Vol. 14, No. 6

INFBCTION AND IMMUNITY, Dec. 1976, p. 1315-1321 Copyright C 1976 American Society for Microbiology

Printed in U.S.A.

Interaction of Inflammatory Cells and Oral Microorganisms III. Modulation of Rabbit Polymorphonuclear Leukocyte Hydrolase Release Response to Actinomyces viscosus and Streptococcus mutans by Immunoglobulins and Complement WILLIAM P. McARTHUR,* PIERRE BAEHNI, 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

In the absence of antiserum, rabbit polymorphonuclear leukocytes (PMNs) released lysosomal enzymes in response to Actinomyces viscosus (19246) but not to Streptococcus mutans (6715). Antibodies had a marked modulating influence on these reactions. PMN hydrolase release was significantly enhanced to both organisms when specific rabbit antiserum and isolated immunoglobulin G (IgG) were included in the incubations. Immune complex F(ab')2 fragments of IgG directed against S. mutans agglutinated bacteria. Immune complexes consisting of S. mutans and F(ab')2 fragments of IgG directed against this organism were not effective as bacteria-IgG complexes in stimulating PMN release. The intensity of the release response to bacteria-IgG complexes was also diminished when PMNs were preincubated with isolated Fc fragments derived from IgG. Fresh serum as a source of complement components had no demonstrable effect on PMN release either alone or in conjunction with antiserum in these experiments. These data may be relevant to the mechanisms and consequences of the interaction of PMNs and plaque bacteria in the pathogenesis of periodontal disease.

Immunoglobulins and complement components have been identified in inflamed gingival tissues and crevicular fluids (1, 3, 4, 22, 28, 31). The various classes of immunoglobulins found in these lesions may, to a large degree, represent antibodies synthesized in response to bacterial antigens originating from dental plaque (3, 22). The local union of antibodies with their respective antigens would result in the formation of immune complexes, and it has been hypothesized that antigen-antibody aggregates might serve as triggers for lysosome release

from polymorphonuclear leukocytes (PMNs) that have emigrated into diseased gingival tissues and crevices (33). The purpose of this study was to gain information on the role of antibody and complement as modulators of PMN enzyme release in response to two different gram-positive plaque isolates, Actinomyces viscosus and Streptococcus mutans. In previous studies we have shown that in the absence of antibody or complement, A. viscosus could serve as a positive inducer for the release of enzymes from rabbit PMNs. On the other hand, S. mutans (36) (grown in the absence of sucrose) failed to provoke such reactions. These microorganisms were selected, therefore, in order to ascertain the effect of

antibody and complement on the response of rabbit PMNs to positive and negative bacterial hydrolase release-inducing stimuli. MATERIALS AND METHODS The general experimental design involved exposing isolated rabbit PMNs to varying concentrations of A. viscosus or S. mutans with or without added specific antibodies or complement. Secretion of PMN lysosomal markers into the culture fluid was monitored at the conclusion of the experiments. Rabbit peritoneal exudate PMNs. PMN-rich (80 to 90%) exudates were induced by intraperitoneal injection of 0.1% glycogen in adult (5.0 to 6.0 kg) New Zealand rabbits. Cells were harvested 4 h later, washed, and suspended (50 x 106 PMNs/ml) in Hanks buffer containing 0.1% gelatin as described elsewhere (36). Propagation of microorganisms. A. viscosus (ATCC 19246) was grown in Trypticase soy broth under aerobic conditions. S. mutans (ATCC 6715) was cultivated aerobically in brain heart infusion (BHI) medium (Difco, Detroit, Mich.). Cultures were incubated at 37°C for 16 to 24 h, harvested by centrifugation, washed with cold, physiological saline, and resuspended in Hanks buffer containing 0.1% gelatin. Bacterial cell counts were made using Petroff-Hausser chambers. Rabbit antisera. New Zealand rabbits (3.0 to 4.0

1315

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McARTHUR, BAEHNI, AND TAICHMAN

kg) were immunized by subcutaneous inoculations of live A. viscosus (19246) (5 x 106 bacteria per injection), S. mutans (6715) (10"' bacteria per injection), or bovine serum albumin (BSA; 10 mg per injection; Pentex Inc., Kankakee, Ill.) emulsified in 1.0 ml of complete Freund adjuvant (Difco, Detroit, Mich.). Each animal received three injections spaced over a 4-week period. Hyperimmune antisera were collected and pooled beginning at 3 days after completing the immunization schedule. Normal rabbit sera were obtained from unimmunized animals. All sera were heated to 56°C for 30 min and stored at -20°C. Portions of antisera to S. mutans or to BSA were precipitated by ammonium sulfate and fractionated by diethylaminoethyl-cellulose chromatography to obtain immunoglobulin G (IgG)-enriched preparations (5). Purity of these fractions was monitored by electrophoresis using goat anti-whole rabbit serum. Samples of anti-S. mutans and anti-BSA IgG were digested with pepsin to yield F(ab')2 fragments (25), which were isolated on a G-150 Sephadex column (26). Removal of the Fc piece from IgG was confirmed by the fact that the appropriate F(ab')2 fragments still agglutinated sheep erythrocytes coated with BSA (5) or with S. mutans culture supernate antigens (T. Dishon, unpublished observation). F(ab')2 did not support complement-mediated (fresh guinea pig serum) immune lysis of sensitized sheep erythrocytes (5). Anti-BSA F(ab')2 fragments also failed to sensitize guinea pigs for passive cutaneous anaphylaxis (37). F(ab')2 preparations were suspended to their original serum volumes in phosphate-buffered saline (0.1 M, pH 7.4). IgG was obtained from normal rabbit serum as described above and digested with papain and purified by the method of Putman et al. (26) to obtain Fc fragments. Purity of the Fc fraction was checked by immunodiffusion against goat anti-rabbit IgG. This preparation was diluted to one-fifth of its original serum volume with buffered saline. Complement sources. Fresh rabbit and guinea pig sera were collected, stored at - 70°C, and used as sources of complement. Preincubation of bacteria with immune reagents. Before exposure to PMNs, varying numbers of A. viscosus or S. mutans (final concentrations ranging from 5, 25, 50, and 100 bacteria per PMN) were preincubated in duplicate at 37°C for 15 min in a total volume of 1.0 ml of Hanks gelatin buffer containing various immune reagents. Polypropylene centrifuge tubes (12.0-ml capacity) were used as culture vessels. A. viscosus was preexposed to whole anti-A. viscosus serum (10%), whole anti-S. mutans serum (10%), normal rabbit serum (10%), or Hanks buffer. S. mutans was preincubated with whole antiS. mutans serum (1 to 10%), whole antisera to BSA (10%) or to A. viscosus (10%), normal rabbit serum (10%), or Hanks buffer. IgG fractions (10%) or F(ab')2 preparations (10%) of anti-S. mutans or antiBSA were also preincubated with S. mutans. Fresh rabbit or guinea pig sera (10%) as sources of complement were added during the preincubation period in certain experiments. Incubation of bacteria with PMNs. One milliliter

INFECT. IMMUN.

of PMNs (50 x 106) was added directly to the tubes containing pretreated microorganisms and incubated at 37°C in a gyratory water bath shaker (150 rpm, model G86, New Brunswick Scientific Co., New Brunswick, N.J.) for 15 or 30 min. Controls consisted of PMNs in Hanks-gelatin buffer alone (i.e., "resting" cells) and PMNs exposed to the various immune reagents in the absence of bacteria. At the end of the experiment, PMNs and bacteria were sedimented and culture supernatants were monitored for extracellular release of PMN enzymes (36). Preincubation of PMNs with Fc fragments of rabbit IgG. PMNs were preincubated with Fc fragments (10% of a stock preparation, which was concentrated to one-fifth of its original serum volume) for 15 min at 4°C. PMNs were then directly added to S. mutans-antiserum complexes for an additional 15 min at 37°C. Leukocytes and bacteria were then removed by centrifugation and culture supernatants were assayed for enzyme activities. Enzyme determinations. Lysozyme, 3-glucuronidase, and cathepsin D were used as representative markers for the extracellular discharge of PMN lysosomal constituents. The biochemical techniques for assaying these hydrolases have been published elsewhere (36). Since the total concentrations of individual hydrolases in a given cell preparation varied from one experiment to another, enzyme release was expressed as a percentage of the total available activity in the PMN cultures. Total activity was measured by dispersing 1.0 ml of the PMN suspensions in 1.0 ml of distilled water and subjecting the preparation to five rapid freeze-thaw cycles (acetone-dry ice). In arriving at the final percentage of enzyme release from cells exposed to various stimuli, the spontaneous liberation of enzymes from resting PMNs placed in Hanks buffer alone was subtracted from the experimental values. Where appropriate (i.e., when immune reagents were included in the incubations), inherent enzyme activities in various reagents were also subtracted from the experimental values. The mean percentage of enzyme release in response to bacteria incubated with various immune reagents was compared to the appropriate controls by using Student's t test. All P values of greater than 0.05 were considered nonsignificant.

RESULTS Effect of whole antiserum and complement on the response of PMNs to A. viscosus. PMN enzyme release was observed in response to varying concentrations ofA. viscosus in Hanks buffer in the absence of added immune reagents (Table 1). A. viscosus pretreated with specific whole antiserum became more potent triggers at all concentrations for PMN release (Table 1). The inclusion of fresh guinea pig or rabbit sera with bacteria and specific antiserum did not enhance release to a greater degree than that recorded for antiserum-bacteria alone (Table 1). Preincubation of A. viscosus with either anti-S. mutans serum or normal rabbit serum

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INTERACTION OF PMNs AND ORAL BACTERIA. III.

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in the presence or absence of added complement failed to modify release above that recorded for A. viscosus in Hanks buffer alone (Table 1). Although data are not presented, none of the immune reagents used in this study stimulated significant PMN release in the absence of added bacteria. Effect of whole antiserum and complement on the response of PMNs to S. mutans. Varying concentrations of S. mutans in Hanks buffer failed to promote PMN enzyme release (Table 2). Bacteria preincubated in specific, whole antiserum were capable of provoking significant PMN reactions (Table 2), but the addition of complement sources to bacteria-antiserum complexes did not further augment release (Table 2). By comparison, nonspecific whole antisera (i.e., anti-A. viscosus) with or without complement did not "convert" S. mutans into a PMN release-inducing stimulus (Table 2). Effect of antibody fractions on the response of PMNs to S. mutans. As with whole anti-S. mutans serum, S. mutans preincubated in IgG derived from this antiserum provoked PMN enzyme release (Table 3). On the other hand, S. mutans pretreated with the F(ab')2 fraction of anti-S. mutans IgG were not potent stimuli for PMN reactions (Table 3). Nonspecific whole rabbit serum and F(ab')2 fragments of antiBSA IgG fraction did not alter the ability of S. mutans to provoke PMNs (Table 3). Effect of Fc fragments on PMN response to S. mutans-IgG complexes. PMN release in response to S. mutans-IgG aggregates was significantly depressed when leukocytes were preincubated with Fc fragments (Table 4). Further, Fc-pretreated PMNs failed to respond to S. mutans in buffer alone (Table 4).

DISCUSSION

sources

Gingivitis and periodontitis represent a series of chronic inflammatory disorders that lead to the progressive destruction of tissues that support teeth in their sockets (35). The onset and continued development of gingivitis and periodontitis has been linked to the accumulation and retention of heterogenous microbial plaques on the cervical surfaces of teeth (20, 32). But the manner in which plaque bacteria or their products promote tissue damage remains poorly defined. A substantial and continuous exudation of PMNs represents one of the hallmarks of periodontal disease. There is evidence to suggest that PMNs liberate lysosomal products in host tissues and in gingival crevices or pockets (6, 8, 9, 18). Several investigators have speculated that PMN lysosome release may represent one potential mechanism of tissue destruction in periodontal disease (2, 10, 34). Lysosome release could occur as a consequence of the interaction of PMNs with whole plaque (N. S. Taichman, W. P. McArthur, and P. Baehni, J. Dent. Res. 55:B213, 1976), and we have previously reported that certain specific, but not all representative, gram-positive and gram-negative plaque bacteria can stimulate PMN enzyme release (36; W. P. McArthur, B. F. Hammond, and N. S. Taichman, J. Dent. Res. 55:B213, 1976). The results presented in the present communication demonstrate that antibodies can exert significant modulating effects on the rabbit PMN response to A. viscosus and S. mutans. A. viscosus was used in our studies because, in the absence of antibody, these microorga-

TABLE 2. Effect of whole antisera" and complement" on PMN response to S. mutans % PMN lysozyme release'

S. mutans preincubated with:" 5e

Hanks buffer Rabbit anti-S. mutans serum (10%) Rabbit anti-S. mutans serum (10%) plus fresh rabbit serum (10%) Rabbit anti-S. mutans serum (10%) plus fresh guinea pig serum (10%) Rabbit anti-A. viscosus serum (10%) Fresh rabbit serum (10%) Fresh guinea pig serum (10%)

25

50

100

1.0 ± 1.0 5.8 ± 3.0 (P < 0.015)f 7.2 + 1.5 (P > 0.05)°

1.2 ± 1.2 11.0 + 4.0 (P < 0.008) 8.0 ± 0.3 (P > 0.05)

2.0 ± 1.0 15.0 ± 1.6 (P < 0.001) 17.0 ± 0.8 (P > 0.05)

2.3 ± 1.2 20.0 ± 2.0 (P < 0.001) 24.0 + 7.0 (P > 0.05)

8.0 ± 3.0 (P > 0.05)"

12.0 ± 4.0 (P > 0.05)

17.0 ± 3.0 (P > 0.05)

23.0 ± 2.0 (P > 0.05)

2.0 ± 1.6

2.4 ± 1.4

3.0 + 1.2

4.0 ± 1.8

1.0 ± 1.0 2.0 ± 1.0

1.6 ± 1.0 3.0 ± 2.0

2.4 + 1.4 4.0 ± 2.0

2.6 ± 1.8 5.0 ± 3.0

a.b.d.e.f.g See Table 1.

Prior to exposure to PMNs, S. mutans were preincubated with various immune reagents for 15 min at 37°C. "

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VOL. 14, 1976

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TABLE 3. Effect of purified mmunoglobulin components on PMN response to S. mutans % PMN enzyme releaseb

S. mutans (50/PMN)a preincubated with: Lysozyme

13-Glucuronidase

Cathepsin D

4.3 + 0.7 3.7 ± 0.6 2.7 ± 0.6 29.0 ± 8.0 8.3 ± 1.8 13.0 ± 1.2 (P < 0.016) (P < 0.007)' (P < 0.001) Rabbit anti-S. mutans IgG (10%) 25.0 + 7.0 19.0 + 4.0 13.0 + 2.8 (P < 0.006) (P < 0.011) (P < 0.006) Rabbit anti-S. mutans F(ab')2 (10%) 7.0 + 4.0 4.0 + 3.6 3.5 + 2.0 (P < 0.35) (P < 0.16) (P < 0.38) Rabbit anti-BSA serum (10%) 4.5 ± 0.7 1.7 ± 0.6 2.5 ± 0.7 Rabbit anti-BSA IgG (10%) 3.5 ± 0.7 3.3 ± 0.6 1.2 ± 0.6 Rabbit anti-BSA F(ab')2 (10%) 2.5 ± 0.7 2.3 ± 0.6 1.0 ± 1.0 a Before exposure to PMNs, S. mutans were preincubated with various immune reagents for 15 min at 370C. b Enzyme release expressed as in Table 1. e P values represent comparison of the mean of the release reactions induced by bacteria preincubated with immune reagents as compared to bacteria preincubated in Hanks buffer alone.

Hanks buffer Rabbit anti-S. mutans serum (10%)

TABLE 4. Effect ofpreincubating PMNs with Fc fragmentsa on enzyme release induced by immune complexes (S. mutans plus anti-S. mutans serum) PMNs preincubated with:b

Hanks buffer

Release-inducing stimulir S. mutans (50/PMN) S. mutans (50/PMN)

% Lysozyme released

1.5 ± 0.4

plus Fc fragments (10%)

anti-S. mutans serum (1%) S. mutans (50/PMN) S. mutans (50/PMN) plus anti-S. mutans serum (1%)

16.5 ± 2.4 1.5 ± 0.3

10.0 + 1.7 (P < 0.005)e Fc fragments obtained by papain digestion of rabbit IgG and concentrated to five times original serum volume. b PMNs preincubated in Hanks or with Fc fragments for 15 min. ' PMNs exposed to bacterial stimuli for 30 min. d Release expressed as a percentage of total available lysozyme activity in PMN suspension. e p values represent comparison of mean release of enzymes from PMNs preincubated with Fc fragments as compared to PMNs preincubated in Hanks buffer alone. a

nisms can provoke PMN lysosome release (36). Preincubation of A. viscosus with hyperimmune, specific antiserum or IgG significantly enhanced PMN release in response to these bacteria. Under the conditions utilized, complement in itself or in the presence of antiserum had no effect on the release reactions. S. mutans was selected because this organism in itself fails to stimulate PMNs. But specific antiserum or IgG antibody can "convert" these bacteria into stimuli capable of provoking PMN release. Once again, complement was without effect in modulating PMN reactions. When S. mutans are grown in medium supplemented with sucrose (36) or preincubated in Hanks buffer containing sucrose, they become capable of provoking PMN release (20a). Under these conditions, S. mutans undergo spontaneous aggregation and are capable of adhering to foreign surfaces (12). These phenomena are

presumably mediated by bacterial synthesis of cell-surface polysaccharides from sucrose. Likewise, preincubation of S. mutans with specific antibodies results in clumping of the organisms. When compared with S. mutans in Hanks buffer alone, both S. mutans-antibody complexes and bacterial aggregates formed in the presence of sucrose are more efficiently phagocytosed by PMNs (P. Baehni, M. A. Listgarten, W. P. McArthur, and N. S. Taichman, J. Dent. Res. 55:B64, 1976). Since PMN enzyme release is often dependent upon ingestion of various particulates (16, 19, 40), it is reasonable to assume that increased phagocytosis of S. mutans in the presence of antibody or polysaccharides accounts for the increased intensity of PMN release under these conditions. The precise mechanism whereby immune aggregation of bacteria promotes phagocytosis and release PMNs is not known, but there is reason to

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McARTHUR, BAEHNI, AND TAICHMAN

believe that the Fc fragment of IgG may be more important than the mere aggregation of the microorganisms. This was shown by the results of experiments with subfractions of IgG. S. mutans pretreated with F(ab'), were aggregated to the same extent as that seen with IgG anti-S. mutans. But F(ab')2-bacteria complexes were significantly less potent in causing PMN release than IgG-bacteria aggregates. Likewise, others have shown that PMN release to BSA-anti-BSA precipitates does not occur to the same degree when complexes contain antibodies devoid of Fc fragments (27). These data suggest that the Fc portion of IgG antibody is a crucial determinant in accounting for the release-inducing properties of immune complexes formed with soluble antigens or with bacteria. Rabbit PMNs possess surface membrane receptors for the Fc pieces of IgG molecules that have interacted with antigens (16). It would appear, therefore, that immune complexes interact via Fc receptors on the PMN as a prerequisite step for the induction of lysosome release (14, 16, 23, 27, 40). This hypothesis gains support in the present study by virtue of experiments showing a depression of enzyme release from PMNs preincubated with isolated Fc fragments. Under these circumstances, it is presumed that the Fc fragments "saturated" Fc receptors on PMN membranes and subsequently inhibited attachment of antibody-bacteria aggregates. As noted earlier, rabbit or guinea pig complement failed to affect release of enzymes from rabbit PMNs exposed to A. viscosus or S. mutans alone or in the presence of antibody. Taichman et al. (38) also found that complement had no demonstrable effect on rabbit PMN release in response to immune complexes at equivalence or in antigen excess. Preliminary data using human rather than rabbit PMNs incubated with plaque suggest that release in this instance in enhanced in the presence of complement (Taichman et al., J. Dent. Res. 55:B213, 1976), which agrees with reports that activated complement components can trigger human PMN enzyme release (13, 15). Although the evidence is indirect, the interaction of PMNs and immune complexes may have some bearing on the pathogenesis of periodontal disease. In this context, PMN immune complex interaction resulting in lysosome release may play a role in the production of tissue injury analogous to that visualized in experimental Arthus reactions (7) or in such clinical inflammatory diseases as rheumatoid arthritis (41). Various immunoglobulins (4, 11, 31), specific antibodies to plaque bacteria (3, 22, 24, 28), and complement components (1, 31)

INFECT. IMMUN.

have been localized in the inflammatory infiltrate and fluid exudate in diseased gingiva.

And it has been hypothesized that the combination of antibodies and plaque antigens may result in the deposition of immune complexes in the affected tissues (11, 21, 29, 30, 39). Experimental gingival and periodontal inflammation can be induced by local formation and deposition of immune complexes in gingival tissues (21, 29, 30, 39). In the present study we have demonstrated that immune complexes formed with bacteria as opposed to soluble protein antigens can trigger PMN lysosome release. It is highly conceivable that plaque bacteria-antibody complexes may be formed in the gingival environment and may thereby stimulate PMN release during the course of periodontal disease.

In vitro studies from our laboratory have shown that whole plaque can provoke PMN lysosome release, but it appears that not all species of plaque bacteria can induce this reaction. In the present study we found that antibodies could not only enhance PMN release in response to a positive, release-inducing organism (i.e., A. viscosus) but could also "convert" a negative PMN stimulus (i.e., S. mutans) into a positive PMN-activating stimulus. In relating these findings to natural disease, it is tempting to speculate that the host's humoral response to plaque bacteria may have a significant bearing on the pathogenesis of tissue injury. If one assumes that PMN lysosomes represent at least one mechanism of tissue damage in periodontal disease, then antibodies can conceivably modulate PMN lysosome release to both "positive" and "negative" plaque bacteria in vivo. ACKNOWLEDGMENTS Grateful thanks are due to Janice Garber and Ann Ste-

phenson for their assistance in the execution of these exper-

iments and H. M. Myers for his critical evaluation of the manuscript. This study was supported by Public Health Service grants DE 02623 and DE 03995 from the National Institute of Dental Research.

LITERATURE CITED 1. Attstrom, R., A. B. Laurel, U. Lahsson, and A. Sjoholm. 1975. Complement factors in gingival crevice material from healthy and inflamed gingiva in humans. J. Periodontal Res. 10:19-27. 2. Attstrom, R., G. Tynelius-Bratthall, and I. Egelberg. 1971. Effect of experimental leukopenia on chronic gingival inflammation in dogs. II. Induction of leukopenia by heterologous anti-neutrophil serum. J. Per-

iodontal Res. 6:200-210. 3. Berglund, S. E. 1971. Immunoglobulins in human gingiva with specificity for oral bacteria. J. Periodontol. 42:546-551.

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INTERACTION OF PMNs AND ORAL BACTERIA. III.

4. Byers, C. W., P. D. Toto, and A. W. Garguilo. 1975. Levels of immunoglobulins IgG, IgA and IgM in human inflamed gingiva. J. Periodontol. 46:387-390. 5. Campbell, D. H., J. S. Garvey, N. E. Cremer, and D. H. Sussdorf. 1970. Methods in immunology, 2nd ed. W. A. Benjamin, Inc., New York. 6. Cimasoni, G. 1974. The crevicular fluid. S. Karger, Basel. 7. Cochrane, C. G. 1968. Immunologic tissue injury mediated by neutrophilic leukocytes. Adv. Immunol. 9:97-162. 8. Cowley, G. C. 1972. Enzyme activity in gingival immunocytes. J. Dent. Res. 51:384-392. 9. Freedman, H. L., M. A. Listgarten, and N. S. Taichman. 1968. Electron microscopic features of chronically inflamed human gingiva. J. Periodontal Res. 3:313-327. 10. Gavin, J. B. 1970. Ultrastructural features of chronic marginal periodontitis. J. Periodontal Res. 5:19-29. 11. Genco, R. J., P. A. Mashimo, G. Krygier, and S. A. Ellison. 1974. Antibody-mediated effects on the periodontium. J. Periodontol. 45:330-337. 12. Gibbons, R. J., and J. van Houte. 1975. Bacterial adherence in oral microbial ecology. Annu. Rev. Microbiol. 29:19-44. 13. Goldstein, I. M., S. Hoffstein, J. Gallin, and G. Weissmann. 1973. Mechanisms of lysosomal enzyme release from human leukocytes. Microtubule assembly and membrane fusion induced by a component of complement. Proc. Natl. Acad. Sci. U.S.A. 70:29192920. 14. Goldstein, I. M. 1976. Polymorphonuclear leukocyte lysosomes and immune tissue injury. Prog. Allergy 20:301-340. 15. Goldstein, I. M., M. Brai, A. G. Osler, and G. Weissmann. 1973. Lysosomal enzyme release from human leukocytes. Mediation by the alternate pathway of complement activation. J. Immunol. 11:33-37. 16. Henson, P. M. 1971. The immunologic release of constituents from neutrophil leukocytes. I. The role of antibody and complement on nonphagocytosable surfaces of phagocytosable particles. J. Immunol. 107:1535-1546. 17. Janoff, A. (ed.). 1972. Symposium on neutrophil proteases as mediators of tissue injury. Am. J. Pathol. 68:539-623. 18. Lange, D., and H. E. Schroeder. 1971. Cytochemistry and ultrastructure of gingival crevice cells. Helv. Odontol. Acta 15:68-86. 19. Leffell, M. S., and J. K. Spitznagel. 1975. Fate of human lactoferrin and myeloperoxidase in phagocytizing human neutrophils: effects of immunoglobulin G subclasses and immune complexes coated on latex beads. Infect. Immun. 12:813-820. 20. Loe, H., E. Theilade, and S. B. Jensen. 1965. Experimental gingivitis in man. J. Periodontol. 36:177-187. 20a. McArthur, W. P., and N. S. Taichman. 1976. 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. Infect. Immun. 14:1309-1314. 21. McDougall, W. A. 1975. The effect of topical antigen on the gingiva of sensitized rabbits. J. Periodontal Res. 9:153-164. 22. Mayron, L. W., and R. J. Loisell. 1973. Bacterial antigens and antibodies in human periodontal tissue. J. Periodontol. 44:164-166.

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23. Messner, R. P., and J. Jelinek. 1970. Receptors for human yG globulin on human neutrophils. J. Clin. Invest. 49:2165-2171. 24. Nisengard, R. J., and E. H. Beutner. 1970. Immunologic studies of periodontal disease. V. IgG type antibodies and skin test responses to Actinomyces and mixed oral flora. J. Periodontol. 41:149-152. 25. Nisonoff, A., L. N. Lipman, F. C. Wissler, and D. L. Woernley. 1960. Separation of univalent fragments from the bivalent rabbit antibody by reduction of disulfide bonds. Arch. Biochem. Biophys. 89:230-244. 26. Putman, F. W., M. Tan, L. T. Lynn, C. W. Easley, and S. Migita. 1962. The cleavage of rabbit gamma globulin by papain. J. Biol. Chem. 237:717-726. 27. Ranadive, N. S., A. N. Sajnani, K. Alimurka, and H. Z. Movat. 1973. Release of basic proteins and lysosomal enzymes from neutrophil leukocytes of the rabbit. Int. Arch. Allergy 45:880-898. 28. Ranney, R. R. 1970. Specific antibody in gingiva and submandibular nodes of monkeys with all?rgic periodontal disease. J. Periodontal Res. 5:1-7. 29. Ranney, R. R., and H. A. Zander. 1970. Allergic periodontal disease in sensitized squirrel monkeys. J. Periodontol. 41:12-21. 30. Rizzo, A. A., and C. T. Mitchell. 1966. Chronic allergic inflammation induced by repeated deposition of antigen in rabbit gingival pockets. Periodontics 4:5-10. 31. Shillitoe, E. J., and T. Lehner. 1972. Immunoglobulins and complement in crevicular fluid, serum and saliva in man. Arch. Oral Biol. 17:241-247. 32. Socransky, S. S. 1970. Relationship of bacteria to the etiology of periodontal disease. J. Dent. Res. 49:203222. 33. Taichman, N. S. 1970. Mediation of inflammation by the polymorphonuclear leukocyte as a sequela of immune reactions. J. Periodontol. 41:228-231. 34. Taichman, N. S., H. L. Freedman, and T. Uriuhara. 1966. Inflammation and tissue injury. I. The response to intradermal injections of human dentogingival plaque in normal and leukopenic rabbits. Arch. Oral Biol. 11:1385-1392. 35. Taichman, N. S., and W. P. McArthur. 1975. Current concepts in periodontal disease. Annu. Rep. Med. Chem. 10:228-239. 36. Taichman, N. S., and W. P. McArthur. 1976. Interaction of inflammatory cells and oral bacteria: release of lysosomal hydrolases from rabbit polymorphonuclear leukocytes exposed to Gram-positive plaque bacteria. Arch. Oral Biol. 21:257-263. 37. Taichman, N. S., H. Z. Movat, M. F. Glyn, and I. Broder. 1971. Further studies on the role of neutrophils in passive cutaneous anaphylaxis of the guinea pig. Immunology 21:623-635. 38. Taichman, N. S., W. Pruzanski, and N. S. Ranadive. 1972. Release of intracellular constituents from rabbit polymorphonuclear leukocytes exposed to soluble and insoluble immune complexes. Int. Arch. Allergy 43:182-195. 39. Terner, C. 1965. Arthus reaction in the oral cavity of laboratory animals. Periodontics 3:18-22. 40. Weissmann, G., R. B. Zurier, P. J. Spieler, and I. M. Goldstein. 1971. Mechanism of lysosomal enzyme release from leukocytes exposed to immune complexes and other particles. J. Exp. Med. 134:149s165s. 41. Zwaifler, N. J. 1970. Further speculation on the pathogenesis of joint inflammation in rheumatoid arthritis. Arthritis Rheum. 13:895-901.

Interaction of inflammatory cells and oral microorganisms. III. Modulation of rabbit polymorphonuclear leukocyte hydrolase release response to Actinomyces viscosus and Streptococcus mutans by immunoglobulins and complement.

In the absence of antiserum, rabbit polymorphonuclear leukocytes (PMNs) released lysosomal enzymes in response to Actinomyces viscosus (19246) but not...
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