Vol. 16, No. 3 Printed in U.S.A.
INFECTION AND IMMUNITY, June 1977, p. 934-937 Copyright © 1977 American Society for Microbiology
Exocytosis of Polymorphonuclear Leukocyte Lysosomal Contents Induced by Dental Plaque ROBERT R. WHITE*
AND
EDWARD H. MONTGOMERY
Departments of Microbiology and Pharmacology, The University of Texas Dental Branch at Houston, Houston, Te-xas 77030 Received for publication 22 December 1976
Rabbit polymorphonuclear leukocytes were incubated with a sonically treated suspension of pooled dental plaque to determine if the plaque would induce release of lysosomal enzymes from the polymorphonuclear leukocytes. Cells incubated with plaque at 37°C released significantly greater amounts of the lysosomal enzymes, 8-glucuronidase and lysozyme, than did cells incubated with plaque at 00C or without plaque at 37°C. This response was both dose and time dependent. Release of the cytoplasmic enzyme lactate dehydrogenase was minimal, and there were no significant differences in lactate dehydrogenase release between cells at 0 and 37°C, or without plaque. These results indicate that dental plaque can induce the selective release of lysosomal enzymes, which could be involved in the periodontal injury produced by dental plaque. During phagocytosis, polymorphonuclear leukocytes (PMNs) release lysosomal constituents into the surrounding medium (12, 15). These compounds have been implicated as mediators of the inflammatory response and may be involved in the ensuing tissue injury (10, 15, 22). Several studies (20; W. P. McArthur, N. S. Taichman, A. Stephenson, and R. Negron, J. Dent. Res. 53:178, 1974) suggest that dental plaque also may be capable of inducing the release of lysosomal constituents, an event that could potentiate the injurious effects of the inflammatory reaction occurring in the diseased periodontium. Taichman et al. (23) reported that intradermal injection of dental plaque into rabbits induced the appearance of lysosomal enzymes, some apparently of leukocytic origin. He and his colleagues also have reported that pure cultures of organisms commonly found in dental plaque can induce lysosomal enzyme release in vitro (McArthur et al., J. Dent. Res. 53:178, 1974; N. S. Taichman, W. P. McArthur, C. C. Tsai, and U. R. Nilsson, J. Dent. Res. 54:64, 1975). The purpose of this study was to determine if suspensions of whole dental plaque could mediate the release of lysosomal enzymes from rabbit PMNs in vitro. MATERIALS AND METHODS Preparation of plaque extracts. Plaque was collected by the dental hygiene students of the University of Texas Dental Branch at Houston. The plaque consisted of both supra- and subgingival material from patients with mild to moderate periodontal disease. Plaque samples were obtained before any other treatments, placed in sterile saline, and frozen until used.
Samples from 20 patients were pooled and centrifuged at 10,000 x g for 10 min to sediment particulate matter. The sediment was suspended in 5 ml of Hanks balanced salt solution (HBSS) (Grand Island Biological Co., Grand Island, N.Y.) and sonically treated for 30 s with a Branson 75 Sonifier at a setting of 4. After standing for 5 min to allow sedimentation of calculus, the suspension was decanted and designated as the "plaque suspension." Such preparations were readily retained in suspension by the gentle shaking procedure utilized during incubation with the PMNs. Initial studies indicated that a 1:50 dilution corresponding to a turbidity of 200 Klett-Summerson colorimeter units caused release of PMN enzymes. Subsequently, all plaque suspensions prepared were diluted to 200 Klett units. The final concentration of plaque proteins and carbohydrates in the incubation media ranged from 60 to 116 yig of protein per ml as measured by the Lowry method (17) and 57 to 180 ,ug of carbohydrate per ml as measured by a modified anthrone procedure (21). Preparation of PMN suspensions. PMNs were obtained according to a modification of the method described by Goldstein et al. (11). Male New Zealand white rabbits were bled by cardiac puncture. The blood was mixed with 0.25 volume of 6% dextran (average molecular weight, 234,000) (Sigma Chemical Co., St. Louis, Mo.) in pyrogen-free physiological saline, and erythrocytes were allowed to sediment at room temperature. The plasma was centrifuged at 150 x g for 10 min, and the cell pellet was retained. Remaining erythrocytes were lysed by suspending the cell pellet in 1 volume of 0.1% saline for 20 s before adjusting the solution to isotonicity by addition of 1 volume of 1.7% saline. The cells were washed once and suspended in HBSS at a concentration of 5 x 100 cells per ml. Differential cell counts performed according to the rapid fixation method of Hirsch and Cohn (16) showed that the preparations 34
LYSOSOMAL ENZYME RELEASE INDUCED BY PLAQUE
VOL. 16, 1977
contained 80 to 85% PMNs. Preparations that contained greater than 5% nonviable cells based on trypan blue exclusion were discarded. All treatments and assays were run in duplicate, and only cells from one rabbit were used in any individual experiment. Evaluation of enzyme release. Two milliliters of the cell suspension was incubated in a shaking water bath (60 strokes/min) at 0 and 37°C with 200 ,ul of plaque suspension or HBSS. After incubation, the cell suspension was divided into two aliquots. One aliquot was centrifuged to remove the PMNs, and the supernatant was assayed for enzymatic activities. The enzyme activity in this supernatant was considered to be the enzyme release induced by plaque. The second aliquot was lysed with 0.2% Triton X-100. The enzyme activity in this lysate was considered to be the total enzyme activity of the PMNs. Lysozyme and 3-glucuronidase were used as lysosomal enzyme markers. Lactate dehydrogenase (LDH) was used as a cytoplasmic marker. Lysozyme was measured according to the method of Dietrich and Bloch (8), /8-glucuronidase according to the method of Fishman (9), and LDH according to the method of Bergmeyer et al. (6). Reagents were obtained from Sigma Chemical Co. Comparison of the relative concentrations of these enzymes was used to determine if the lysosomal enzymes were released during a phagocytic process or as a result of cell lysis.
RESULTS Figure 1 shows the results obtained after incubation of the PMNs for 60 min with the plaque suspension at 0 and 370C and with HBSS at 370C. The enzyme activity released from the PMNs is expressed as the percentage of total enzyme activity present in the lysate of these cells. Each point represents the mean from six animals. Mean values for the release of en-
935
zymes in the presence of plaque at 37°C were
7% for LDH, 14.8% for f3-glucuronidase, and 42% for lysozyme. Incubation with plaque at 37°C caused significant (P < 0.001) increases in ,8-glucuronidase and lysozyme, as compared with incubation either with plaque at 0°C or with HBSS at 3700. Small levels of extracellular LDH were found after each of the three treatments, indicating a minimal degree of cell lysis. However, there was no significant increase in LDH activity when the cells were incubated with plaque at 3700 as compared with incubation with HBSS at 3700. The increase in lysosomal enzyme activities seen by incubation with plaque at 37°C is not the result of cell lysis, but rather it is a selective release of lysosomal enzymes mediated by plaque. All plaque preparations gave quantitatively similar results. Figure 2 shows the enzymatic activity released from PMN cells incubated with plaque suspensions at 3700 for 15, 30, and 60 min. Each point represents the mean of five animals. Again, enzyme activity is expressed as a percentage of total activity. Plaque-mediated release of the lysosomal indicators 18-glucuronidase and lysozyme increased in a relatively linear fashion with time, each value being sig60L YS tA
-a
LU
w
5.
LDH=LACTIC DEHYDROGENASE B6= i- GLUCUROMIDASE LYS=LYSOZYME
4
Ca
a1-
a30a.
BG
Or3
20LOH
I
ILIHIUGILYSI
PLAGUE4-C
ILDHI *6 ILYSI HBSS-3rC
NDj Br. ILYS_
PLAaUE-3rC
FIG. 1. Release of cellular enzymes induced by incubation of PMNs in a suspension ofdental plaque at 0 and 37C or in HBSS at 37°C for 60 min. Each point represents the mean of six animals. The vertical bars indicate the standard errors of the means.
15
30
60
TIMPE (MINUTES) FIG. 2. Release of cellular enzymes by PMNs incubated in a dental plaque suspension for varying periods of time. Each point represents the mean of five animals. The vertical bars indicate the standard errors of the means.
936
WHITE AND MONTGOMERY
INFECCT. IMMUN.
nificantly (P < 0.05) higher than that for the previous time interval. At 60 min plaque induced the release of 13.8% of the total cellular ,3-glucuronidase activity and 54.5% of the total lysozyme activity. There was no significant change in LDH activity during this time period, showing that incubation with plaque did not cause cell lysis and that the linear increase in extracellular lysosomal enzymes was due to a selective release of lysosomal contents mediated by plaque. Figure 3 shows the percentage of total enzymatic activity released after 60 min of incubation as a function of plaque concentration. The undiluted preparation of plaque contained 116 ,ug of protein per ml and 84 ,ug of carbohydrate per ml. Each point represents the mean of five animals. The enzyme values are expressed as the difference from the control values for incubation with HBSS at 370C and thus represent the effects of plaque concentration only. The release of both /3-glucuronidase and lysozyme from the PMNs increased significantly (P < 0.02) as the concentration of plaque was increased from 1/16 dilution of the initial concentration up to the undiluted concentration. A small, but significant (P < 0.05), increase in LDH activity occurred when the plaque concentration was increased from a 1/16 to a 1/4 dilution. LDH levels were the same for the 1/4 dilution and the undiluted plaque extract, showing that cell lysis contributed only mini35 -
-
LYSzLYSOZYME 30-
_
C6= -GLUCUROUIDASE
LYS
LDHLNACTIC DEHYDROGENASE 20
/
INFECTION AND IMMUNITY, June 1977, p. 934-937 Copyright © 1977 American Society for Microbiology
Exocytosis of Polymorphonuclear Leukocyte Lysosomal Contents Induced by Dental Plaque ROBERT R. WHITE*
AND
EDWARD H. MONTGOMERY
Departments of Microbiology and Pharmacology, The University of Texas Dental Branch at Houston, Houston, Te-xas 77030 Received for publication 22 December 1976
Rabbit polymorphonuclear leukocytes were incubated with a sonically treated suspension of pooled dental plaque to determine if the plaque would induce release of lysosomal enzymes from the polymorphonuclear leukocytes. Cells incubated with plaque at 37°C released significantly greater amounts of the lysosomal enzymes, 8-glucuronidase and lysozyme, than did cells incubated with plaque at 00C or without plaque at 37°C. This response was both dose and time dependent. Release of the cytoplasmic enzyme lactate dehydrogenase was minimal, and there were no significant differences in lactate dehydrogenase release between cells at 0 and 37°C, or without plaque. These results indicate that dental plaque can induce the selective release of lysosomal enzymes, which could be involved in the periodontal injury produced by dental plaque. During phagocytosis, polymorphonuclear leukocytes (PMNs) release lysosomal constituents into the surrounding medium (12, 15). These compounds have been implicated as mediators of the inflammatory response and may be involved in the ensuing tissue injury (10, 15, 22). Several studies (20; W. P. McArthur, N. S. Taichman, A. Stephenson, and R. Negron, J. Dent. Res. 53:178, 1974) suggest that dental plaque also may be capable of inducing the release of lysosomal constituents, an event that could potentiate the injurious effects of the inflammatory reaction occurring in the diseased periodontium. Taichman et al. (23) reported that intradermal injection of dental plaque into rabbits induced the appearance of lysosomal enzymes, some apparently of leukocytic origin. He and his colleagues also have reported that pure cultures of organisms commonly found in dental plaque can induce lysosomal enzyme release in vitro (McArthur et al., J. Dent. Res. 53:178, 1974; N. S. Taichman, W. P. McArthur, C. C. Tsai, and U. R. Nilsson, J. Dent. Res. 54:64, 1975). The purpose of this study was to determine if suspensions of whole dental plaque could mediate the release of lysosomal enzymes from rabbit PMNs in vitro. MATERIALS AND METHODS Preparation of plaque extracts. Plaque was collected by the dental hygiene students of the University of Texas Dental Branch at Houston. The plaque consisted of both supra- and subgingival material from patients with mild to moderate periodontal disease. Plaque samples were obtained before any other treatments, placed in sterile saline, and frozen until used.
Samples from 20 patients were pooled and centrifuged at 10,000 x g for 10 min to sediment particulate matter. The sediment was suspended in 5 ml of Hanks balanced salt solution (HBSS) (Grand Island Biological Co., Grand Island, N.Y.) and sonically treated for 30 s with a Branson 75 Sonifier at a setting of 4. After standing for 5 min to allow sedimentation of calculus, the suspension was decanted and designated as the "plaque suspension." Such preparations were readily retained in suspension by the gentle shaking procedure utilized during incubation with the PMNs. Initial studies indicated that a 1:50 dilution corresponding to a turbidity of 200 Klett-Summerson colorimeter units caused release of PMN enzymes. Subsequently, all plaque suspensions prepared were diluted to 200 Klett units. The final concentration of plaque proteins and carbohydrates in the incubation media ranged from 60 to 116 yig of protein per ml as measured by the Lowry method (17) and 57 to 180 ,ug of carbohydrate per ml as measured by a modified anthrone procedure (21). Preparation of PMN suspensions. PMNs were obtained according to a modification of the method described by Goldstein et al. (11). Male New Zealand white rabbits were bled by cardiac puncture. The blood was mixed with 0.25 volume of 6% dextran (average molecular weight, 234,000) (Sigma Chemical Co., St. Louis, Mo.) in pyrogen-free physiological saline, and erythrocytes were allowed to sediment at room temperature. The plasma was centrifuged at 150 x g for 10 min, and the cell pellet was retained. Remaining erythrocytes were lysed by suspending the cell pellet in 1 volume of 0.1% saline for 20 s before adjusting the solution to isotonicity by addition of 1 volume of 1.7% saline. The cells were washed once and suspended in HBSS at a concentration of 5 x 100 cells per ml. Differential cell counts performed according to the rapid fixation method of Hirsch and Cohn (16) showed that the preparations 34
LYSOSOMAL ENZYME RELEASE INDUCED BY PLAQUE
VOL. 16, 1977
contained 80 to 85% PMNs. Preparations that contained greater than 5% nonviable cells based on trypan blue exclusion were discarded. All treatments and assays were run in duplicate, and only cells from one rabbit were used in any individual experiment. Evaluation of enzyme release. Two milliliters of the cell suspension was incubated in a shaking water bath (60 strokes/min) at 0 and 37°C with 200 ,ul of plaque suspension or HBSS. After incubation, the cell suspension was divided into two aliquots. One aliquot was centrifuged to remove the PMNs, and the supernatant was assayed for enzymatic activities. The enzyme activity in this supernatant was considered to be the enzyme release induced by plaque. The second aliquot was lysed with 0.2% Triton X-100. The enzyme activity in this lysate was considered to be the total enzyme activity of the PMNs. Lysozyme and 3-glucuronidase were used as lysosomal enzyme markers. Lactate dehydrogenase (LDH) was used as a cytoplasmic marker. Lysozyme was measured according to the method of Dietrich and Bloch (8), /8-glucuronidase according to the method of Fishman (9), and LDH according to the method of Bergmeyer et al. (6). Reagents were obtained from Sigma Chemical Co. Comparison of the relative concentrations of these enzymes was used to determine if the lysosomal enzymes were released during a phagocytic process or as a result of cell lysis.
RESULTS Figure 1 shows the results obtained after incubation of the PMNs for 60 min with the plaque suspension at 0 and 370C and with HBSS at 370C. The enzyme activity released from the PMNs is expressed as the percentage of total enzyme activity present in the lysate of these cells. Each point represents the mean from six animals. Mean values for the release of en-
935
zymes in the presence of plaque at 37°C were
7% for LDH, 14.8% for f3-glucuronidase, and 42% for lysozyme. Incubation with plaque at 37°C caused significant (P < 0.001) increases in ,8-glucuronidase and lysozyme, as compared with incubation either with plaque at 0°C or with HBSS at 3700. Small levels of extracellular LDH were found after each of the three treatments, indicating a minimal degree of cell lysis. However, there was no significant increase in LDH activity when the cells were incubated with plaque at 3700 as compared with incubation with HBSS at 3700. The increase in lysosomal enzyme activities seen by incubation with plaque at 37°C is not the result of cell lysis, but rather it is a selective release of lysosomal enzymes mediated by plaque. All plaque preparations gave quantitatively similar results. Figure 2 shows the enzymatic activity released from PMN cells incubated with plaque suspensions at 3700 for 15, 30, and 60 min. Each point represents the mean of five animals. Again, enzyme activity is expressed as a percentage of total activity. Plaque-mediated release of the lysosomal indicators 18-glucuronidase and lysozyme increased in a relatively linear fashion with time, each value being sig60L YS tA
-a
LU
w
5.
LDH=LACTIC DEHYDROGENASE B6= i- GLUCUROMIDASE LYS=LYSOZYME
4
Ca
a1-
a30a.
BG
Or3
20LOH
I
ILIHIUGILYSI
PLAGUE4-C
ILDHI *6 ILYSI HBSS-3rC
NDj Br. ILYS_
PLAaUE-3rC
FIG. 1. Release of cellular enzymes induced by incubation of PMNs in a suspension ofdental plaque at 0 and 37C or in HBSS at 37°C for 60 min. Each point represents the mean of six animals. The vertical bars indicate the standard errors of the means.
15
30
60
TIMPE (MINUTES) FIG. 2. Release of cellular enzymes by PMNs incubated in a dental plaque suspension for varying periods of time. Each point represents the mean of five animals. The vertical bars indicate the standard errors of the means.
936
WHITE AND MONTGOMERY
INFECCT. IMMUN.
nificantly (P < 0.05) higher than that for the previous time interval. At 60 min plaque induced the release of 13.8% of the total cellular ,3-glucuronidase activity and 54.5% of the total lysozyme activity. There was no significant change in LDH activity during this time period, showing that incubation with plaque did not cause cell lysis and that the linear increase in extracellular lysosomal enzymes was due to a selective release of lysosomal contents mediated by plaque. Figure 3 shows the percentage of total enzymatic activity released after 60 min of incubation as a function of plaque concentration. The undiluted preparation of plaque contained 116 ,ug of protein per ml and 84 ,ug of carbohydrate per ml. Each point represents the mean of five animals. The enzyme values are expressed as the difference from the control values for incubation with HBSS at 370C and thus represent the effects of plaque concentration only. The release of both /3-glucuronidase and lysozyme from the PMNs increased significantly (P < 0.02) as the concentration of plaque was increased from 1/16 dilution of the initial concentration up to the undiluted concentration. A small, but significant (P < 0.05), increase in LDH activity occurred when the plaque concentration was increased from a 1/16 to a 1/4 dilution. LDH levels were the same for the 1/4 dilution and the undiluted plaque extract, showing that cell lysis contributed only mini35 -
-
LYSzLYSOZYME 30-
_
C6= -GLUCUROUIDASE
LYS
LDHLNACTIC DEHYDROGENASE 20
/
