Vol. 14, No. 6 Printed in U.S.A.
INFECTION AND IMMUNITY, Dec. 1976, p. 1309-1314 Copyright C) 1976 American Society for Microbiology
Interaction of Inflammatory Cells and Oral Microorganisms II. Modulation of Rabbit Polymorphonuclear Leukocyte Hydrolase Release by Polysaccharides in Response to Streptococcus mutans and Streptococcus sanguis WILLIAM P. McARTHUR* AND NORTON S. TAICHMAN Department ofPathology* and Center for Oral Health Research, School of Dental Medicine, University of Pennsylvania, Philadelphia, Pennsylvania 19174
Received for publication 8 July 1976
The release of lysosomal hydrolases from polymorphonuclear leukocytes (PMNs) has been postulated in the pathogenesis of tissue injury in periodontal disease. In the present study, lysosomal enzyme release was monitored from rabbit peritoneal exudate PMNs exposed to Streptococcus mutans or Streptococcus sanguis. S. mutans grown in brain heart infusion (BHI) broth failed to promote significant PMN enzyme release. S. sanguis grown in BHI broth, although more effective than S. mutans, was a weak stimulus for promotion of PMN hydrolase release. Preincubation of washed, viable S. mutans in sucrose or in different-molecular-weight dextrans resulted in the ability of the organisms to provoke PMN release reactions. This effect could not be demonstrated with boiled or trypsinized S. mutans or with viable S. sanguis. However, when grown in BHI broth supplemented with sucrose, but not with glucose, both S. mutans and S. sanguis triggered discharge of PMN enzymes. The mechanism(s) whereby dextran or sucrose modulates PMN-bacterial interaction may in some manner be related to promotion of microbial adhesiveness or aggregation by dextran and by bacterial synthesis of glucans from sucrose.
The initiation and progression of gingivitis and periodontitis in humans and experimental animals appears to be primarily dependent upon the accumulation of microbial plaques on crowns of teeth in the gingival sulcus area (3, 17). But the manner in which the constituents of plaque cause injury to host tissues remains poorly defined (21). The release of hydrolytic enzymes from polymorphonuclear leukocytes (PMNs) that have interacted with plaque bacteria or with their products may conceivably be one injurious pathway. We have shown that isolated rabbit PMNs liberate intracellular enzymes when exposed to certain gram-positive plaque bacteria (22). These PMN-stimulating bacteria also appear to be capable of synthesizing extracellular polysaccharides that would allow them to form adherent plaques on teeth (8, 9). Furthermore, these microorganisms can cause periodontal disease syndromes upon intraoral inoculation in conventional or gnotobiotic laboratory animals (3, 18). The ability of certain specific plaque bacteria to elaborate extracellular polysaccharides may help to explain their capacity to promote PMN enzyme release (22). Streptococcus mutans (ATCC 6715) grown in brain heart infusion broth in the absence of sucrose do not form 1309
glucans and fail to stimulate PMNs. But when sucrose is added to the growth medium, S. mutans form glucans and subsequently cause PMNs to release enzymes. The purpose of the present study was to gain more information on the ability of polysaccharides to "convert" S. mutans from nonrelease-inducing organisms (i.e., when grown in sucrose-free medium) to stimuli capable of provoking PMN enzyme release. MATERIALS AND METHODS PMNs. Peritoneal exudates were induced in adult rabbits with glycogen, and cells were harvested and washed as previously described (22). PMNs (50 x 106/ml) were suspended in Hanks buffer supplemented with 0.1% gelatin. Propagation of microorganisms. Microorganisms used in this study were generously supplied by B. F. Hammond (University of Pennsylvania, School of Dental Medicine, Philadelphia). S. mutans (ATCC 6715) and S. sanguis (ATCC 6913) were grown aerobically in brain heart infusion broth (Difco, Detroit, Mich.) in the absence of added sucrose (BHI) or in broth supplemented with 1% sucrose (BHI-SUC). All cultures were incubated at 37°C for 18 to 24 h with rotation. Bacteria were harvested by centrifugation at 10,000 x g for 15 min at 4°C, washed with cold 0.9% saline, and resuspended in Hanks bal-
1310
McARTHUR AND TAICHMAN
anced salt solution containing 0.1% gelatin. Bacterial culture supernatants were saved and stored at -20°C until used. Cell concentrations were determined by diluting stock bacterial suspensions and counting in Petroff-Hausser chambers. Bacteria were freshly harvested for each experiment. Pretreatment of bacteria before exposure to PMNs. Washed, viable S. mutans or S. sanguis grown in broth supplemented with sucrose were added directly to PMNs (5, 25, 50, 100, and 200 bacteria/PMN). Identical concentrations of bacteria grown in the absence of sucrose were either added directly to PMNs or preincubated with a variety of agents before being added to PMNs. The pretreatment procedure involved incubating bacterial suspensions in 5% sucrose, maltose, or glucose for 30 min at 37°C in a total volume of 2.9 ml of Hanks gelatin buffer. Similarly, bacteria were preincubated with dextrans (final concentration of 0.1 M) of varying molecular weight (40,000 to 500,000; Sigma Chemical Co., St. Louis, Mo.). In addition, S. mutans grown without sucrose were preexposed to undiluted. sterile BHI and BHI-SUC broth or to BHI and BHI-SUC post-growth culture supernatant from 24-h S. mutans cultures. Controls consisted of bacteria preincubated in Hanks gelatin buffer alone. After pretreatment, bacteria were washed by centrifugation and resuspended in 1.0 ml of Hanks buffer. Incubation of PMNs with bacteria. Microorganisms (5 to 200/PMN) were incubated with PMNs (50 x 106) in a total volume of 2.0 ml of Hanks gelatin buffer in stoppered 12-ml polypropylene centrifuge tubes. Serum was not added to the cultures. Duplicate tubes were set up for each bacterium/ PMN ratio tested and were continuously agitated at 150 rpm in a gyratory water bath shaker (model G86, New Brunswick Scientific Co., New Brunswick, N.J.) at 37°C. The incubation period was varied from 5 to 60 min but usually lasted 30 min. PMNs placed in Hanks gelatin buffer without bacteria served as unstimulated controls. At the conclusion of the experiment, PMNs and bacteria were sedimented by centrifugation, and the culture supernates were assayed for hydrolase activities (22). Enzyme determinations. The procedures used for the quantitation of lysozyme, /3-glucuronidase, and lactate dehydrogenase released from PMNs have been described elsewhere (22). Enzyme release was expressed as a percentage of the total available activity in the PMN suspensions minus spontaneous enzyme release from control PMNs placed in buffer without bacteria. Additional procedures will be described below.
RESULTS PMN hydrolase release in response to bacteria grown in the absence or presence of sucrose. When grown in EHI broth not supplemented with sucrose, various concentrations of S. mutans failed to cause significant release of lysozyme or ,3-glucuronidase from PMNs (Fig. 1 and 2). S. sanguis was a weak stimulus for enzyme release. In sections to follow, these
INFECT. IMMUN.30
O 20
w. 10 .
5
25
50 S. mutans Per PMN
100
FIG. 1. PMN release of lysosomal enzymes after a 30-min exposure to varying concentrations of S. mutans grown in different culture media. Symbols: -) Lysozyme; (---) -glucuronidase; (0) BHI broth; (0) BHI broth supplemented with 1% sucrose; (A) BHI broth supplemented with 1% glucose.
40,
//': .- ~- --o-*-
30
0
0
20
E c
w
0t 10, 0
0
.; 5
~~-A
A
50
25 S. sonquis
100
Per PMN
FIG. 2. PMN release of lysosomal enzymes after a 30-min exposure to varying concentrations of S. sanguis grown in different culture media. Symbols are as in Fig. 1.
weak or non-release-inducing bacteria will be referred to as S. mutans (NR) and S. sanguis (NR). When such organisms were grown in BHI broth supplemented with sucrose, they triggered vigorous PMN enzyme release (Fig. 1 and 2). It should be mentioned that organisms cultivated in BHI broth supplemented with 1% glucose did not cause greater PMN release than bacteria grown in BHI (Fig. 1 and 2). Release of lactate dehydrogenase from PMNs did not occur under any circumstances used in the present study.
PMN hydrolase release in response to S. mutans (NR) preincubated with culture media or growth culture supernatants. Although S. mutans (NR) failed to promote PMN release,
VOL. 14, 1976
INTERACTION OF PMNs AND ORAL BACTERIA. II.
preincubation in uninoculated BHI-SUC broth or in inoculated BHI-SUC growth culture supernatant "converted" this organism into a PMN release inducer (Table 1). BHI growth culture supematants were ineffective in this regard. Extensive dialysis of BHI-SUC growth culture broth or supernatants against phosphate-buffer saline (0.1 M, pH 7.2, 4°C) removed the abilities of these fluids to enhance the induction of PMN enzyme release by S. mutans (NR). Activity of dialyzed materials to convert S. mutans (NR) could be restored by the addition of sucrose (1%, final concentration) to the dialysate. None of the various media tested promoted a release response when added directly (up to 10% of final volume) to PMN cultures in the absence of bacteria.
1311
PMN hydrolase release in response to S. mutans (NR) or S. sanguis (NR) pretreated
with various sugars. Preincubation of varying concentrations of washed viable S. mutans (NR) in Hanks buffer containing final concentrations of 5% sucrose significantly enhanced the potential of this organism to induce PMN enzyme release (Fig. 1; Table 2). Although only the data utilizing a ratio of 100 S. mutans per PMN are illustrated, preincubation with sucrose of 25, 50, and 200 bacteria per PMN also resulted in a significantly enhanced PMN release over that of S. mutans alone at the same ratios. Bacteria preincubated in buffer alone or in buffer containing 5% maltose or 5% dextrose did not stimulate PMN release (Table 2). Boiling (30 min), trypsinization (0.25% tryp-
TABLE 1. PMN hydrolase release in response to S. mutans ( 00 per PMN) preincubated with various culture broths and growth culture supernatantsa Percent enzyme releasec
S. mutans (NR)b preincubated in:
Lysozyme 3.3 + 2.7
pd
3-Glucuronidase 1.3 ± 0.4 1.0 ± 0 7.1 ± 1.8 1.0 ± 0 6.8 ± 2.5
pd
Hanks buffer BHI broth 2.8 ± 1.5 .NS NS BHI broth plus sucrose 11.0 ± 2.3
INFECTION AND IMMUNITY, Dec. 1976, p. 1309-1314 Copyright C) 1976 American Society for Microbiology
Interaction of Inflammatory Cells and Oral Microorganisms II. Modulation of Rabbit Polymorphonuclear Leukocyte Hydrolase Release by Polysaccharides in Response to Streptococcus mutans and Streptococcus sanguis WILLIAM P. McARTHUR* AND NORTON S. TAICHMAN Department ofPathology* and Center for Oral Health Research, School of Dental Medicine, University of Pennsylvania, Philadelphia, Pennsylvania 19174
Received for publication 8 July 1976
The release of lysosomal hydrolases from polymorphonuclear leukocytes (PMNs) has been postulated in the pathogenesis of tissue injury in periodontal disease. In the present study, lysosomal enzyme release was monitored from rabbit peritoneal exudate PMNs exposed to Streptococcus mutans or Streptococcus sanguis. S. mutans grown in brain heart infusion (BHI) broth failed to promote significant PMN enzyme release. S. sanguis grown in BHI broth, although more effective than S. mutans, was a weak stimulus for promotion of PMN hydrolase release. Preincubation of washed, viable S. mutans in sucrose or in different-molecular-weight dextrans resulted in the ability of the organisms to provoke PMN release reactions. This effect could not be demonstrated with boiled or trypsinized S. mutans or with viable S. sanguis. However, when grown in BHI broth supplemented with sucrose, but not with glucose, both S. mutans and S. sanguis triggered discharge of PMN enzymes. The mechanism(s) whereby dextran or sucrose modulates PMN-bacterial interaction may in some manner be related to promotion of microbial adhesiveness or aggregation by dextran and by bacterial synthesis of glucans from sucrose.
The initiation and progression of gingivitis and periodontitis in humans and experimental animals appears to be primarily dependent upon the accumulation of microbial plaques on crowns of teeth in the gingival sulcus area (3, 17). But the manner in which the constituents of plaque cause injury to host tissues remains poorly defined (21). The release of hydrolytic enzymes from polymorphonuclear leukocytes (PMNs) that have interacted with plaque bacteria or with their products may conceivably be one injurious pathway. We have shown that isolated rabbit PMNs liberate intracellular enzymes when exposed to certain gram-positive plaque bacteria (22). These PMN-stimulating bacteria also appear to be capable of synthesizing extracellular polysaccharides that would allow them to form adherent plaques on teeth (8, 9). Furthermore, these microorganisms can cause periodontal disease syndromes upon intraoral inoculation in conventional or gnotobiotic laboratory animals (3, 18). The ability of certain specific plaque bacteria to elaborate extracellular polysaccharides may help to explain their capacity to promote PMN enzyme release (22). Streptococcus mutans (ATCC 6715) grown in brain heart infusion broth in the absence of sucrose do not form 1309
glucans and fail to stimulate PMNs. But when sucrose is added to the growth medium, S. mutans form glucans and subsequently cause PMNs to release enzymes. The purpose of the present study was to gain more information on the ability of polysaccharides to "convert" S. mutans from nonrelease-inducing organisms (i.e., when grown in sucrose-free medium) to stimuli capable of provoking PMN enzyme release. MATERIALS AND METHODS PMNs. Peritoneal exudates were induced in adult rabbits with glycogen, and cells were harvested and washed as previously described (22). PMNs (50 x 106/ml) were suspended in Hanks buffer supplemented with 0.1% gelatin. Propagation of microorganisms. Microorganisms used in this study were generously supplied by B. F. Hammond (University of Pennsylvania, School of Dental Medicine, Philadelphia). S. mutans (ATCC 6715) and S. sanguis (ATCC 6913) were grown aerobically in brain heart infusion broth (Difco, Detroit, Mich.) in the absence of added sucrose (BHI) or in broth supplemented with 1% sucrose (BHI-SUC). All cultures were incubated at 37°C for 18 to 24 h with rotation. Bacteria were harvested by centrifugation at 10,000 x g for 15 min at 4°C, washed with cold 0.9% saline, and resuspended in Hanks bal-
1310
McARTHUR AND TAICHMAN
anced salt solution containing 0.1% gelatin. Bacterial culture supernatants were saved and stored at -20°C until used. Cell concentrations were determined by diluting stock bacterial suspensions and counting in Petroff-Hausser chambers. Bacteria were freshly harvested for each experiment. Pretreatment of bacteria before exposure to PMNs. Washed, viable S. mutans or S. sanguis grown in broth supplemented with sucrose were added directly to PMNs (5, 25, 50, 100, and 200 bacteria/PMN). Identical concentrations of bacteria grown in the absence of sucrose were either added directly to PMNs or preincubated with a variety of agents before being added to PMNs. The pretreatment procedure involved incubating bacterial suspensions in 5% sucrose, maltose, or glucose for 30 min at 37°C in a total volume of 2.9 ml of Hanks gelatin buffer. Similarly, bacteria were preincubated with dextrans (final concentration of 0.1 M) of varying molecular weight (40,000 to 500,000; Sigma Chemical Co., St. Louis, Mo.). In addition, S. mutans grown without sucrose were preexposed to undiluted. sterile BHI and BHI-SUC broth or to BHI and BHI-SUC post-growth culture supernatant from 24-h S. mutans cultures. Controls consisted of bacteria preincubated in Hanks gelatin buffer alone. After pretreatment, bacteria were washed by centrifugation and resuspended in 1.0 ml of Hanks buffer. Incubation of PMNs with bacteria. Microorganisms (5 to 200/PMN) were incubated with PMNs (50 x 106) in a total volume of 2.0 ml of Hanks gelatin buffer in stoppered 12-ml polypropylene centrifuge tubes. Serum was not added to the cultures. Duplicate tubes were set up for each bacterium/ PMN ratio tested and were continuously agitated at 150 rpm in a gyratory water bath shaker (model G86, New Brunswick Scientific Co., New Brunswick, N.J.) at 37°C. The incubation period was varied from 5 to 60 min but usually lasted 30 min. PMNs placed in Hanks gelatin buffer without bacteria served as unstimulated controls. At the conclusion of the experiment, PMNs and bacteria were sedimented by centrifugation, and the culture supernates were assayed for hydrolase activities (22). Enzyme determinations. The procedures used for the quantitation of lysozyme, /3-glucuronidase, and lactate dehydrogenase released from PMNs have been described elsewhere (22). Enzyme release was expressed as a percentage of the total available activity in the PMN suspensions minus spontaneous enzyme release from control PMNs placed in buffer without bacteria. Additional procedures will be described below.
RESULTS PMN hydrolase release in response to bacteria grown in the absence or presence of sucrose. When grown in EHI broth not supplemented with sucrose, various concentrations of S. mutans failed to cause significant release of lysozyme or ,3-glucuronidase from PMNs (Fig. 1 and 2). S. sanguis was a weak stimulus for enzyme release. In sections to follow, these
INFECT. IMMUN.30
O 20
w. 10 .
5
25
50 S. mutans Per PMN
100
FIG. 1. PMN release of lysosomal enzymes after a 30-min exposure to varying concentrations of S. mutans grown in different culture media. Symbols: -) Lysozyme; (---) -glucuronidase; (0) BHI broth; (0) BHI broth supplemented with 1% sucrose; (A) BHI broth supplemented with 1% glucose.
40,
//': .- ~- --o-*-
30
0
0
20
E c
w
0t 10, 0
0
.; 5
~~-A
A
50
25 S. sonquis
100
Per PMN
FIG. 2. PMN release of lysosomal enzymes after a 30-min exposure to varying concentrations of S. sanguis grown in different culture media. Symbols are as in Fig. 1.
weak or non-release-inducing bacteria will be referred to as S. mutans (NR) and S. sanguis (NR). When such organisms were grown in BHI broth supplemented with sucrose, they triggered vigorous PMN enzyme release (Fig. 1 and 2). It should be mentioned that organisms cultivated in BHI broth supplemented with 1% glucose did not cause greater PMN release than bacteria grown in BHI (Fig. 1 and 2). Release of lactate dehydrogenase from PMNs did not occur under any circumstances used in the present study.
PMN hydrolase release in response to S. mutans (NR) preincubated with culture media or growth culture supernatants. Although S. mutans (NR) failed to promote PMN release,
VOL. 14, 1976
INTERACTION OF PMNs AND ORAL BACTERIA. II.
preincubation in uninoculated BHI-SUC broth or in inoculated BHI-SUC growth culture supernatant "converted" this organism into a PMN release inducer (Table 1). BHI growth culture supematants were ineffective in this regard. Extensive dialysis of BHI-SUC growth culture broth or supernatants against phosphate-buffer saline (0.1 M, pH 7.2, 4°C) removed the abilities of these fluids to enhance the induction of PMN enzyme release by S. mutans (NR). Activity of dialyzed materials to convert S. mutans (NR) could be restored by the addition of sucrose (1%, final concentration) to the dialysate. None of the various media tested promoted a release response when added directly (up to 10% of final volume) to PMN cultures in the absence of bacteria.
1311
PMN hydrolase release in response to S. mutans (NR) or S. sanguis (NR) pretreated
with various sugars. Preincubation of varying concentrations of washed viable S. mutans (NR) in Hanks buffer containing final concentrations of 5% sucrose significantly enhanced the potential of this organism to induce PMN enzyme release (Fig. 1; Table 2). Although only the data utilizing a ratio of 100 S. mutans per PMN are illustrated, preincubation with sucrose of 25, 50, and 200 bacteria per PMN also resulted in a significantly enhanced PMN release over that of S. mutans alone at the same ratios. Bacteria preincubated in buffer alone or in buffer containing 5% maltose or 5% dextrose did not stimulate PMN release (Table 2). Boiling (30 min), trypsinization (0.25% tryp-
TABLE 1. PMN hydrolase release in response to S. mutans ( 00 per PMN) preincubated with various culture broths and growth culture supernatantsa Percent enzyme releasec
S. mutans (NR)b preincubated in:
Lysozyme 3.3 + 2.7
pd
3-Glucuronidase 1.3 ± 0.4 1.0 ± 0 7.1 ± 1.8 1.0 ± 0 6.8 ± 2.5
pd
Hanks buffer BHI broth 2.8 ± 1.5 .NS NS BHI broth plus sucrose 11.0 ± 2.3
