. and Cimasoni G. 1975. The relative amounts of free and latent acid hydrolases in homogenates of human gingiva. Archs oral Biol. 20, 521-526. Hasegawa K., Cimasoni G. and Vuagnat P. 1975. Inflamed gingivae contain more free lysosomal enzyme. Experientia 31, 765-766. Ishikawa I. and Cimasoni G. 1977. Isolation of cathepsin D from human leucocytes. Biochim. biophys. Acta 480, 228-240. Ishikawa I., Cimasoni G. and Ahmad-Zadeh C. 1972. Possible role of lysosomal enzymes in the pathogenesis of periodontitis: a study on cathepsin D in human gingival fluid. Archs oral Biof. 17, 11 I-I 17. Lange D. 1967. Uber das Vorkommen von Zellen in der menschlichen Zahnfleischtaschenfliissigkeit und ihre zytochemischen Reaktionen. D. zahniirztl. Ztg. 22, 836-847. Liie H. 1967. The gingival index, the plaque index and the retention index system. J. Periodont. 38, 61G-616. L6e H., Theilade, E. and Jensen S. B. 1965. Experimental gingivitis in man. J. Periodont. 36, 177-187. Lowry 0. H., Rosebrough N. J., Farr A. L. and Randall R. J. 1951. Protein measurement with Folin reagent. J. biol. Chem. 193, 265-275. Oppenheim F. G. 1970. Preliminary observations on the presence and origin of serum albumin in human saliva. Helv. odont. Actd 14, l&17. Page R. and Schroeder H. 1976. Pathoeenesis of inflammat%y periodontal disease. A summayy of current work. Lab. lnuest. 33, 235-249. Raeste A. M. 1972. Degeneration of oral leucocytes. Stand. J. dent. Res. 80, 285-291. Ross R. 1969. Wound healing. Science Am. 220, 4c-50. SchiGtt C. R. and Liie H. 1970. The origin and variation in number of leucocytes in the human saliva. J. periodont. Res. 5, 36-41. Skapski H. and Lehner T. 1975. Cellular investigation of crevicular fluid in man with normal gingiva. Internat. Ass. for Dent. Res. Preprinted abstracts. 53rd General Meeting. Abstract L256. Valazza A., Matter J., Ogilvie A. and Cimasoni G. 1972. Fluide gingival inflammation gingivale, profondeur des poches et perte osseuse. R.M.S.O. 82, 8tk-88.