/. Periodontal Res. 12: 30-36, 1977
Monocyte chemotactic activity in human dental plaque LEIF HELLDEN
Department of Periodontology, University of Goteborg, Goteborg, Sweden The monocyte chemotacfic activity elaborated by soluble dental plaque material and by various fractions of the plaque material was studied in vitro with the modified Boyden chamber technique. Monocytes were harvested from the peritoneal cavity of rabbits after intraperitoneal injection of a 1 % suspension of hydrolysed starch. The plaque material was separated in Sephadex® gel columns (G-25 and G-200). Three fractions differing from each other in molecular size were selected from the protein peaks in the chromatograms. The results showed that dental plaque material contains factors with a chemotactic effect on monocytes. Examination of various fractions of dental plaque material revealed that the major part of the monocyte chemotactic activity resided in a fraction with an appoximate molecular size of 10,000-70,000. (Accepted for publication April 8, 1976)
Inlfoductlon
Chemotactic factors in dental plaque are assumed to enhance the nuoiber of leukocytes in the junctional epithelium and gingival crevices when plaque is aUowed to accumulate on the feeth (Schroeder 1970, Tempel et al. 1970, Hellden 1973). This hypothesis is supported by the fact that dental plaque contains factors which can penetrate the junctional epithelium (Kahnberg, Llndhe & Hellden 1976) and attract neutrophil leukocytes (e.g. Tempel ef aL 1970, Miller, Umana & Folke 1971, Lindhe & Hellden 1972, Hellden & Lindhe 1973, Kraal & Loesche 1974). Schroeder (1973) showed that the junctional epithelium contains not only neutrophil leukocytes, but also a relatively large number of Monocytes. Whether the presence of monocytes in the
junctional epithelium is caused by attracting factors released from the dental plaque is not yet known. The aim of the present series of experiments was to find out if human dental plaque contains factors chemotactic for rabbit monocytes (Experiment I) and whether monocyte chemotactic factors occur in various fractions of the plaque (Experiment 11). Experiment I: Maierial and Methods
Plaque: Supragingival human dental plaque was collected in the way described by Lindhe & Hellden (1972). Care was taken to avvoid sampling from the area of the gingival crevice and to avoid contamination with saliva. The plaque samples collected
M O N O C Y T E
C H E M O T A C T I C
from various individuals were pooled, suspended in 9 ml of Gey's medium* (pH 7.2), homogenized with a Branson® sonifier B-12 (Danburg, USA) for 60 seconds at 20 kHz and then centrifuged at 12,100 g^ for 30 minutes at 4°C. The supernatant (plaque extract) was separated from the ceUular pellet and then sterile fUtered through a 0.45 [iM Millipore® filter (Millipore filter Corp., New Bedford; USA). The protein content of the plaque extract was determined according to Lowry et al. (1951) with tyrosine as standard. The extract was stored at — 20°C until used. Monocytes. Rabbit mononuclear ceils were obtained after intraperitoneal stimulation by a 1 % suspension of hydrolysed starch as described by Ward (1968). The cell-containing exudate was washed (250 ^^ for 10 minutes at 4°C) three times in Gey's medium and then resuspended in Gey's medium containing 2 % bovine serum albumin. The suspension contained 3.3 X 10" cells per ml. Approximately 85 % of the cells cotdd be classified as mononuclear. Measurement of chemotactic activity: The chemotactic activity of the plaque extract was determined with the modified Boyden chamber technique (Snyderman et al. 1969, Lindhe & Hellden 1972). Millipore® filters with 5 um and 8 [xm pore size were used. The Boyden chambers were incubated at 37°C for five and eight hours, respectively. Gey's medium was used as negative control substance and casein (2 mg per ml; Casein "nach Hammarsten"; Merch, Darmstadt, W. Germany; Keller & Sorkin 1968) was used as positive control substance to check the reliability of the test system. The number of Boyden cham-
ACTIVITY
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31
ber tests with filters of different pore sizes and incubation times is given in Table 1. Mononuclear cells on the lower surface of the filter were counted and the results were expressed as fhe mean number of cells counted in 100 randomly selected fields (see Lindhe & Hellden 1972). Statistical analysis: The results were statistically analysed with the use of Students' t-test. Results
The amount of extractable protein in the plaque extract was 251 ^g/ml. Table 2 shows that soluble human dental plaque contains factors with the ability to attract rabbit monocytes. Experiment IL Material anil Methods
Plaque: Supragingival plaque was sampled and treated in the same way as in Experiment I with the difference that the plaque material was suspended in 5 ml of 0.05 M ammonium bicarbonate buffer (pH 8). After the extraction procedure tbe plaque extract was lyophilized. The dried material was redissolved in 2 ml ammonium bicarbonate buffer, and the protein concentration was assessed (Lowry et al. 1951; tyrosine as standard) after which the material was subjected to gel chromatography.
Table t Number of Boyden chamber tests performed in Experiment I.
Material tested
* Gey's medium: 8.000 g NaCl, 2.000 g Glucose, 0.375 g KCl, 0.275 g CaClj, 0.210 g MgClj, 0.150 g NajHPO,, 0.025 g KH^PO^, 0.250 g NaHCOs; Dissolved to 1,000 ml in redist.water (Gey 1945 according to Paul /1970/).
PLAQUE
Pteque extract Gey's medium (neg. control) Caseio (pos. control)
Fiiter pore size 8 nm incEJbation times hrs.
Filter pore size 5' ^m incubation time 8 hrs.
HELLDgN
32
Table 2
Chemotactic activity for rabbit monocytes elaborated by extracts from human dental plaque (tested in Boydan chambers). Materiai tested
Fiiter pore size 8 ^m incubation tjme 5 hrs.
Plaque extract
X S.E. 145.3 ± 18.2 ~l
Gey's medium (neg. controi:) Casein (pos. controf)
34.8 ± 8.0 132.5 ± 26.7
Fiiter pore size 5 fim incubation time B hrs. X S.E. 472. ± 82.5
P < 0.001 J
Chromatography: Fractionatioo of the water soluble substances was made by chromatography in Sephadex® gels (Pharmacia Fine Chemical; Uppsala, Sweden) at 4°C. A two step procedure was used. The plaque extract was first applied to a Sephadex G-25 column (2.7 X 31.5 cm). The column was equilibrated and eluted with 0.05 M ammonium bicarbonate. A descending elution technique was used. The flow rate was 0.2 ml/min and fractions of 3 ml were collected. In each fraction the conductivity was measured with a conductivity meter (Radiometer Copenhagen CDM 2c). The protein distribution was determined by reading the optical density at 220 nm in a spectrophotometer (Beckman DB-GT). Material from the various series of fractions, representing protein peaks in the chromatogram or containing charged substances, were then pooled separately and made up to a uniform final volume by addition of 0.05 M. amnaonium bicarbonate before being lyophilized. The lyophilized void volume material was then dissolved in 2 ml of the ammonium bicarbonate buffer, pH 8,, and applied to a Sephadex® G-200 column (1.6 x 30 cm). The column was eluted with 0.05 M ammonium bicarbonate. Fractions of 1.5 ml were collected at a flow rate of 0.2 ml/ min. Each fraction was analysed for protein by measuring the optical density at 220 nm in a spectrophotometer (Beckman DB-GT). Before lyophilization the content in the
P < 0.001 24.0' ± 4.0 232.5 ± 30.3
tubes representing different protein peaks in the chromatograms was pooled separately, and made up to a uniform final volume by addition of 0.05 M ammonium bicarbonate in the same way as the fractions pooled from the G-25 separation. To remove possible residues of ammonium bicarbonate, all the freeze-dried material was redissolved in water and lyophilized three times. The lyophilized fractions of soluble plaque material were then stored at —20°C until used. To estimate the molecular size of the different plaque fractions cytochrome C (mol. wt. 13,000; Nutritional Biochemicals Corp., Cleveland, USA) and human serum albumin (mol. wt. 58,000; Fraction V, Sigma Corp; USA) were run in the Sephadex® columns prior to fraetionation of the plaque extracts. Protein determination: The protein content of the isolated fractions of the plaque material was determined with the method of Lowry et al. (1951) with tyrosine as standard. Measurement of chemotactic activity: Each of the lyophilized fractions was dissolved in 6 ml of Gey's medium and then sterile filtered through a 0.45 jim MiUipore® filter. The chemotactic activity was determined in the same way as in Experiment I with the exception that only filters with a pore size of 5 [im were used. The Boyden chambers were incubated for eight hours. The cell suspension in the upper
MONOCYTE
CHEMOTACTIC
Fig.iA
ACTIVITY
IN
PLAOUE
33
were performed with each of the plaque fractions. In these tests plaque substances of equal concentration were placed both above and below the filters in the Boyden chambers, as recommended by Kraal & Loesche (1974). Statistical analyses. The statistical evaluation was based on single factor analysis of variance. The Newman-Keul's test was used to assess the differences among the means.
2Dl
0:5
Results
BED VOLUME G-200
Fi9.1B 2.0
1.0
O 0.5'
25
50
BED VOLUME Fig. 1. SephadeK G-25 (fig. 1A) and G-2D0 (fig. IB) chromatography of soiuble dentai pJaque mai'Sriai (twvo-step procedure). Elution was performed witfi 0.05 M ammoni'um bicarbonate (pH B). From tfie peaks 'in tiie chromatograms tiiree fractions '[designated A, B and C) were selected for the Boyden chamber experiments. The retention voiumes O'f the reference substanoes are indicated by arrows.
compartment of the Boyden chambers contained 2.2 X lO'i cells/ml of which approximately 85 % could be classified as monoeytes. To exclude the possibility of cell migration through the filters being the result of random movement, two additional tests
Chromatography. Chromatography on Sephadex gels (Sephadex® G-25 and G-200) resulted in two chromatograms (Fig. lA, IB). Three series of fractions were selected from the protein and conductivity peaks in the chromatograms. For convenience the peaks in the chromatograms were designated A, B and C. Fraction A contained substanoes with low, and fraction C substances with high molecular size. The elution volumes of cytochrome C and HSA (human seram albumin) are given in the chromatograms. The elution volumes of the reference substances indicated that fraction A contained substances with molecular weight of < 13,000 and fraction C > 68,000. Protein determination. Table 3 gives the amount of extractable protein in different fractions after gel filtration. The protein recovery was 93 %. Measurement of chemotactic activity. Table 4 presents the results from the Boy-
Tabie 3 Protein determination according to Lowry et al. (1951). Materiai tested Piaque fraction A Plaque fraction B Plaque fraction C
Protein ^g/mi 169 150
34 Table 4
Chemotactic activity for rabbit monocytes elaborated by different fractions of soluble dentai plaque (tested in Boyden chambers). Number of monocytes counted on the iower side of filter
Materiai tested
Pfaque fraction A Piaque fraoti'O'n B Piaque fractfon C Gey's medium {neg. contr.) Casein (pos. controi}
X 25.0 47.0 23.1 8.0 34.9
+ ± ± ± +
S.E. 1.07 3.00 2.09 0.63 3.21
There are' hi'ghly signifioani differences between all means except for fraction A vs. C.
den chamber experiments. The statistical analysis revealed differences between all the test groups and the controls. Significant differences were found also between plaque fraction B, on one hand, and fractions A and C on the other. When plaque material was placed both in the upper and lower compartment of the Boyden chambers no positive chemotaxis of monocytes could be demonstrated. The mean number of monocytes which had migrated through the Millipore® filters was 2.3 ± 0.92 (S.B.). Discussion
The results from the present study show that microbiai dental plaque contains substances which in vitro attract rabbit monocytes. When different fractions were tested regarding their chemotactic activity it was found that all the plaque fractions tested attracted mononuclear leukocytes. The findings were thus in line with earlier published data concerning the capacity of dental plaque to attract neutrophilic Ieukoc34es (Tenapel et al. 1970, Miller et al. 1971, Lindhe & Helldeo 1972, Hellden, Ericson & Lindhe 1973). However, the molecular size of the plaqne fraction that induced the major part
of the inonoc3fte chemotactic activity was larger than that of the corresponding neutrophil chemotactic factor (Hellden et al. 1973). The increased number of neutrophil leukocytes in the junctional epithelium and in the gingival sulcus when plaque is allowed to accumulate on the teeth (Attstrom & Egelberg 1971, Payne et al. 1975) have been ascribed to depend on neutrophilic chemtactic activity elaborated by substances within the dental plaque (Schroeder 1970, Tempel et al. 1970, Attstrom 1971, Hellden 1973). The results of the present investigation indicate that also the presence of monocytes in the junctional epithelium (Schroeder 1973) and gingival sulcus (Attstrom 1970) may be the result of chemotactic stimulation from plaque. Autoradiographic and histologic studies have shown that horseradish peroxidase (mol.wt. 40.000; McDougall 1971, 1972), albumin (mol. wt. 68,000; Tolo 1971) and endotoxin (Schwartz, Stinson & Parker 1972; Ranney & Montgomery 1973) can penetrate the junctional epithelium in the rabbit, pig and dog. Furthermore, Kahnberg et al. (1976) demonstrated an increased number of neutrophils and monocytes in the junctional epithelium in dogs following topical application of soluble dental plaque extract to healthy marginal gingivae. Hence, it seems reasonable to assume that factors chemotactic for monocytes can penetrate the junctional epithelium barrier. In the present study no attempt was made to find out which substances in the plaque elaborated the monocyte chemotactic activity. Wllkinsson, O'Neill & Wapshaw (1973) and Ward (1968) reported that mononuclear cells were attracted by various strains of anaerobic corynebacteria and by peptides from Str. pneumoniae. There is thus reason to assume that microbiai products might he responsible for the activity found also in the present study. It cannot be excluded, however, that some of the activity observed
M O N O C Y T S
C H E M O T A C T I C
may have been induced by complement componetits present within the. dental plaque material (Mergenhagen & Hook 1972, Shillitoe & Lehner 1972, Attstrom et al. 1975). Acknowledgment
The research assistance given by Mrs. Yvonne Sundin is gratefully acknowledged.
References
Attstrom, R. 1970. Presence of leukocytes in the crevices of healthy and chronically inflamed gingivae. J. Periodontal Res. 5: 42—47. Attstrom, R. 1971. Studies on neutrophil polymorph'Onuclear leukocytes at the dento-gingival junction in gingival health and disease. J. Periodontal Res. supplement No. 8. Attstrom, R. & Egelberg, I. 1971. Presence of leukocytes within the gingival crevices during developing gingivitis in dogs. /. Periodontal Res. 6: 110-114. Attstrom, R., Laurell, A.-B., Larsson, U. & Sjoholm, A. 1975. Complement factors in gingival crevice material from healthy and inflamed gingiva in humans.}. Periodontal Res. l'O: 19-27. Hellden,L. & Lindhe,!. 1973. Enhanced emigration of crevicular leukocytes mediated by factors in human dental plaque. Scand. J. Dent. Res. 81: 123-129. Hellden, L., Ericson, T. & Lindhe, I. 1973. Neutrophil chemotactic substances in different fractions of soluble dental plaque material Scand. J. Dent. Res. 81: 276-284. Hellden, L. 1973. On chemotactic factors in the dento-gingival region. Thesis. University of Goteborg; Sweden. Kahnberg, K. E., Lindhe, J. & Hellden, L. 1976. Initial gingivitis induced by topical application of plaque extract. A histometric study. /. Periodontal Res. Accepted for publication. Keller, H. V. & Sorkin, E. 1968. Chemotaxis of leukocytes. Experientia. 24: 641-652. Kraal, J. H. & Loesche, W. 1. 1974. Rabbit polymorphonuclear leukocyte migration in vitro in response to dental plaque. J. Periodontal Res. 9: 1-9. Lindhe, J. & Hellden, L. 1972. Nentrophil chemotactic activity elaborated by human dental plaque. /. Periodontal Res. 7: 297-303.
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P L A Q U E
35
Lowry, O. H., Rosebrough, N. I., Farr, A. L. '& Randall, R.J. 1951. Protein measurement with the Folin Phenol reagent. J. biol. Chem. 193: 265-275. McDougall, W. A. 1971. Penetration pathways of a topically applied fO'reign protein into rat gingiva. J. Periodontal Res. 6: 89-99. McDougall, W. A. 1972. Ultrastnictural localization of antibody to an antigen applied topically to rabbit gingiva. J. Periodontal Res. T. 304-314. Miller, R.L., Umana, C.R. & Folke, L. 1971. Chemotactic ability of dental plaque upon homologous and heterologous human PMN's. I.A.D.R. Meeting. Abstract No 244. p. 113. Paul, J. 1970. Cell and tissue culture. 4th ed.. Chap. VI, p. 91. Edinburgh and London. E. and S. Livingstone. Payne, W. A., Page, R. C, Ogilvie, A. L. & Hall, W. B. 1975. Histopathologic features of the initial and early stages of experimental gingivitis in man. J. Periodontal Res. 10: 51-64. Ranney, R. R. & Montgomery, E. H. 1973. Vascular leakage resulting from topical application of endotoxin to the gingiva of the beagle dog. Archs. Oral Biol. 18: 963-970. Schroeder, H. E. 1970. 'Quantitative parameters of early human gingival inflammation. Arch. Oral Biol. IS: 383-400. Schroeder, H. E. 1973. Transmigration and infiltration of leukocytes in human junctional epithelium. Hetv. Odont. Acta 17: 6-18. Schwartz, J., Stinson, F. L. & Parker, R. B. 1972. The passage of tritiated bacterial endotoxin across intact gingival crevicular epithelium. /. Periodontol. 43: 270-276. Shillitoe, E. 1. & Lehner, T. 1972. Immunoglobulins and complement in crevicular fluid, serum and saliva in man. Arch. Oral Biol. 17: 241-247. Snyderman, R., Shin, H. S., Phillips, 1. K., Gewurz, H. & Mergenhagen, S. E. 1969. A neutrophil chemotactic factor derived from C'5 upon interaction of guinea pig serum with endotoxin. i. Immunol 103: 413-422. Tempel, T. R., Snyderman, R., lordan, H. V. & Mergenhagen, S. E. 1970. Factors from saliva and oral bacteria, chemotactic for polymorphonuclear leukocytts: Their possible role in gingival inflammation. /. Periodontol. 41: 71-79. Tolo, K. I. 1971. A study of permeability of gingival pocket epithelium to albumin in guinea pigs and norwegian pigs. Arch. Oral Biol 16: 881-888.
36
^
Ward, P. A- 1968. Chemotaxis of mononuclear cells. /. Experimental Med. 128: 1201-1219. Wilkinson, P. C, O'Neill, G. I. & Wapshaw, K. G. 1973. Role of anaerobic coryneforms iB Address: Avdelning for Parodontologi Lantmannivagen 111 461 OS TrOllhdttan Sweden
specific and nonspecific immunological reactions. II. Production of a chemotactic ftictor specific for macrophages. Immunology 24: 997-1006.
Monocyte chemotactic activity in human dental plaque LEIF HELLDEN
Department of Periodontology, University of Goteborg, Goteborg, Sweden The monocyte chemotacfic activity elaborated by soluble dental plaque material and by various fractions of the plaque material was studied in vitro with the modified Boyden chamber technique. Monocytes were harvested from the peritoneal cavity of rabbits after intraperitoneal injection of a 1 % suspension of hydrolysed starch. The plaque material was separated in Sephadex® gel columns (G-25 and G-200). Three fractions differing from each other in molecular size were selected from the protein peaks in the chromatograms. The results showed that dental plaque material contains factors with a chemotactic effect on monocytes. Examination of various fractions of dental plaque material revealed that the major part of the monocyte chemotactic activity resided in a fraction with an appoximate molecular size of 10,000-70,000. (Accepted for publication April 8, 1976)
Inlfoductlon
Chemotactic factors in dental plaque are assumed to enhance the nuoiber of leukocytes in the junctional epithelium and gingival crevices when plaque is aUowed to accumulate on the feeth (Schroeder 1970, Tempel et al. 1970, Hellden 1973). This hypothesis is supported by the fact that dental plaque contains factors which can penetrate the junctional epithelium (Kahnberg, Llndhe & Hellden 1976) and attract neutrophil leukocytes (e.g. Tempel ef aL 1970, Miller, Umana & Folke 1971, Lindhe & Hellden 1972, Hellden & Lindhe 1973, Kraal & Loesche 1974). Schroeder (1973) showed that the junctional epithelium contains not only neutrophil leukocytes, but also a relatively large number of Monocytes. Whether the presence of monocytes in the
junctional epithelium is caused by attracting factors released from the dental plaque is not yet known. The aim of the present series of experiments was to find out if human dental plaque contains factors chemotactic for rabbit monocytes (Experiment I) and whether monocyte chemotactic factors occur in various fractions of the plaque (Experiment 11). Experiment I: Maierial and Methods
Plaque: Supragingival human dental plaque was collected in the way described by Lindhe & Hellden (1972). Care was taken to avvoid sampling from the area of the gingival crevice and to avoid contamination with saliva. The plaque samples collected
M O N O C Y T E
C H E M O T A C T I C
from various individuals were pooled, suspended in 9 ml of Gey's medium* (pH 7.2), homogenized with a Branson® sonifier B-12 (Danburg, USA) for 60 seconds at 20 kHz and then centrifuged at 12,100 g^ for 30 minutes at 4°C. The supernatant (plaque extract) was separated from the ceUular pellet and then sterile fUtered through a 0.45 [iM Millipore® filter (Millipore filter Corp., New Bedford; USA). The protein content of the plaque extract was determined according to Lowry et al. (1951) with tyrosine as standard. The extract was stored at — 20°C until used. Monocytes. Rabbit mononuclear ceils were obtained after intraperitoneal stimulation by a 1 % suspension of hydrolysed starch as described by Ward (1968). The cell-containing exudate was washed (250 ^^ for 10 minutes at 4°C) three times in Gey's medium and then resuspended in Gey's medium containing 2 % bovine serum albumin. The suspension contained 3.3 X 10" cells per ml. Approximately 85 % of the cells cotdd be classified as mononuclear. Measurement of chemotactic activity: The chemotactic activity of the plaque extract was determined with the modified Boyden chamber technique (Snyderman et al. 1969, Lindhe & Hellden 1972). Millipore® filters with 5 um and 8 [xm pore size were used. The Boyden chambers were incubated at 37°C for five and eight hours, respectively. Gey's medium was used as negative control substance and casein (2 mg per ml; Casein "nach Hammarsten"; Merch, Darmstadt, W. Germany; Keller & Sorkin 1968) was used as positive control substance to check the reliability of the test system. The number of Boyden cham-
ACTIVITY
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ber tests with filters of different pore sizes and incubation times is given in Table 1. Mononuclear cells on the lower surface of the filter were counted and the results were expressed as fhe mean number of cells counted in 100 randomly selected fields (see Lindhe & Hellden 1972). Statistical analysis: The results were statistically analysed with the use of Students' t-test. Results
The amount of extractable protein in the plaque extract was 251 ^g/ml. Table 2 shows that soluble human dental plaque contains factors with the ability to attract rabbit monocytes. Experiment IL Material anil Methods
Plaque: Supragingival plaque was sampled and treated in the same way as in Experiment I with the difference that the plaque material was suspended in 5 ml of 0.05 M ammonium bicarbonate buffer (pH 8). After the extraction procedure tbe plaque extract was lyophilized. The dried material was redissolved in 2 ml ammonium bicarbonate buffer, and the protein concentration was assessed (Lowry et al. 1951; tyrosine as standard) after which the material was subjected to gel chromatography.
Table t Number of Boyden chamber tests performed in Experiment I.
Material tested
* Gey's medium: 8.000 g NaCl, 2.000 g Glucose, 0.375 g KCl, 0.275 g CaClj, 0.210 g MgClj, 0.150 g NajHPO,, 0.025 g KH^PO^, 0.250 g NaHCOs; Dissolved to 1,000 ml in redist.water (Gey 1945 according to Paul /1970/).
PLAQUE
Pteque extract Gey's medium (neg. control) Caseio (pos. control)
Fiiter pore size 8 nm incEJbation times hrs.
Filter pore size 5' ^m incubation time 8 hrs.
HELLDgN
32
Table 2
Chemotactic activity for rabbit monocytes elaborated by extracts from human dental plaque (tested in Boydan chambers). Materiai tested
Fiiter pore size 8 ^m incubation tjme 5 hrs.
Plaque extract
X S.E. 145.3 ± 18.2 ~l
Gey's medium (neg. controi:) Casein (pos. controf)
34.8 ± 8.0 132.5 ± 26.7
Fiiter pore size 5 fim incubation time B hrs. X S.E. 472. ± 82.5
P < 0.001 J
Chromatography: Fractionatioo of the water soluble substances was made by chromatography in Sephadex® gels (Pharmacia Fine Chemical; Uppsala, Sweden) at 4°C. A two step procedure was used. The plaque extract was first applied to a Sephadex G-25 column (2.7 X 31.5 cm). The column was equilibrated and eluted with 0.05 M ammonium bicarbonate. A descending elution technique was used. The flow rate was 0.2 ml/min and fractions of 3 ml were collected. In each fraction the conductivity was measured with a conductivity meter (Radiometer Copenhagen CDM 2c). The protein distribution was determined by reading the optical density at 220 nm in a spectrophotometer (Beckman DB-GT). Material from the various series of fractions, representing protein peaks in the chromatogram or containing charged substances, were then pooled separately and made up to a uniform final volume by addition of 0.05 M. amnaonium bicarbonate before being lyophilized. The lyophilized void volume material was then dissolved in 2 ml of the ammonium bicarbonate buffer, pH 8,, and applied to a Sephadex® G-200 column (1.6 x 30 cm). The column was eluted with 0.05 M ammonium bicarbonate. Fractions of 1.5 ml were collected at a flow rate of 0.2 ml/ min. Each fraction was analysed for protein by measuring the optical density at 220 nm in a spectrophotometer (Beckman DB-GT). Before lyophilization the content in the
P < 0.001 24.0' ± 4.0 232.5 ± 30.3
tubes representing different protein peaks in the chromatograms was pooled separately, and made up to a uniform final volume by addition of 0.05 M ammonium bicarbonate in the same way as the fractions pooled from the G-25 separation. To remove possible residues of ammonium bicarbonate, all the freeze-dried material was redissolved in water and lyophilized three times. The lyophilized fractions of soluble plaque material were then stored at —20°C until used. To estimate the molecular size of the different plaque fractions cytochrome C (mol. wt. 13,000; Nutritional Biochemicals Corp., Cleveland, USA) and human serum albumin (mol. wt. 58,000; Fraction V, Sigma Corp; USA) were run in the Sephadex® columns prior to fraetionation of the plaque extracts. Protein determination: The protein content of the isolated fractions of the plaque material was determined with the method of Lowry et al. (1951) with tyrosine as standard. Measurement of chemotactic activity: Each of the lyophilized fractions was dissolved in 6 ml of Gey's medium and then sterile filtered through a 0.45 jim MiUipore® filter. The chemotactic activity was determined in the same way as in Experiment I with the exception that only filters with a pore size of 5 [im were used. The Boyden chambers were incubated for eight hours. The cell suspension in the upper
MONOCYTE
CHEMOTACTIC
Fig.iA
ACTIVITY
IN
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33
were performed with each of the plaque fractions. In these tests plaque substances of equal concentration were placed both above and below the filters in the Boyden chambers, as recommended by Kraal & Loesche (1974). Statistical analyses. The statistical evaluation was based on single factor analysis of variance. The Newman-Keul's test was used to assess the differences among the means.
2Dl
0:5
Results
BED VOLUME G-200
Fi9.1B 2.0
1.0
O 0.5'
25
50
BED VOLUME Fig. 1. SephadeK G-25 (fig. 1A) and G-2D0 (fig. IB) chromatography of soiuble dentai pJaque mai'Sriai (twvo-step procedure). Elution was performed witfi 0.05 M ammoni'um bicarbonate (pH B). From tfie peaks 'in tiie chromatograms tiiree fractions '[designated A, B and C) were selected for the Boyden chamber experiments. The retention voiumes O'f the reference substanoes are indicated by arrows.
compartment of the Boyden chambers contained 2.2 X lO'i cells/ml of which approximately 85 % could be classified as monoeytes. To exclude the possibility of cell migration through the filters being the result of random movement, two additional tests
Chromatography. Chromatography on Sephadex gels (Sephadex® G-25 and G-200) resulted in two chromatograms (Fig. lA, IB). Three series of fractions were selected from the protein and conductivity peaks in the chromatograms. For convenience the peaks in the chromatograms were designated A, B and C. Fraction A contained substanoes with low, and fraction C substances with high molecular size. The elution volumes of cytochrome C and HSA (human seram albumin) are given in the chromatograms. The elution volumes of the reference substances indicated that fraction A contained substances with molecular weight of < 13,000 and fraction C > 68,000. Protein determination. Table 3 gives the amount of extractable protein in different fractions after gel filtration. The protein recovery was 93 %. Measurement of chemotactic activity. Table 4 presents the results from the Boy-
Tabie 3 Protein determination according to Lowry et al. (1951). Materiai tested Piaque fraction A Plaque fraction B Plaque fraction C
Protein ^g/mi 169 150
34 Table 4
Chemotactic activity for rabbit monocytes elaborated by different fractions of soluble dentai plaque (tested in Boyden chambers). Number of monocytes counted on the iower side of filter
Materiai tested
Pfaque fraction A Piaque fraoti'O'n B Piaque fractfon C Gey's medium {neg. contr.) Casein (pos. controi}
X 25.0 47.0 23.1 8.0 34.9
+ ± ± ± +
S.E. 1.07 3.00 2.09 0.63 3.21
There are' hi'ghly signifioani differences between all means except for fraction A vs. C.
den chamber experiments. The statistical analysis revealed differences between all the test groups and the controls. Significant differences were found also between plaque fraction B, on one hand, and fractions A and C on the other. When plaque material was placed both in the upper and lower compartment of the Boyden chambers no positive chemotaxis of monocytes could be demonstrated. The mean number of monocytes which had migrated through the Millipore® filters was 2.3 ± 0.92 (S.B.). Discussion
The results from the present study show that microbiai dental plaque contains substances which in vitro attract rabbit monocytes. When different fractions were tested regarding their chemotactic activity it was found that all the plaque fractions tested attracted mononuclear leukocytes. The findings were thus in line with earlier published data concerning the capacity of dental plaque to attract neutrophilic Ieukoc34es (Tenapel et al. 1970, Miller et al. 1971, Lindhe & Helldeo 1972, Hellden, Ericson & Lindhe 1973). However, the molecular size of the plaqne fraction that induced the major part
of the inonoc3fte chemotactic activity was larger than that of the corresponding neutrophil chemotactic factor (Hellden et al. 1973). The increased number of neutrophil leukocytes in the junctional epithelium and in the gingival sulcus when plaque is allowed to accumulate on the teeth (Attstrom & Egelberg 1971, Payne et al. 1975) have been ascribed to depend on neutrophilic chemtactic activity elaborated by substances within the dental plaque (Schroeder 1970, Tempel et al. 1970, Attstrom 1971, Hellden 1973). The results of the present investigation indicate that also the presence of monocytes in the junctional epithelium (Schroeder 1973) and gingival sulcus (Attstrom 1970) may be the result of chemotactic stimulation from plaque. Autoradiographic and histologic studies have shown that horseradish peroxidase (mol.wt. 40.000; McDougall 1971, 1972), albumin (mol. wt. 68,000; Tolo 1971) and endotoxin (Schwartz, Stinson & Parker 1972; Ranney & Montgomery 1973) can penetrate the junctional epithelium in the rabbit, pig and dog. Furthermore, Kahnberg et al. (1976) demonstrated an increased number of neutrophils and monocytes in the junctional epithelium in dogs following topical application of soluble dental plaque extract to healthy marginal gingivae. Hence, it seems reasonable to assume that factors chemotactic for monocytes can penetrate the junctional epithelium barrier. In the present study no attempt was made to find out which substances in the plaque elaborated the monocyte chemotactic activity. Wllkinsson, O'Neill & Wapshaw (1973) and Ward (1968) reported that mononuclear cells were attracted by various strains of anaerobic corynebacteria and by peptides from Str. pneumoniae. There is thus reason to assume that microbiai products might he responsible for the activity found also in the present study. It cannot be excluded, however, that some of the activity observed
M O N O C Y T S
C H E M O T A C T I C
may have been induced by complement componetits present within the. dental plaque material (Mergenhagen & Hook 1972, Shillitoe & Lehner 1972, Attstrom et al. 1975). Acknowledgment
The research assistance given by Mrs. Yvonne Sundin is gratefully acknowledged.
References
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Ward, P. A- 1968. Chemotaxis of mononuclear cells. /. Experimental Med. 128: 1201-1219. Wilkinson, P. C, O'Neill, G. I. & Wapshaw, K. G. 1973. Role of anaerobic coryneforms iB Address: Avdelning for Parodontologi Lantmannivagen 111 461 OS TrOllhdttan Sweden
specific and nonspecific immunological reactions. II. Production of a chemotactic ftictor specific for macrophages. Immunology 24: 997-1006.
