The origins of acid phosphatase in human gingival fluid

Archs oral Biol.

Vol. 13, pp. 553-558, 1968.

Pergamon Press.

Printed in Cit. Britain.

THE ORIGINS OF ACID PHOSPHATASE IN HUMAN GINGIVAL FLUID T. SUEDA and G. CIMASONI

Institut de Mtdecine dentaire, Universitt de Geneve, Switzerland Summary-The origin of acid phosphatase, which has recently been shown to occur in very high levels in human gingival fluid, has been investigated by cytochemical and quantitative techniques. Frozen sections of cellular sediments and smears were prepared from human crevicular fluid; stained with the coupling azo dye technique for acid phosphatase, they showed the occurrence of lysosomal enzyme in the epithelial cells and leucocytes. Enzyme activity was also found at the sites of bacterial accumulation. Repeated freezing and thawing of whole gingival fluid caused a striking increase of acid phosphatase, as measured in the supematant by a quantitative technique. Filtration of the fluid through suitable millipore filters eliminated both the epithelial and blood cells. After freezing and thawing, the filtered gingival fluid showed an increase of acid phosphatase activity, thus confirming that part of the enzyme is produced by bacteria.

RECENTWORKin this laboratory has shown the occurrence, in human gingival fluid, of high levels of acid phosphatase (SUEDA, CIMASONIand HELD, 1967). The concentration of the hydrolytic enzyme could not be correlated with the intensity of periodontal inflammation. However, it was confirmed that the intensity of the flow of the gingival fluid was related to pocket depth (BRILL and BJORN,1959; MANN, 1963). As pointed out in the previous publication, the extracellular acid phosphatase found in crevicular fluid could represent an accumulation of lysosomal enzyme from the desquamating epithelial cells of the crevicular epithelium or from connective tissue cells. It could also have a bacterial origin (HEPPEL, 1967). In the present work, an attempt has been made to study the sources of the hydrolytic enzyme, with particular emphasis on the possible bacterial origin. EXPERIMENTAL

METHODS

Crevicular fluid was collected from a series of patients with extensive gingival inflammation and periodontitis. The collection was performed by using capillary tubes, having an i.d. of 0.2 mm, in a manner similar to that described by BRANDTZAEG and MANN (1964). Cytochemical investigations

The fluid from four patients was pooled in four plastic microtubes, which were centrifuged for 10 min at 5000 rev/min. Each tube was then transferred to a cryostat (Kryotom-Gefrieranlage WKF) at -25°C. Its tip was removed and mounted on the microtome chuck, with the aid of solid CO, and water. Sections 7-10 ~1thick were obtained of the tip of the plastic tube with its contents. The sections were picked up on 553

T. SUEDA AND G.

554

CIMASONI

warm histological slides and kept in the cryostat, the plastic of the tube being discarded at the moment of thawing. Smears were prepared with a few ~1 of fluid from each patient. Sections and smears were stained with haematoxylin and eosin, a 0.02 per cent solution of amido-black (WUNDERLY,1959), the Gram stain and the standard coupling azo dye technique for acid phosphatase (PEARSE,1960). In a few cases, sections were stained in series of three, so that an estimate of the cellular distribution of acid phosphatase could be obtained. Fractionation and quantitative observations Samples of 30-35 ~1 of fluid were collected from seven supplemental patients. Three aliquots were taken from each sample and handled according to the procedure outlined in Fig. 1. The first aliquot (10 ~1) was treated in a manner identical to that described in the previous publication (SUEDAet al. 1967); it was centrifuged for 10 min at 5ooO rev/min, and the supernatant used for the quantitative determination of acid phosphatase after dilution in O-09 M citrate buffer at pH 4.9 (RICH-ERICH,COLOMBO and WEBER,1962). The second aliquot (5 ~1) was submitted, after dilution, to repeated

freezing and thawing, followed by centrifugation and enzyme determination in the supernatant (fraction 2). The third aliquot (15 ~1) was diluted in physiological saline (O-9 per cent W/V), and filtered, with the aid of an hypodermic syringe, through a millipore titer having a diameter of 1 cm and a pore size of 1 IL,mounted on a “swinny” filter holder (Millipore Filter Corp., Bedford, Mass, USA.). The total amount of saline used for dilution and Gingival fluid IO /bl

Centrifuged

5 pl top layers added to 45 ~1 citrate buffer

x 10)

Fraction 1

to 75 ~1

15 ~1 added

citrate buffer I Four times frozen and thawed

saline solution I Filtered on millipore filter

I Centrifuged

I 25 ~‘1

40 pl top layers

Enzyme determination Enzyme determination (Dilution

1

5 ~1 added to 45 pl

(Dilution

Fraction 2

x 10)

Centrifuged I 20 ~1 supematant added to 30 ~1 citrate buffer I

20 ~1 added to 30 ~1 citrate buffer I Four times frozen and thawed I Centrifuged I 40 ~1 supematant

Enzyme determination (Dilution X 15)

Enzyme determination (Dilution x 15)

Fraction 3

Fraction 4

FIG. 1. Fractionation of a sample of gingival fluid.

THE

ORIGINS

OF ACID

PHOSPHATASE

IN HUhfAN

GINGIVAL

5.55

FLUID

washing was 75 ~1. Smears were prepared with a few ~1 of the filtered solution; after staining with haematoxylin and eosin and with the Gram technique, they showed the presence of bacteria but were devoid of epithelial and blood cells. Of the filtered solution, 25 ~1 was centrifuged and the enzyme determined in the supernatant (fraction 3). Another 20 ~1 was further diluted and submitted to freezing and thawing, prior to centrifugation and determination of acid phosphatase in the supernatant (fraction 4). As shown in Fig. 1, the dilution factors were known for each step of the fractionation. RESULTS

Cytochemical investigations

Sections of the microtube tips, stained with amido-black, showed the presence of a cellular residue and a supernatant in the centrifuged samples of human gingival fluid. Abundant leucocytes and desquamated epithelial cells, often disposed in two fairly distinct layers, were visible in the cellular residues (Figs. 2 and 5). Figures 2, 3 and 4 show three serial sections of a cellular sediment; they are stained respectively with haematoxylin and eosin, with the Gram technique and with the azo dye technique for acid phosphatase. The supernatant was lost by these staining procedures. Gram-positive and Gram-negative bacteria were often concentrated at the bottom of the microtube and in some isolated clumps (Fig. 3). As for the acid phosphatase, a marked activity of the enzyme could be observed in the layers of leucocytes and in the epithelial cells (Figs. 4 and 5). As expected, the enzyme was seen in lysosome-like particles, as visible on both sections (Figs. 5 and 6) and smears (Fig. 7). Bacterial clumps were invariably associated with high concentrations of acid phosphatase (Figs. 3, 4 and 6). Quantitative observations

The activities of total acid phosphatase, expressed as PM of substrate hydrolysedl min by 1000 ml of pure gingival fluid, in the four fractions, are shown in Table 1. Only fractions 1 and 2 were available from the last two patients. TABLE

1.

A CTMTIES

OF TOTAL

ACID

PHOSPHATASE*

OF HUMAN

GINGNAL

R-4 THE FLUID

SUPERNATANT

OF FOUR

FRACTIONS

cgf)

Patient No.

Fraction 1 (whole gf)

Fraction 2 (whole gf, frozen and thawed)

Fraction 3 (filtered gf)

Fraction 4 (filtered gf, frozen and thawed)

1

105.6

258.4

101.7

143.4

; 4

106.8 79.7 162.2

240.2 162.5 334.0

70.3 98.0 151.8

112.5 150.1 242.1

: 7

117.0 94.4 76.6

246.4 175.5 273.3

91.8 -

148.6 -

l

JJ moles p-nitrophenyl-phosphate/min

per 1000 ml.

556

T. SUEDAAND

G.

CIMASONI

The supernatant of whole gingival fluid, after freezing and thawing (fraction 2), consistently contained 2-3 times more acid phosphatase than that of fluid which had not undergone such a procedure (fraction 1). The activities of acid phosphatase in the filtered solution (fraction 3) were similar to those found in fraction 1. Finally, as shown in Table 1, freezing and thawing of filtered crevicular fluid (fraction 4) caused a constant and significant increase of acid phosphatase activity measured in the supernatant. DISCUSSION

The present findings confirm that gingival fluid contains leucocytes and desquamated epithelial cells (WEINSTEINand MANDEL, 1964). Lysosome-like particles can be shown in both these types of cells (Figs. 5,6 and 7). As pointed out by DE DUVE (1963), the lysosomes found in the leucocytes are packed with hydrolytic enzymes, which play a role in the digestion of engulfed particles and bacteria. As for the epithelium, a recent communication (LANGEand CAMILLERI,1967) has shown that the cells of the gingival cuff contain enlarged lysosomes, suggesting the possibility that these cells undergo autolysis with the liberation of acid hydrolases. The results reported in this paper confirm this possibility and indicate that a major source of the acid phosphatase in the gingival fluid is represented by epithelial cells and leucocytes. Indeed, as shown in Table 1, the values of enzyme activity found in the filtered fraction after freezing and thawing (fraction 4) were lower than those obtained after freezing and thawing of whole gingival fluid (fraction 2). Sections of cellular residues and smears showed the presence, in human crevicular fluid, of abundant Gram-positive and Gram-negative bacteria, and allowed the demonstration of acid phosphatase at the sites of bacterial accumulation (Figs. 3, 4 and 6). This cytochemical finding could be confirmed by the quantitative results obtained after filtration of gingival fluid through millipore filters: repeated freezing and thawing of filtered crevicular fluid, in the absence of epithelial and blood cells, caused a significant increase in the acid phosphatase activity found in the supernatant (Table 1). The subject of release of enzymes from bacteria has recently been reviewed (HEPPEL, 1967); preparations of cell membranes of Gram-positive organisms contain a number of firmly bound enzymes. Furthermore, a whole family of hydrolytic enzymes, including non-specific acid phosphatase, is found within and near the cell surface of Gram-negative bacteria. In general, enzyme activity measured with intact organisms is less than that observed with equivalent amounts of cell extracts. In cultures of bacteria isolated from gingival crevices, enzymes, such as polysaccharidases (SCHULTZ-HAUDT and SCIIERP, 1955) and collagenase (GIBBONSand MACDONALD,1961) have been demonstrated. Moreover, proteolytic enzymes of bacterial origin were recently shown in human gingival fluid (K~~RBER and SCHAUER, 1967). The results of the present research suggest that part of the acid phosphatase found in gingival fluid is of bacterial origin. Acid phosphatase, whether from ruptured

THE ORIGINSOF ACIDPHOSPHATASE IN HUMANGINGIVALFLUlD

557

lysosomes, or secreted by bacteria, is active on a variety of phosphate esters, which are found in cellular and extracellular components (SCHMIDT,1961); it is able to reduce the mutual adhesiveness of cultured epithelial cells (BERWICK and COMAN, 1962) and its role in the formation of a pathological periodontal pocket could therefore be very significant. Acknowledgements-This work was supported in part by a grant of the Swiss ARPA. The authors are grateful to Dr. C. AHMAD-ZADEH, Institute of Hygiene, Medical School, University of Geneva, for his helpful BARBARA WIPPERF~~RTHfor her skilful technical assistance.

suggestions

and

to Miss

R&mm&-L’origine de la phosphatase acide, qui se trouve en quantitC Blevee dans le liquide gingival humain, a et.5ttudibz par des techniques cytochimiques et quantitatives. Des coupes par con&lation de sediments cellulaires et de frottis de liquide provenant du 1isCrCgingival humain sont colorCes par la technique de coloration B l’azo pour la phosphatase acide. L’enzyme lysosomique est situ6 dans les cellules &pithCliales et les leucocytes, ainsi qu’au niveau des agrkgats bactkriens. Une congClation et un rkhauffement rkp&&s de liquide gingival total provoquent une augmentation nette en pbosphatase acide, ainsi que le dkmontre 1’3naIyse quantitative du liquide surnageant. La filtration du liquide & travers des filtres millipores adapt& Climine les globules sanguins et les cellules 6pith6liales. Apr& congklation et rkhauffement, le liquide gingival filtrC prbsente toujours une augmentation en phosphatase acide, demontrant ainsi qu’une partie de l’enzyme constitue un produit bactCrien. Zusammenfassung-Mit Hilfe zytochemischer und quantitativer Methoden wurde die Herkunft der sauren Phosphatase untersucht, die kiirzlich in sehr grossen Mengen in der menschlichen Gingiva-Fliissigkeit nachgewiesen worden ist. Es wurden Gefrierschnitte zellulLer Sedimente und Beliige aus dem fltissigen Inhalt der Zahnfleischfurche des Menschen angefertigt und mit Hilfe der gekoppelten Azo-Technik fiir saure Phosphatase angefgrbt. Sie zeigten das Vorkommen von lysosomalem Enzym in den epithelialen Zellen und Leukozvten. Die Enzvmaktivitat wurde such an den Stellen bakterieller Anhlufungen gefunden. Wiederhbltes Einfrieren und Auftauen der gesamten Gingivafliissigkeit rief einen starken Anstieg der sauren Phosphatase hervor, wie im uberstand mit Hilfe einer quantitativen Technik nachgewiesen werden konnte. Die Filtration der Fliissigkeit durch geeignete feinstporige Filter trennten sowohl epitheliale als such Blutzellen ab. Nach Frieren und Auftauen 1ieBdie gefilterte Gingivafliissigkeit einen Anstieg der AktivitPt der sauren Phosphatase erkennen und bestatigte damit, da13 ein Teil des Enzyms von Bakterien produziert wird. REFERENCES BERWICK,L. and &MAN, D. R. 1962. Some chemical factors in cellular adhesion and stickiness. Cancer Res. 22,928-981. BRANDTZAEG,P. and MANN, W. V. 1964. A comparative study of the lysosome activity of human gingival pocket fluid, serum and saliva. Acta odont. stand. 22,441-4X BRILL, N. and BJORN, H. 1959. Passage of tissue fluid into human gingival pockets. Acta odont. stand. 17,l l-21. DE DUVE, C. 1963. The lysosome. Scient. Am. 208,64-72. GIBBONS,R. J. and MACDONALD,J. B. 1961. Degradation of collagenous substrates by Bacteroides melaninogenicus. J. Bact. 81, 614-621. HEPPEL, L. A. 1967. Selective release of enzymes from bacteria. Science, N. Y. 156, 1451-1455. K~~RBER,E. and SCHAUER,R. 1967. Bestimmung von Enzymaktivitlten im Sekret von Zahnfleischtaschen. D. zahtirztl. Z. 22, 332-337. LANGE, D. and CAMILLERI,G. E. 1967. Cytochemical demonstration of lysosomes in exfoliated epithelial cells of the gingival cuff. J. dent. Res. 46, 625.

558

T. SUEDA AND G. CIMASONI

MANN, W. V. 1963. The correlation of gingivitis, pocket depth and exudate from the gingival crevice. J. Periodont.

34, 319-381.

PEARSE,A. G. E. 1960. Histochemistry: Theoretical and Applied, p. 882 (2nd ed.) Churchill, London. RICHTERICH,R., COLOMBO,J. P. and WEBER,H. 1962. Ultramikromethoden im Klinischen Laboratorium. VII. Bestimmung der sauren Prostata-Phosphatase. Schweiz. Med. Wschr. 92,1496-1500. SCHMIDT,G. 1961. Non specific acid phosphomonoesterases. In: The Enzymes (Vol. 5). (Edited by BOYER,P. D., LARDY,H. and MYRBACK,K.), Chap. 2, pp. 37-47. Academic Press, New York. SCHULTZ-HAUDT.S. and SCHERP.H. W. 1965. Production of hyaluronidase and beta nlucuronidase by viridans streptococci isolated from gingival crevices. J. d&t. Res. 34, 924-929. SUEDA,T., Q~SONI, G. and HEID, A. J. 1967. High levels of acid phosphatase in human gingival fluid. Archs oral Biol. 12, 1205-1207. WEINSTEIN,E. and MANDEL,I. D. 1964. The fluid of the gingival sulcus. Periodontics 2,147-153. W~ERLY, CH. 1959. Paper electrophoresis. In: Electrophoresis, Theory, Methodr and Applicationr (Edited by BIER, M.) p. 200. Academic Press, New York.

PLATE 1 FIG. 2. Section of frozen cellular sediment. Most leucocytes are confined to the top layers. Epithelial cells are seen in the lower part of the centrifugation residue. Haematoxylin and eosin. x 26. FIG. 3. Frozen cellular sediment. Serial section to Fig. 2. The arrow indicates a clump of bacteria in the upper right part of the section. Gram. x 26. FIG. 4. Frozen cellular sediment. Serial section to Figs. 2 and 3. Acid phosphatase activity can be seen in both the layers of leucocytes and epithelial cells. Abundant azo dye is also found in association with the bacterial clump shown in Fig. 3. Numbered arrows refer to the regions from which Figs. 5 and 6 were made. x 26. FIG. 5. Higher magnification of area outlined in Fig. 4. Note abundant like particles in leucocytes (L) and epithelial cells (E). x 650.

lysosome-

FIG. 6. Magnified view from area outlined in Fig. 4. Abundant diffuse azo dye is seen at the site of bacterial accumulation (B) and in cellular lysosomal particles. x 400. FIG. 7. Smear prepared with human crevicular fluid. Both epithelial cells (E) and leucocytes (L) contain lysosomes. Azo dye technique for acid phosphatase. x 480.

THE ORIGINS

OF ACID

PHOSPHATASE

IN HUMAN

GINGIVAL

FLUID

PLATE

1

A.O.B. f.p. 558