Archs oral Bid. Vol. 24. pp. 369 lo 377 Pergamon Press Ltd 1979. Prmted m Great Bntain
IMMUNO-ELECTRON MICROSCOPIC STUDY OF ANTIGENIC SURFACE COMPONENTS OF ACTINOMYCES NAESLUNDII IN HUMAN DENTAL PLAQUE P. R. GARANT*, MOON-IL CHO*, V. IACONO~ and G. J. SHEMAkAt *Department of Oral Biology and Pathology and iDepartment of Periodontics, School of Dental Medicine, Health Sciences Center, State University of New York at Stony Brook, Stony Brook, NY 11794, U.S.A.
Summary-Rabbit antiserum to formalin-killed Actinomyces naeslundii I was used to investigate the ultrastructural location and distribution of the antigenic sites of A. naeslundii. Antigenie sites were identified by an indirect technique using goat anti-rabbit IgG coupled to horseradish peroxidase, also used to identify individual cells of A. naeslundii in a mixed bacterial population and in freshly isolated dental plaque. The bound antibody and associated reaction product were visible in ultrathin sections as an electron-dense amorphous material (lO&lSOnm thick) in juxtaposition to the bacterial cell wall. The location of the immunoreactants suggested that the antigens are distributed superficially and evenly over the entire bacterial cell surface. Bridge-like extensions of the immuno-reactants connected adjacent cells, suggesting that a limited amount of antigen& material might extend out from the cell wall to provide structural continuity with similar material on adjacent cells.
at 37°C in brain-heart infusion medium (BHI-Difco Laboratories, Detroit, Mich.) supplemented with 0.3 per cent glucose. The cells were harvested by centrifugation (16,3OOg, lOmin, 4°C) and washed twice in phosphate-buffered saline (PBS-O.15 M sodium chloride, 0.02 M sodium phosphate, pH 7.5). PBS-washed cells were used for immuno-electron microscopic studies. Some preparations were formalin killed (Taubman and Smith, 1974) and used in immunization procedures. The following organisms were used for detection of A. naeslundii in mixed bacterial suspensions: Staphylococcus aureus and Escherichia coli, both donated by Dr. J. Pollock and grown in BHI, Streptococcus salivarius 4TCC No. 25975, also grown in BHI, Lactobacillus casei ATCC No. 25180-B, grown in Rogosa medium, and Fusobacteria fusiforme ATCC No. 23726, grown in BHI plus 0.5 per cent thioglycollate. Standard suspensions of bacteria (lO’jm1) were inoculated into 10ml of media and grown at 37°C for 18-24 h. Mixtures of the six species of bacteria, including A. naeslundii I, were prepared by combining 1 ml suspensions of washed cells of each species. The combined cells were then pelleted and washed 3 times in PBS prior to forming the final pellet for immunocytochemical procedures. Supragingival plaque samples were obtained from a healthy donor and from one subject with severe gingivitis by gentle scaling with a curette and immediately pre-fixed in a saturated picric acid solution containing 4 per cent paraformaldehyde at 4°C.
INTRODUCTION
One aspect of the current hypothesis of the pathogenesis of periodontal disease is the immunologic response of the host to bacterial (plaque) antigens; it is therefore important to identify the major surface antigens of oral bacteria and to show the presence of such antigens in tissues and cells of the host. Evidence that serum antibodies (Nisengard and Beutner, 1970; Gilmour and Nisengard, 1974) and cellmediated immune mechanisms (Ivanyi and Lehner, 1970; Ivanyi, Wilton and Lehner, 1972; Horton, Leiken and Oppenheim, 1972; Horton, Oppenheim and Mergenhagen, 1974; Mackler et al., 1974) are directed against plaque bacteria indicates that antigen penetration into host tissues probably occurs in periodontal disease. It would be helpful to ascertain the distribution and extent of antigenic localization in gingival and periodontal tissues in order to correlate antigen penetration and binding to the host cells with local tissue destruction. We have used immunocytochemical procedures to localize surface antigens of Actinomyces naeslundii on its cell surface coat and to identify Actinomyces in human dental plaque. These studies were carried out in order to establish the specificity and reliability of the method as preliminary steps in the localization of surface antigens in the oral tissues and cells of rats mono-infected with A. naeslundii. Socransky, Hubersak and Propas (1970) and Garant (1976) showed that rats develop advanced periodontitis when mono-infected with A. naeslundii which has been implicated as an agent in human periodontal disease (Gilmour and Nisengard, 1974; Baker et al., 1976). MATERIALS Preparation
of bacterial
A. naeslundii
AND
Preparation
METHODS
cultures
I, was routinely
and human plaque
grown
of antisera
A male new Zealand White rabbit was injected with 3 ml of vaccine consisting of 10” formalin-killed A. naeslundii I in 1.5 ml PBS emulsified in an equal volume of Complete Freund’s Adjuvant (Difco). The
for 2&24 h 369
370
P. R.
Garant et al.
vaccine was administered into a hind foot-pad and subcutaneously in the dorsum of the neck. Booster injections were administered 2 weeks after the first injection and periodically thereafter to maintain high titres of antibody. The rabbit was bled one week after each booster injection and the sera obtained were heated at 56°C for 31 min to inactivate complement. All sera were tested in immunodiffusion and immunoelectrophoretic analyses for antibody activity (Iacono, Katiyar and Shemaka, 1976a). Sera that reacted similarly were pooled. Normal rabbit serum was obtained prior to any immunization procedures. Bacterial cell, plaque and section preparation Pelleted, PBS-washed A. naeslundii I and mixed cultures were pre-fixed in either 1 per cent glutaraldehyde in 0.1 M phosphate buffer, pH 7.4, for 2 h at room temperature, or in saturated picric acid containing 4 per cent paraformaldehyde for 4 h at 4°C (Lai, Listgarten and Rosan, 1975a). Plaque samples were prefixed in 4 per cent paraformaldehyde at time of collection (see above). The fixed samples were washed 4 times in PBS and stored overnight in PBS at 4°C. Samples selected for polyethylene glycol (PEG) embedding were first dehydrated in a graded series of ethanol (total time 3 h) and then placed in 100 per cent PEG (2 changes, 1 h each) at 39°C in a vacuum oven. The samples were then placed in gelatin capsules containing 100 per cent PEG and hardened at 4°C. Blocks were stored in a desiccator at 4°C until sectioned. Sections (15-30pM thick) of the PEG embedded samples were cut on a portable rotary microtome in a cold room. The sections were floated on 5 per cent glycerol in PBS for 70min and then transferred with a plastic loop to PBS where they were kept for 40min with a change of solution. The sections were then transferred to gelatin-coated slides and air dried before being immersed in staining jars filled with PBS. After 30 min in the PBS, the sections were ready for immunocytochemical studies. Immunocytochemical procedures (A) For sectioned samples: Aliquots (1.5 ml) of anti-A. naeslundii I serum (diluted 1: 1 with PBS) or normal rabbit serum were pipetted on the slides which were kept moistened in closed Petri dishes. The slides were then incubated for 1 h with replacement of fresh antiserum or normal rabbit serum at 20min intervals. The sections were then washed by slow agitation for 1 h in staining jars containing PBS which was changed every 20min. Incubation with horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG (Cappel Labs., Downingtown, PA.) was then performed in moistened Petri dishes for 1 h with two changes of the fresh conjugate. Washing in PBS was again carried out prior to fixation of the samples in 5 per cent glutaraldehyde in 0.1 M phosphate buffer, pH 7.4 at 4°C for 1 h. The fixed sections were washed (3 x 10min) in 0.1 M phosphate buffer (pH 7.4) containing 4.5 per cent sucrose and stored overnight at 4°C in the same solution. The samples were then washed (15 min) in 0.05 M trisHC1 buffer, pH 7.6 and pre-incubated in 3,3-diamino benzidine (DAB, 80 mg) in 100 ml 0.05 M tris-HCl buffer, (pH 7.6) for 1 h, then incubated for
5 min in the DAB solution containing 0.005 per cent HzOz. The samples were rinsed in 0.05 M tris_HCl (15 min), postfixed in 1 per cent 0~0, in 0.1 M veronal acetate buffer for 30 min, and washed in the same buffer for 15 min. The fixed sections were then scraped from the slides, dehydrated in a graded series of ethanol and embedded in Epon. (B) For chopped samples: Washed and pre-fixed pellets of either pure or mixed bacteria and plaque samples were chopped into small pieces, approximately 0.5 mm x 0.1 mm, under a dissecting microscope. All immunocytochemical procedures were the same as for the PEG-sectioned samples with the exception that centrifugation and pipetting were used for all fluid changing and rinsing steps. All steps were carried out in 15 ml centrifuge tubes containing 1 ml of the appropriate solution. After completion of the immunocytochemical processes, post-fixation and dehydration, the final pellets were embedded in Epon directly in the 15 ml centrifuge tubes. Electron microscopy Ultrathin sections were cut on a Sorvall MTB-2 ultramicrotome equipped with a diamond knife. Some sections were doubly stained with uranyl acetate and lead citrate. The sections were examined in a JEOL-100B electron microscope. Table 1 summarizes the various experimental and control specimens which were finally produced and examined ultrastructurally. RESULTS
The immunochemical staining was more intense at the peripheral zone of both the pelleted bacteria and the 15 and 30 PM sections of the PEG-embedded material. This was apparent as a brown HRP-positive margin around the PEG sections or the 1 PM Epon sections of the pelleted samples, when viewed unstained in the light microscope. The central areas of pelleted bacteria, plaque and the thicker PEG sections were usually devoid of any reaction. This was clearly established when viewing unstained ultrathin sections in the electron microscope. A. naeslundii I which was treated with HRP-conjugated goat anti-rabbit IgG (Fig. 1) or with normal rabbit serum (Fig. 2), followed by incubation in DAB and H,02 showed no cell-wall-associated material (Fig. 3). The DAB did, however, increase the electron density of the innermost 8-1Onm region of the cell wall. When the bacteria were reacted with rabbit antisera to A. naes[undii I followed by HRP-conjugated goat anti-rabbit IgG and DAB plus H,Oz an electrondense halo of immuno-reactants was deposited around each cell. This material was visible in unstained as well as stained sections (Fig. 4). The uniform distribution of the immuno-reactants suggested that the antigens were located superficially and evenly distributed over the entire bacterial cell surface. The bound antibody and associated reaction product were visible as an electron-dense amorphous material (3s50nm thick) juxtaposed to the bacterial cell wall (Figs. 5 and 6). Bridge-like extensions of the amorphous immuno-reactants connected adjacent bacterial cells (Fig. 5). The presence of these structures suggests that a limited amount of antigenic material might extend out from the cell wall to provide struc-
Immuno-electron Table
1. Summary
of immunocytochemical
Antisera to A. naeslundii I I. Pure culture Experimental Control (A) Control (B) Control (C) 11. Mixed species Experimental 111. Plaque Experimental Control (a) Control (b) Control (c)
microscopy of A. naeslundii
staining and preparatory human plaque Normal rabbit serum
-
procedures
371 applied
HRP-conjugated goat anti-rabbit IgG
DAB + H202
to cultures
Heavy metal staining
-t -
+ -
+ + + -
+ + + +
+&+&+&+&-
+
-
+
+
+&-
+ -
+
+ + + -
+ + + +
+&f&+8z+&-
-
tural continuity with similar material on adjacent cells. In mixed cultures treated with the full complement of immuno-reactants the cells of A. naeslundii I appeared as small clusters (Fig. 7). Each A. naeslundii I cell was clearly demarcated by the halo of amorphous material (Fig. 8). Other Gram-positive organisms sometimes showed a small amount of loosely bound amorphous material resembling the immuno-reactants (Fig. 9). Actinomyces cells were clearly demarcated in the plaque samples by the circumferential band of adherent immuno-reactants (Figs. 10-12). Similar bands were not present on other Gram-positive and Gramnegative organisms. Actinomyces cells were usually present in small clusters or microcolonies (Fig. 10). Adjacent cells were frequently connected by strands of material which were “stained” by the immunoreactants. Plaque, in contrast with the pelleted pure and mixed cultures, contained greater amounts of interbacterial debris or matrix material (Fig. 11). Such substances were difficult to separate from the immuno-reactants in stained sections but were not electron dense when viewed in unstained ultrathin sections. Prefixation of cells and plaque in either 1 per cent glutaraldehyde or in 4 per cent paraformaldehyde did not appear to have any noticeable effect on the reactivity of the antigens. In both cases, equally dense halos of immuno-reactants were deposited, suggesting that mild glutaraldehyde fixation does not block the antigenic sites. Thorough washing of the glutaraldehyde-fixed tissues was necessary to minimize inactivation of the antigenic sites. DISCUSSION
Similar immunocytochemical techniques have been used to show surface antigens of Streptococcus sanguis (Lai et al., 1975a) and Streptococcus mutans (Iacono et al., 1976b; Grenier et al., 1977). Furthermore, the distribution of Strep. sanguis in plaque was shown at the EM level by Lai et al. (1975b). The PEG method gives reproducible results and is especially useful when correct tissue orientation must be main-
and
tained or when serial sections are to be studied (Lai et al., 1975b). Our study was not concerned with preserving the original orientation of the plaque but only with localizing Actinomyces. Our study and those referred to show that unlabelled rabbit antibody or antiserum together with HRP-conjugated anti-rabbit IgG is a reliable method of identifying specific bacteria in human plaque. Haugen, Helgeland and Grov (1975) used the immuno-peroxidase technique to localize bacterial antigens directly upon ultrathin sections of bacteria embedded in low-viscosity plastic. Our attempts to use those techniques have failed; we could not prevent non-specific staining of the ultrathin sections. The use of the Fab fraction of purified antibody, to A. naeslundii may prove more successful. Direct localization of antigen on ultrathin sections would be useful because sections of special interest from a variety of tissue blocks could be reacted with a minimum amount of immunoglobulin. The Haugen et a/. method was used by Erlandsen, Parsons and Taylor, (1973) to localize lysozyme in human Paneth cells. Kraehenbuhl and Jamieson (1972) in a review of the methods for localizing intracellular antigens by immuno-electron microscopy, reported that albumin embedding techniques can be very useful in the surface localization of antigens on ultrathin sections. The reaction of antiserum directed against A. naeslundii I with surface components of Actinomyces viscosus T14 (Iacono et al., 1976) was expected, as Gerencser and Stark (1976) have suggested that the catalase positive A. oiscosus should be classified as a variety or subspecies of A. naeslundii. Therefore, the cells localized in the plaque samples with the anti-A. naeslundii I serum could be representative strains of one or more of the several species of Actinomyces (Bowden, Hardie and Fillery, 1976). The immunoreactant-staining interconnecting strands of material between adjacent Actinomyces cells in both cultures and plaque samples are similar to fibrils detected in electron micrographs of Actinomyces by Girard and Jacius (1974). Girard and Jacius (1974) suggested that the fibrillar coatings may mediate attachment of Actinomyces to the tooth surface and may account for their haemagglutinating activity (Rolla and Kilian,
P. R. Gal rant et ul.
312
1977). The small clusters we observed of Actinomyces cells in whole plaque samples apparently connected by fibrillar bridges suggests a role for these fibrils in interbacterial aggregation. The nature of the fibrillar antigens has yet to be determined. However, Birdsell and Fischlschweiger (1977) observed fibrils only on virulent strains of A. uiscosus T14. Virulent strains contain 6-deoxytalose (6-DOT)-containing antigen in their cell walls (Hammond Steel and Peindl, 1976). The ultrastructural orientation of this antigen at the cell surface has not been determined. We found that 6-DOT antigens are readily released from stock suspensions of A. oiscosus T14 by incubating with a neutral buffer and, furthermore, Iacono et al. (1977) detected them in culture media. These antigens may occupy a superficial position at the cell surface and, therefore, may serve as a structural component in the surface fibrils. They may also be constituents of the observed interbacterial debris surrounding the Actinomyces cell in plaque (Fig. 11). Localization studies we have in progress with Fab fractions of monospecific antibody to surface components of Actinomyces should elucidate these matters. Acknowledyements~This research was supported by the National Institute of Dental Research, N.I.H., Research Grant Nos. ROl DE 03745 and DE 04168. We thank Ms. Mary Lee and Mr. J. Loffredo for their technical assistance. The help of Mrs. B. Sykes in preparation of the manuscript is greatly appreciated.
REFERENCES
Baker J. J., Chan S. P., Socransky S. S., Oppenheim J. J. and Mergenhagen S. E. 1976. Importance of Actinomyces and certain gram-negative anaerobic organisms in the transformation of lymphocytes from patients with periodontal disease. Infect. Immun. 13, 1363-1368. Birdsell D. C. and Fischlschweiger W. 1977. Comparative ultrastructure of Actinomyces uiscosus. J. dent. Res. 56B, 117. Bowden G. H., Hardie J. M. and Fillery E. D. 1976. Antigens from Actinomyces species and their value in identification. J. dent. Res. 55 (Suppl. A), 192-204. Erlandsen S. L., Parsons J. A. and Taylor T. D. 1973. Ultrastructural immunocytochemical localization of lysozyme in the Paneth cells of man. J. Histochem. Cytothem. 22,401-413. Garant P. R. 1976. An electron microscopic study of the periodontal tissues of germfree rats and rats monoinfected with Actinomyces naeslundii. .I. periodont. Res. (Suppl. 15) l-79. Gerencser M. A. and Stark J. M. 1976. Serological identification of Acrinomyces using fluorescent antibody techniques. J. denr. Res. 55 (Suppl. A), 184-191. Gilmour M. M. and Nisengard R. J. 1974. Interactions between serum titers to filamentous bacteria and their relationship to human periodontal disease. Archs oral Biol. 19, 959-968. Girard A. E. and Jacius B. H. 1974. Ultrastructure of Actinomyces uiscosus and Actinomyces naeslundii. Archs oral Biol. 19, 71-79.
Grenier E. M., Gray R. H., Loesche W. J. and Eveland W. C. 1977. Microcapsules on Streptococcus mutans serotypes by electron microscopy. J. dent. Res. 56, 16C176. Hammond B. F., Steel C. F. and Peindl K. S. 1976. Antigens and surface components associated with virulence of Actinomyces uiscosus. J. dent. Res. 55 (Suppl. A), 19-25. Haugen A., Helgeland S. and Grov A. 1975. Localization of antigens in thin sections of bacteria by the immunoperoxidase technique. Acta path. microbial. stand. (B) 83, 79-90. Horton J. E., Leiken S. and Oppenheim J. J. 1972. Human lymphoproliferative reaction to saliva and dental plaque deposits: an in vitro correlation with periodontal disease. J. Periodont. 43, 522-527. Horton J. E., Oppenheim J. J. and Mergenhagen S. E. 1974. A role for cell-mediated immunity in the pathogenesis of periodontal disease. J. Periodont. 45, 351-360. Iacono V. J., Katiyar V. N. and Shemaka G. J. 1976a. Analysis of antibody directed against surface antigens of Actinomyces species. J. Dent. Res. 55 (Suppl. B), 222. Iacono V. J., Levine M. J., Shemaka G. J. and Katiyar V. N. 1977. Characterization of surface antigens of Actinomyces viscosus T14. J. Dent. Res. 56 (Suppl. A), 120. Iacono V. J., Taubman M. A., Smith D. J., Garant P. R. and Pollock J. J. 1976b. Structure and function of the type specific polysaccharide of Streptococcus mutans 6715. Immunology Abstracts (Special Suppl. Immunological Aspects of Dental Caries) 75-90. Ivanvi L. and Lehner T. 1970. Stimulation of lvmohocvte transformation by bacterial antigens in paiieits with Deriodontal disease. Archs oral Biol. 15. 1089%1096. Ivanyi L., Wilton J. M. A. and Lehner T. 1972. Cellmediated immunity in periodontal disease: cytotoxicity, migration inhibition, and lymphocyte transformation studies. Immunology 22, 141-145. Kraehenbuhl J. P. and Jamieson J. D. 1972. Localization of intracellular antigens by immunoelectron microscopy.. . Int. Rev. exp. Path.-12, 1:53. Lai C. H.. Listearten M. A. and Rosan B. 1975a. Immunoelectron m&oscopic identification and localization of Streptococcus sanguis with peroxidase-labelled antibody: Localization of surface antigens in pure cultures. Infect. Immun. 11, 193-199. Lai C. H., Listgarten M. A. and Rosan B. 1975b. Immunoelectron microscopic identification and localization of Streptococcus sanguis with peroxidase-labelled antibody: Localization, of Streptococcus sanguis in intact dental plaque. Infect. Immun. 11, 200-210. Mackler B. F., Altman L. C., Wahl S., Rosenstreich D. L., Oppenheim J. J. and Mergenhagen S. E. 1974. Blastogenesis and lymphokine synthesis by T and B lymphocytes from patients with periodontal disease. Infect. Imman. 10, 844~850. Nisengard R. J. and Beutner E. H. 1970. Immunologic studies of periodontal disease. V. IgG type antibodies and skin test responses to Actinomyces and mixed flora. J. Periodont. 41, 149-152. Rolla G. and Kilian M. 1977. Haemaaglutination activity of plaque-forming bacteria. Caries I&. 11, 85-89. Socranskv S. S.. Hubersak C. and Prooas D. 1970. Induction oi periodontal destruction in gnotobiotic rats by a human oral strain of Actinomyces naeslundii. Archs oral Biol. 15, 993-995. Taubman M. A. and Smith D. J. 1974. Etfects of local immunization with Streptococcus mutans on induction of salivary IgA antibody and experimental dental caries in rats. Infecf. Immun. 9, 1079-1091.
Immuno-electron microscopy of A. naeslundii
Plates l-3 overleaf.
373
P. R. Garant
314
Plate
et al.
1.
Fig. I. Pure culture of A. naeslundii incubated with HRP-conjugated plus H,O,. Note the lack of any cell-wall-associated material. Uranyl x 27,000
goat anti-rabbit IgG and DAB acetate and lead citrate stained.
Fig. 2. Pure culture of A. naeslundii incubated with normal rabbit serum, HRP-conjugated goat antirabbit IgG followed by DAB and HzO,. Slight traces of cell-wall-associated amorphous material can be seen (unlabelled arrows). Uranyl acetate and lead citrate stained. x 19,500 Fig. 3. Pure culture of A. naeslundii incubated only in DAB and H,O,. Note absence of any adherent amorphous material. Uranyl acetate and lead citrate stained. x 26,000 Fig. 4. Pure culture of A. naeslundii incubated with antiserum, HRP-conjugated goat anti-rabbit IgG and DAB plus H,Oz. Note the dense layer of amorphous material surrounding each cell, Uranyl acetate and lead citrate stained. x 26,000 Plate 2. Figs. 5 and 6. Pure culture of A. naeslundii treated with antiserum, HRP-conjugated goat anti-rabbit IgG and DAB plus H202. Clearly defined halos of immuno-reactants (IR) are seen as dense layers approximately 30-50nm adjacent to the cell walls (cw). Note the connecting strands (arrowheads) of the immuno-reactants. Uranyl acetate and lead citrate stained. Fig. 5, x 53,000; Fig. 6, x 96,000 Fig. 7. Mixture of bacteria stained with the full complement of immuno-reactants. Note the clear demarcation of A. naeslundii (A) by the dense band of immuno-reactants. The Acrinomyces are present in small clusters. Lesser numbers of Gram-negative organisms and other Gram-positive organisms are present. Uranyl acetate and lead citrate stained. x 10,000 Figs. 8 and 9. Higher magnification micrographs demonstrating the dense halo of immuno-reactant material around the A. naeslundii cells and the absence of such material around the Gram-negative organisms. Gram-positive organisms (Fig. 9) sometimes had surface associated dense materials (arrows). These materials were usually separated from the cell wall by a narrow clear space and were not electron dense in unstained sections. Uranyl acetate and lead citrate stained. Fig. 8. x 22,500; Fig. 9, x 20,000 Plate
3
Fig. 10. Human plaque stained with the full complement of immuno-reactants. Note large aggregate of Actinomyces (An) cells demarcated by dense halos. Uranyl acetate and lead citrate stained. x 16,000 Fig. 1 I. Human (An) are clearly material toward
plaque stained with the full complement of immuno-reactants. The Actinomyces cells defined by the cell-wall-associated dense layer. Note the radiation of similar dense adjacent cells (arrows). Numerous Gram-negative bacteria are not stained. Uranyl acetate and lead citrate stained. x 24,000
Fig. 12. High magnification of Actinomyces cells depicting the dense band of immuno-reactants mately 3&50nm thick (IR). Note absence of such material on adjacent Gram-negative Uranyl acetate and lead citrate stained. x 64,000
approxiorganisms.
Immuno-electron
microscopy of A. naeslundii
! / I
I
1
Plate 1.
375
316
P. R. Garant et al.
Plate 2.
Immuno-electron
microscopy of A. nacshrndii
Plate 3.
377
IMMUNO-ELECTRON MICROSCOPIC STUDY OF ANTIGENIC SURFACE COMPONENTS OF ACTINOMYCES NAESLUNDII IN HUMAN DENTAL PLAQUE P. R. GARANT*, MOON-IL CHO*, V. IACONO~ and G. J. SHEMAkAt *Department of Oral Biology and Pathology and iDepartment of Periodontics, School of Dental Medicine, Health Sciences Center, State University of New York at Stony Brook, Stony Brook, NY 11794, U.S.A.
Summary-Rabbit antiserum to formalin-killed Actinomyces naeslundii I was used to investigate the ultrastructural location and distribution of the antigenic sites of A. naeslundii. Antigenie sites were identified by an indirect technique using goat anti-rabbit IgG coupled to horseradish peroxidase, also used to identify individual cells of A. naeslundii in a mixed bacterial population and in freshly isolated dental plaque. The bound antibody and associated reaction product were visible in ultrathin sections as an electron-dense amorphous material (lO&lSOnm thick) in juxtaposition to the bacterial cell wall. The location of the immunoreactants suggested that the antigens are distributed superficially and evenly over the entire bacterial cell surface. Bridge-like extensions of the immuno-reactants connected adjacent cells, suggesting that a limited amount of antigen& material might extend out from the cell wall to provide structural continuity with similar material on adjacent cells.
at 37°C in brain-heart infusion medium (BHI-Difco Laboratories, Detroit, Mich.) supplemented with 0.3 per cent glucose. The cells were harvested by centrifugation (16,3OOg, lOmin, 4°C) and washed twice in phosphate-buffered saline (PBS-O.15 M sodium chloride, 0.02 M sodium phosphate, pH 7.5). PBS-washed cells were used for immuno-electron microscopic studies. Some preparations were formalin killed (Taubman and Smith, 1974) and used in immunization procedures. The following organisms were used for detection of A. naeslundii in mixed bacterial suspensions: Staphylococcus aureus and Escherichia coli, both donated by Dr. J. Pollock and grown in BHI, Streptococcus salivarius 4TCC No. 25975, also grown in BHI, Lactobacillus casei ATCC No. 25180-B, grown in Rogosa medium, and Fusobacteria fusiforme ATCC No. 23726, grown in BHI plus 0.5 per cent thioglycollate. Standard suspensions of bacteria (lO’jm1) were inoculated into 10ml of media and grown at 37°C for 18-24 h. Mixtures of the six species of bacteria, including A. naeslundii I, were prepared by combining 1 ml suspensions of washed cells of each species. The combined cells were then pelleted and washed 3 times in PBS prior to forming the final pellet for immunocytochemical procedures. Supragingival plaque samples were obtained from a healthy donor and from one subject with severe gingivitis by gentle scaling with a curette and immediately pre-fixed in a saturated picric acid solution containing 4 per cent paraformaldehyde at 4°C.
INTRODUCTION
One aspect of the current hypothesis of the pathogenesis of periodontal disease is the immunologic response of the host to bacterial (plaque) antigens; it is therefore important to identify the major surface antigens of oral bacteria and to show the presence of such antigens in tissues and cells of the host. Evidence that serum antibodies (Nisengard and Beutner, 1970; Gilmour and Nisengard, 1974) and cellmediated immune mechanisms (Ivanyi and Lehner, 1970; Ivanyi, Wilton and Lehner, 1972; Horton, Leiken and Oppenheim, 1972; Horton, Oppenheim and Mergenhagen, 1974; Mackler et al., 1974) are directed against plaque bacteria indicates that antigen penetration into host tissues probably occurs in periodontal disease. It would be helpful to ascertain the distribution and extent of antigenic localization in gingival and periodontal tissues in order to correlate antigen penetration and binding to the host cells with local tissue destruction. We have used immunocytochemical procedures to localize surface antigens of Actinomyces naeslundii on its cell surface coat and to identify Actinomyces in human dental plaque. These studies were carried out in order to establish the specificity and reliability of the method as preliminary steps in the localization of surface antigens in the oral tissues and cells of rats mono-infected with A. naeslundii. Socransky, Hubersak and Propas (1970) and Garant (1976) showed that rats develop advanced periodontitis when mono-infected with A. naeslundii which has been implicated as an agent in human periodontal disease (Gilmour and Nisengard, 1974; Baker et al., 1976). MATERIALS Preparation
of bacterial
A. naeslundii
AND
Preparation
METHODS
cultures
I, was routinely
and human plaque
grown
of antisera
A male new Zealand White rabbit was injected with 3 ml of vaccine consisting of 10” formalin-killed A. naeslundii I in 1.5 ml PBS emulsified in an equal volume of Complete Freund’s Adjuvant (Difco). The
for 2&24 h 369
370
P. R.
Garant et al.
vaccine was administered into a hind foot-pad and subcutaneously in the dorsum of the neck. Booster injections were administered 2 weeks after the first injection and periodically thereafter to maintain high titres of antibody. The rabbit was bled one week after each booster injection and the sera obtained were heated at 56°C for 31 min to inactivate complement. All sera were tested in immunodiffusion and immunoelectrophoretic analyses for antibody activity (Iacono, Katiyar and Shemaka, 1976a). Sera that reacted similarly were pooled. Normal rabbit serum was obtained prior to any immunization procedures. Bacterial cell, plaque and section preparation Pelleted, PBS-washed A. naeslundii I and mixed cultures were pre-fixed in either 1 per cent glutaraldehyde in 0.1 M phosphate buffer, pH 7.4, for 2 h at room temperature, or in saturated picric acid containing 4 per cent paraformaldehyde for 4 h at 4°C (Lai, Listgarten and Rosan, 1975a). Plaque samples were prefixed in 4 per cent paraformaldehyde at time of collection (see above). The fixed samples were washed 4 times in PBS and stored overnight in PBS at 4°C. Samples selected for polyethylene glycol (PEG) embedding were first dehydrated in a graded series of ethanol (total time 3 h) and then placed in 100 per cent PEG (2 changes, 1 h each) at 39°C in a vacuum oven. The samples were then placed in gelatin capsules containing 100 per cent PEG and hardened at 4°C. Blocks were stored in a desiccator at 4°C until sectioned. Sections (15-30pM thick) of the PEG embedded samples were cut on a portable rotary microtome in a cold room. The sections were floated on 5 per cent glycerol in PBS for 70min and then transferred with a plastic loop to PBS where they were kept for 40min with a change of solution. The sections were then transferred to gelatin-coated slides and air dried before being immersed in staining jars filled with PBS. After 30 min in the PBS, the sections were ready for immunocytochemical studies. Immunocytochemical procedures (A) For sectioned samples: Aliquots (1.5 ml) of anti-A. naeslundii I serum (diluted 1: 1 with PBS) or normal rabbit serum were pipetted on the slides which were kept moistened in closed Petri dishes. The slides were then incubated for 1 h with replacement of fresh antiserum or normal rabbit serum at 20min intervals. The sections were then washed by slow agitation for 1 h in staining jars containing PBS which was changed every 20min. Incubation with horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG (Cappel Labs., Downingtown, PA.) was then performed in moistened Petri dishes for 1 h with two changes of the fresh conjugate. Washing in PBS was again carried out prior to fixation of the samples in 5 per cent glutaraldehyde in 0.1 M phosphate buffer, pH 7.4 at 4°C for 1 h. The fixed sections were washed (3 x 10min) in 0.1 M phosphate buffer (pH 7.4) containing 4.5 per cent sucrose and stored overnight at 4°C in the same solution. The samples were then washed (15 min) in 0.05 M trisHC1 buffer, pH 7.6 and pre-incubated in 3,3-diamino benzidine (DAB, 80 mg) in 100 ml 0.05 M tris-HCl buffer, (pH 7.6) for 1 h, then incubated for
5 min in the DAB solution containing 0.005 per cent HzOz. The samples were rinsed in 0.05 M tris_HCl (15 min), postfixed in 1 per cent 0~0, in 0.1 M veronal acetate buffer for 30 min, and washed in the same buffer for 15 min. The fixed sections were then scraped from the slides, dehydrated in a graded series of ethanol and embedded in Epon. (B) For chopped samples: Washed and pre-fixed pellets of either pure or mixed bacteria and plaque samples were chopped into small pieces, approximately 0.5 mm x 0.1 mm, under a dissecting microscope. All immunocytochemical procedures were the same as for the PEG-sectioned samples with the exception that centrifugation and pipetting were used for all fluid changing and rinsing steps. All steps were carried out in 15 ml centrifuge tubes containing 1 ml of the appropriate solution. After completion of the immunocytochemical processes, post-fixation and dehydration, the final pellets were embedded in Epon directly in the 15 ml centrifuge tubes. Electron microscopy Ultrathin sections were cut on a Sorvall MTB-2 ultramicrotome equipped with a diamond knife. Some sections were doubly stained with uranyl acetate and lead citrate. The sections were examined in a JEOL-100B electron microscope. Table 1 summarizes the various experimental and control specimens which were finally produced and examined ultrastructurally. RESULTS
The immunochemical staining was more intense at the peripheral zone of both the pelleted bacteria and the 15 and 30 PM sections of the PEG-embedded material. This was apparent as a brown HRP-positive margin around the PEG sections or the 1 PM Epon sections of the pelleted samples, when viewed unstained in the light microscope. The central areas of pelleted bacteria, plaque and the thicker PEG sections were usually devoid of any reaction. This was clearly established when viewing unstained ultrathin sections in the electron microscope. A. naeslundii I which was treated with HRP-conjugated goat anti-rabbit IgG (Fig. 1) or with normal rabbit serum (Fig. 2), followed by incubation in DAB and H,02 showed no cell-wall-associated material (Fig. 3). The DAB did, however, increase the electron density of the innermost 8-1Onm region of the cell wall. When the bacteria were reacted with rabbit antisera to A. naes[undii I followed by HRP-conjugated goat anti-rabbit IgG and DAB plus H,Oz an electrondense halo of immuno-reactants was deposited around each cell. This material was visible in unstained as well as stained sections (Fig. 4). The uniform distribution of the immuno-reactants suggested that the antigens were located superficially and evenly distributed over the entire bacterial cell surface. The bound antibody and associated reaction product were visible as an electron-dense amorphous material (3s50nm thick) juxtaposed to the bacterial cell wall (Figs. 5 and 6). Bridge-like extensions of the amorphous immuno-reactants connected adjacent bacterial cells (Fig. 5). The presence of these structures suggests that a limited amount of antigenic material might extend out from the cell wall to provide struc-
Immuno-electron Table
1. Summary
of immunocytochemical
Antisera to A. naeslundii I I. Pure culture Experimental Control (A) Control (B) Control (C) 11. Mixed species Experimental 111. Plaque Experimental Control (a) Control (b) Control (c)
microscopy of A. naeslundii
staining and preparatory human plaque Normal rabbit serum
-
procedures
371 applied
HRP-conjugated goat anti-rabbit IgG
DAB + H202
to cultures
Heavy metal staining
-t -
+ -
+ + + -
+ + + +
+&+&+&+&-
+
-
+
+
+&-
+ -
+
+ + + -
+ + + +
+&f&+8z+&-
-
tural continuity with similar material on adjacent cells. In mixed cultures treated with the full complement of immuno-reactants the cells of A. naeslundii I appeared as small clusters (Fig. 7). Each A. naeslundii I cell was clearly demarcated by the halo of amorphous material (Fig. 8). Other Gram-positive organisms sometimes showed a small amount of loosely bound amorphous material resembling the immuno-reactants (Fig. 9). Actinomyces cells were clearly demarcated in the plaque samples by the circumferential band of adherent immuno-reactants (Figs. 10-12). Similar bands were not present on other Gram-positive and Gramnegative organisms. Actinomyces cells were usually present in small clusters or microcolonies (Fig. 10). Adjacent cells were frequently connected by strands of material which were “stained” by the immunoreactants. Plaque, in contrast with the pelleted pure and mixed cultures, contained greater amounts of interbacterial debris or matrix material (Fig. 11). Such substances were difficult to separate from the immuno-reactants in stained sections but were not electron dense when viewed in unstained ultrathin sections. Prefixation of cells and plaque in either 1 per cent glutaraldehyde or in 4 per cent paraformaldehyde did not appear to have any noticeable effect on the reactivity of the antigens. In both cases, equally dense halos of immuno-reactants were deposited, suggesting that mild glutaraldehyde fixation does not block the antigenic sites. Thorough washing of the glutaraldehyde-fixed tissues was necessary to minimize inactivation of the antigenic sites. DISCUSSION
Similar immunocytochemical techniques have been used to show surface antigens of Streptococcus sanguis (Lai et al., 1975a) and Streptococcus mutans (Iacono et al., 1976b; Grenier et al., 1977). Furthermore, the distribution of Strep. sanguis in plaque was shown at the EM level by Lai et al. (1975b). The PEG method gives reproducible results and is especially useful when correct tissue orientation must be main-
and
tained or when serial sections are to be studied (Lai et al., 1975b). Our study was not concerned with preserving the original orientation of the plaque but only with localizing Actinomyces. Our study and those referred to show that unlabelled rabbit antibody or antiserum together with HRP-conjugated anti-rabbit IgG is a reliable method of identifying specific bacteria in human plaque. Haugen, Helgeland and Grov (1975) used the immuno-peroxidase technique to localize bacterial antigens directly upon ultrathin sections of bacteria embedded in low-viscosity plastic. Our attempts to use those techniques have failed; we could not prevent non-specific staining of the ultrathin sections. The use of the Fab fraction of purified antibody, to A. naeslundii may prove more successful. Direct localization of antigen on ultrathin sections would be useful because sections of special interest from a variety of tissue blocks could be reacted with a minimum amount of immunoglobulin. The Haugen et a/. method was used by Erlandsen, Parsons and Taylor, (1973) to localize lysozyme in human Paneth cells. Kraehenbuhl and Jamieson (1972) in a review of the methods for localizing intracellular antigens by immuno-electron microscopy, reported that albumin embedding techniques can be very useful in the surface localization of antigens on ultrathin sections. The reaction of antiserum directed against A. naeslundii I with surface components of Actinomyces viscosus T14 (Iacono et al., 1976) was expected, as Gerencser and Stark (1976) have suggested that the catalase positive A. oiscosus should be classified as a variety or subspecies of A. naeslundii. Therefore, the cells localized in the plaque samples with the anti-A. naeslundii I serum could be representative strains of one or more of the several species of Actinomyces (Bowden, Hardie and Fillery, 1976). The immunoreactant-staining interconnecting strands of material between adjacent Actinomyces cells in both cultures and plaque samples are similar to fibrils detected in electron micrographs of Actinomyces by Girard and Jacius (1974). Girard and Jacius (1974) suggested that the fibrillar coatings may mediate attachment of Actinomyces to the tooth surface and may account for their haemagglutinating activity (Rolla and Kilian,
P. R. Gal rant et ul.
312
1977). The small clusters we observed of Actinomyces cells in whole plaque samples apparently connected by fibrillar bridges suggests a role for these fibrils in interbacterial aggregation. The nature of the fibrillar antigens has yet to be determined. However, Birdsell and Fischlschweiger (1977) observed fibrils only on virulent strains of A. uiscosus T14. Virulent strains contain 6-deoxytalose (6-DOT)-containing antigen in their cell walls (Hammond Steel and Peindl, 1976). The ultrastructural orientation of this antigen at the cell surface has not been determined. We found that 6-DOT antigens are readily released from stock suspensions of A. oiscosus T14 by incubating with a neutral buffer and, furthermore, Iacono et al. (1977) detected them in culture media. These antigens may occupy a superficial position at the cell surface and, therefore, may serve as a structural component in the surface fibrils. They may also be constituents of the observed interbacterial debris surrounding the Actinomyces cell in plaque (Fig. 11). Localization studies we have in progress with Fab fractions of monospecific antibody to surface components of Actinomyces should elucidate these matters. Acknowledyements~This research was supported by the National Institute of Dental Research, N.I.H., Research Grant Nos. ROl DE 03745 and DE 04168. We thank Ms. Mary Lee and Mr. J. Loffredo for their technical assistance. The help of Mrs. B. Sykes in preparation of the manuscript is greatly appreciated.
REFERENCES
Baker J. J., Chan S. P., Socransky S. S., Oppenheim J. J. and Mergenhagen S. E. 1976. Importance of Actinomyces and certain gram-negative anaerobic organisms in the transformation of lymphocytes from patients with periodontal disease. Infect. Immun. 13, 1363-1368. Birdsell D. C. and Fischlschweiger W. 1977. Comparative ultrastructure of Actinomyces uiscosus. J. dent. Res. 56B, 117. Bowden G. H., Hardie J. M. and Fillery E. D. 1976. Antigens from Actinomyces species and their value in identification. J. dent. Res. 55 (Suppl. A), 192-204. Erlandsen S. L., Parsons J. A. and Taylor T. D. 1973. Ultrastructural immunocytochemical localization of lysozyme in the Paneth cells of man. J. Histochem. Cytothem. 22,401-413. Garant P. R. 1976. An electron microscopic study of the periodontal tissues of germfree rats and rats monoinfected with Actinomyces naeslundii. .I. periodont. Res. (Suppl. 15) l-79. Gerencser M. A. and Stark J. M. 1976. Serological identification of Acrinomyces using fluorescent antibody techniques. J. denr. Res. 55 (Suppl. A), 184-191. Gilmour M. M. and Nisengard R. J. 1974. Interactions between serum titers to filamentous bacteria and their relationship to human periodontal disease. Archs oral Biol. 19, 959-968. Girard A. E. and Jacius B. H. 1974. Ultrastructure of Actinomyces uiscosus and Actinomyces naeslundii. Archs oral Biol. 19, 71-79.
Grenier E. M., Gray R. H., Loesche W. J. and Eveland W. C. 1977. Microcapsules on Streptococcus mutans serotypes by electron microscopy. J. dent. Res. 56, 16C176. Hammond B. F., Steel C. F. and Peindl K. S. 1976. Antigens and surface components associated with virulence of Actinomyces uiscosus. J. dent. Res. 55 (Suppl. A), 19-25. Haugen A., Helgeland S. and Grov A. 1975. Localization of antigens in thin sections of bacteria by the immunoperoxidase technique. Acta path. microbial. stand. (B) 83, 79-90. Horton J. E., Leiken S. and Oppenheim J. J. 1972. Human lymphoproliferative reaction to saliva and dental plaque deposits: an in vitro correlation with periodontal disease. J. Periodont. 43, 522-527. Horton J. E., Oppenheim J. J. and Mergenhagen S. E. 1974. A role for cell-mediated immunity in the pathogenesis of periodontal disease. J. Periodont. 45, 351-360. Iacono V. J., Katiyar V. N. and Shemaka G. J. 1976a. Analysis of antibody directed against surface antigens of Actinomyces species. J. Dent. Res. 55 (Suppl. B), 222. Iacono V. J., Levine M. J., Shemaka G. J. and Katiyar V. N. 1977. Characterization of surface antigens of Actinomyces viscosus T14. J. Dent. Res. 56 (Suppl. A), 120. Iacono V. J., Taubman M. A., Smith D. J., Garant P. R. and Pollock J. J. 1976b. Structure and function of the type specific polysaccharide of Streptococcus mutans 6715. Immunology Abstracts (Special Suppl. Immunological Aspects of Dental Caries) 75-90. Ivanvi L. and Lehner T. 1970. Stimulation of lvmohocvte transformation by bacterial antigens in paiieits with Deriodontal disease. Archs oral Biol. 15. 1089%1096. Ivanyi L., Wilton J. M. A. and Lehner T. 1972. Cellmediated immunity in periodontal disease: cytotoxicity, migration inhibition, and lymphocyte transformation studies. Immunology 22, 141-145. Kraehenbuhl J. P. and Jamieson J. D. 1972. Localization of intracellular antigens by immunoelectron microscopy.. . Int. Rev. exp. Path.-12, 1:53. Lai C. H.. Listearten M. A. and Rosan B. 1975a. Immunoelectron m&oscopic identification and localization of Streptococcus sanguis with peroxidase-labelled antibody: Localization of surface antigens in pure cultures. Infect. Immun. 11, 193-199. Lai C. H., Listgarten M. A. and Rosan B. 1975b. Immunoelectron microscopic identification and localization of Streptococcus sanguis with peroxidase-labelled antibody: Localization, of Streptococcus sanguis in intact dental plaque. Infect. Immun. 11, 200-210. Mackler B. F., Altman L. C., Wahl S., Rosenstreich D. L., Oppenheim J. J. and Mergenhagen S. E. 1974. Blastogenesis and lymphokine synthesis by T and B lymphocytes from patients with periodontal disease. Infect. Imman. 10, 844~850. Nisengard R. J. and Beutner E. H. 1970. Immunologic studies of periodontal disease. V. IgG type antibodies and skin test responses to Actinomyces and mixed flora. J. Periodont. 41, 149-152. Rolla G. and Kilian M. 1977. Haemaaglutination activity of plaque-forming bacteria. Caries I&. 11, 85-89. Socranskv S. S.. Hubersak C. and Prooas D. 1970. Induction oi periodontal destruction in gnotobiotic rats by a human oral strain of Actinomyces naeslundii. Archs oral Biol. 15, 993-995. Taubman M. A. and Smith D. J. 1974. Etfects of local immunization with Streptococcus mutans on induction of salivary IgA antibody and experimental dental caries in rats. Infecf. Immun. 9, 1079-1091.
Immuno-electron microscopy of A. naeslundii
Plates l-3 overleaf.
373
P. R. Garant
314
Plate
et al.
1.
Fig. I. Pure culture of A. naeslundii incubated with HRP-conjugated plus H,O,. Note the lack of any cell-wall-associated material. Uranyl x 27,000
goat anti-rabbit IgG and DAB acetate and lead citrate stained.
Fig. 2. Pure culture of A. naeslundii incubated with normal rabbit serum, HRP-conjugated goat antirabbit IgG followed by DAB and HzO,. Slight traces of cell-wall-associated amorphous material can be seen (unlabelled arrows). Uranyl acetate and lead citrate stained. x 19,500 Fig. 3. Pure culture of A. naeslundii incubated only in DAB and H,O,. Note absence of any adherent amorphous material. Uranyl acetate and lead citrate stained. x 26,000 Fig. 4. Pure culture of A. naeslundii incubated with antiserum, HRP-conjugated goat anti-rabbit IgG and DAB plus H,Oz. Note the dense layer of amorphous material surrounding each cell, Uranyl acetate and lead citrate stained. x 26,000 Plate 2. Figs. 5 and 6. Pure culture of A. naeslundii treated with antiserum, HRP-conjugated goat anti-rabbit IgG and DAB plus H202. Clearly defined halos of immuno-reactants (IR) are seen as dense layers approximately 30-50nm adjacent to the cell walls (cw). Note the connecting strands (arrowheads) of the immuno-reactants. Uranyl acetate and lead citrate stained. Fig. 5, x 53,000; Fig. 6, x 96,000 Fig. 7. Mixture of bacteria stained with the full complement of immuno-reactants. Note the clear demarcation of A. naeslundii (A) by the dense band of immuno-reactants. The Acrinomyces are present in small clusters. Lesser numbers of Gram-negative organisms and other Gram-positive organisms are present. Uranyl acetate and lead citrate stained. x 10,000 Figs. 8 and 9. Higher magnification micrographs demonstrating the dense halo of immuno-reactant material around the A. naeslundii cells and the absence of such material around the Gram-negative organisms. Gram-positive organisms (Fig. 9) sometimes had surface associated dense materials (arrows). These materials were usually separated from the cell wall by a narrow clear space and were not electron dense in unstained sections. Uranyl acetate and lead citrate stained. Fig. 8. x 22,500; Fig. 9, x 20,000 Plate
3
Fig. 10. Human plaque stained with the full complement of immuno-reactants. Note large aggregate of Actinomyces (An) cells demarcated by dense halos. Uranyl acetate and lead citrate stained. x 16,000 Fig. 1 I. Human (An) are clearly material toward
plaque stained with the full complement of immuno-reactants. The Actinomyces cells defined by the cell-wall-associated dense layer. Note the radiation of similar dense adjacent cells (arrows). Numerous Gram-negative bacteria are not stained. Uranyl acetate and lead citrate stained. x 24,000
Fig. 12. High magnification of Actinomyces cells depicting the dense band of immuno-reactants mately 3&50nm thick (IR). Note absence of such material on adjacent Gram-negative Uranyl acetate and lead citrate stained. x 64,000
approxiorganisms.
Immuno-electron
microscopy of A. naeslundii
! / I
I
1
Plate 1.
375
316
P. R. Garant et al.
Plate 2.
Immuno-electron
microscopy of A. nacshrndii
Plate 3.
377
