/. Periodotttal Res. 13: 232-239, 1978
Cell mediated immune responses to plaque antigens during experimental gingivitis in man FREDERIC N . SMrm, NIKLAUS P. LANG AND HARALD A. LOE
University of Michigan Dental Research Institute, University of Berne, Switzerland, School of Dental Medicine, University of Connecticut Health Center, School of Dental Medicine The purpose of the present investigation was to evaluate the ability of antigens of human dental plaque microorganisms to stimulate peripheral blood lymphocytes (PBL) from humans during the development and recovery from experimental gingivitis. Eleven healthy dental students (22-36 years) participated in the study. At the start of the experiment, they exhibited clean teeth and clinically healthy gingivae. They then abstained from all oral hygiene measures for four weeks according to the model proposed by Loe et al. (1965). At the start, after 1, 2, 3 and 4 weeks of abolished oral hygiene and 2 weeks following reinstitution of oral hygiene, the oral conditions were assessed, using the criteria of the Plaque Index system (Loe & Silness 1964) and the Gingival Index system (Loe & Silness 1963). At each examination triplicate rnicrocultures, each containing 2 X 10"' viable PBL in .2 ml TC 199 with glutamine and antibiotics containing 1 0 % fetal calf serum, were stimulated with four concentrations of sonicates from V. akalescerts, F. mtcleatttm, B. melattinogenictts, A. viscostis, A. miesltttidii, S. .mnguis and pooled dental plaque all obtained from a previously performed experimental gingivitis. The cultures were incubated for 78 hours at 37°C in 5 % CO,. ''H-thymidine was added for the final 8 hours. Only A. visco.siis was able to stimulate PBL lo undergo blastogenesis during the development of experimental gingivitis. A.viscosus stimulated the PBL of 3 subjects after one week but all subjects after 2 weeks of abolished oral hygiene. The stimulation indices (SI) remained > 2.5 for the rest of the experimental period. Following 2 weeks of reinstituted oral hygiene, the SI reached preexperimental levels. These results suggest that A. viscosus is capable of inducing a specific cellular immune response during the development of experimental gingivitis and may be important as a causative plaque constituent. Further research is indicated to study this association. (Accepted for pttblicatioti October 4, 1977)
The potential influence of periodontal inflammation on systemic factors has received renewed interest with the finding that peripheral blood lymphocytes (PBL) from patients proliferate upon challenge with dental plaque antigens. Stimulation of PBL's by
certain bacterial antigens has been correi
lated wtth the degree of existing periodontal disease (Ivanyi & Lehner 1970, Horton, Lciken & Oppenheim 1972). However^ oilier investigations present conflicting findings (Kiger, Wright & Creamer 1974, Lang & Smith 1977, Smith & Lang 1977). Nevertheless, there is evidence which suggests
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that iti vilfo extracts of laboratory grown bacteria or pooled plaque are capable of stimulating both itt vitfo blastogenesis and lymphokine production of PBL from patients with periodontal inflammation. However, a direct correlation between the severity of periodontal disease and the magnitude of in vitro lymphocyte aclivation is controversial. Longitudinal studies in which patients with periodontal disease are followed over a definite period of time are currently needed to more clearly define the association of lymphocyte blastogenesis with the otiset and progression ol: this widespread disease. The experimental gingivitis studies (Loe, Theilade & Jensen 1965) are potential models with which to examine the early pathogenesis of periodontal inflammation. The pui"pose of this study was to evaluate the ability of certain commonly encountered human plaque antigens to transform PBL's in humans during the onset and recovery from experimental gingivitis. Material and Methods
Three female and eight male dental students aged 22 to 36 years participated in this study. Each received a thorough prophylaxis and oral hygietie instructioti before the beginning of the experiment. Clean teeth and healthy gingivae were required of each student at the start of the research period. The students then abstained from all oral hygiene measures for four weeks, allowing dental plaque to accumulate and gingivitis to develop (Loe et al. 1965). Thorough oral hygiene measures were reinstituted after the fourth week. At the beginning of the study (week zero), after 1, 2, 3 and 4 weeks of no oral hygiene, and two weeks after reinstitution of oral hygiene (week six), plaque was assessed according to the Plaque Index system (PI I) (Silness & Loe 1964) while gingival health was evaluated by the Gingival Index system (Gl)
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(Loe & Silness 1963). At each scoring period, 35 ml of peripheral blood was drawn in a heparinized syringe from each subject and sedimented lor 90 min. The leukocyte-rich plasma was then removed, centrifuged and washed three times in Hanks balanced salt solution (Difco). The number of viable cells was determined by the trypan blue dye exclusion test. Either PHA-P (Difco) in concentrations of 0.5, 2.5 and 5.0 ng/ml or four concentrations of plaque bacterial antigen extracts (see below) were added to triplicate microcultures each containing 2 X lO"' PBL in 0.2 ml of tissue culture medium 199 (GIBCO) stipplemented with 10 % fetal calf serum (GIBCO) (Lang & Smith 1977). The cultures were then incubated for 78 hours at 37°C in a humid 95 % air and 5 % CO2 environment. For the final 8 hours 0.1 [^ic [methyl--''H] thymidine (specific activity 6.7 Ci/mmol; New England Nuclear Corp.) was added to each culture to measure DNA synthesis. The cells were harvested, precipitated on glass fiber filters with 10 % trichloroacetic acid, washed, dried and counted by liquid scintillation. The CPM were recorded and corrected for chemical quenching. Stimulation indexes (SI) were calculated by dividing the mean disintegrations per minute of the stimulated cultures by the mean disintegrations per minute of unstimulated saline controls. For each subject at each examination interval peak SI NO ORAL
Fig. 1. Mean Plaque Indices (PI I) and mean Gingival Indices (GI) during onset (week 0-4) and recovery (week 6) from experimental gingivitis.
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SI 400. 300200.
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iMeon I S.E.
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p < 0.05
Fig. 2. Peak stimulation indices (SI) for ali subjects and mean peak Si for the group foilowing stimulation of PBL with PiHA-P during onset and recovery from experimentai gingivitis.
of the four concentrations of each antigen and PHA-P were obtained. Only SI values greater than 2.5 were considered significantly elevated (Lang & Smith 1977). Mean peak SI vaiues were then obtained for each week. Weekly differences were compared by paired t-tests. Bacterial antigens were obtained by extracting cultures of plaque bacteria isolated during a previous experimental gingivitis study in humans. These organisms, Veillonella alcalescens, Fusobacterlum nucleatutn, Bacteroides melanitiogenicus, Actinotnyces viscosus, Actinomyces naeslundii attd Streptococcus sanguis, were chosen since they represented the predominant cultivatable flora after a period of three weeks with no oral hygiene (Loesche & Syed 1975). The organisms were serially diluted, isolated and recultured. Gram stains of the cultures were examinated in order to assure that there was no contaiiiination. The pure cultures and pooled homologous plaque were prepared as antigenic sonicates according to the method deseribed by Lang & Smith (1977). The antigen preparations were diluted in PBS at four concentrations and checked for sterility by anaerobic and aerobic culturing on sheep blood agar for 72 hours at 37 °C. The saline controls demonstrated background CPM averaging 48 (range 29-68).
Fig. 3. Peak stimulation indices (SI) for ali subjects and mean peak Si for the group following st imulation of PBL with A. viscosus during onset and recovery from experimentai gingivitis.
During the four weeks of plaque accumulation (no oral hygiene), all subjeets showed the development of gingival inflammation (Fig. 1). By the end of four weeks with no oral hygiene the average Plaque Index (PI I) score was 2.2 (S.D. 0.16) while the average Gingival Index (GI) score was 1.45 (S.D. 0.19). Two weeks after reinstitution of oral hygiene the PI I and GI returned to preexperimental levels of 0.14 (S.D. 0.13) and 0.18 (S.D. 0.14), respectively (Fig. 1). Table 1
Stimulation Indices of all subjects after stimulation of PBL by A. viscosus. Paired t-tests indicate changes from week to week (0 to 1, 1 to 2, etc.) Week 31 32
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41 42 43 44 51 52 53 54 Mean S.D. t-test
0 1.75 1.86 1.76 1.55 1.62 1.49 1.56 1.02 1.63 1.43 1.88
1 3.49 3.10 1.05 3.83 1.85 1.53 2.20 1.51 1.27 1.02 0.86
2 4.69 2.62 2.87 3.57 3.05 2.86 4.97 3.06 5.17 4.34 4.47
2.91 3.47 3.12 3.20 4.24 3.73 3.61 3.00 4.16 3.83 3.50
3.40 3.32 3.32 3.73 3.12 2.76 2.91 4.15 4.18 3.20 3.84
1.60 1.32 1.24 0.98 1.09 1.29 1.80 1.55 1.31 1.49 1.72
1.59 1.97 3.78 3.52 3.35 1.39 0.24 1.04 0.95 0.44 0.50 0.25 1.21 4 .12* 0.89 0.80 13.31*
* statistically significant (p < 0.01)
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p < 0.05
I Mton £E.
Poolad P aquf JMean S.E. p 2.5. The increase in mean peak SI between
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Mfon S.E. P 2.5 occurred, the mean peak SI for all other antigens did not show a statistically significant increase at any time during the experimental period (Fig. 4). No individuals displayed elevated Si's when the plaque load was low (Weeks zero and six). No elevated individual SI were seen after stimulation with S. satiguis (Fig. 4a). Only two slightly elevated SI were seen after B. tnelatiinogenictts stimulation (2.5 < S I < 2.9) (Fig. 4b), while three very slightly increased SI (2.5 < SI < 2.6) were seen after stimulation with pooled plaque (Fig. 4c). There were sporadically elevated SI after stimulation with F. tiucleatutn (7) (Fig. 4d) and V. alcalesceti.s (6) (Fig. 4e). Nine individual weekly SI were elevated after A. tiaeslutidii stimulation
AN = A. naeslundll S = S. sanguis P = three week old pooled plaque
(Fig. 41). No distinct individual subject pattern for these sporadic elevations in SI could be determined. However, they clustered iti the second to fourth week when plaque load was heaviest. Figure 5 summarizes the results of the mean peak SI for all antigens tested. Discussion
During the four week period of plaque accumulation gingival inflammation developed in all subjects. This is consistent with the original (Loe et al. 1965) and numerous other reports utilizing the human experimental gingivitis procedure. This model provides the opportunity to study the effects on the systemic cellular immune response of veiy early inflammatory gingival changes brought about solely by the accumulation of dental plaque (Loe 1971). Lehner et al. (1976) found that a second four week experimental gingivitis resulted in an elevated lymphocyte SI. This occurred
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earlier, was of a greater magnitude and lasted longer than the SI seen in the same volunteers during the first four weeks of experimental gingivitis. However, it can never be known how many previous episodes of gingival inflammation this or any other population has experienced, and the differences between primary and anamnestic responses cannot be resolved. Tiie significance of the randomly elevated SI seen in the present study (for antigens other than A. viscostt.'i) cannot be determined. With the exception of elevated SI to A. naeslundii in two of the subjects, the elevated SI rarely occurred in the same individual and rarely lasted more than one week. Since all SI returned to pre-experimental levels after two weeks of reinstituted oral hygiene (week six), the early signs of disease as represented in this model seem to be entirely reversible as assessed by this immunological test. Thus, the clinically observed reversibility of gingivitis appears to be paralleled by reversibility of a systemic immunological parameter. The distinct elevation in PBL response after stimulation with A. viscosus correlates with observations on the association between Actittomyces species and developing periodontal disease (Loesche 1975, Williams, Pantalone & Sheiris 1976). During the first week of experimental gingivitis, the number of viable isolates of Actitiotnyces in the plaque increased by a factor of 65 (Loesche 1975). This corresponded to an increase of from 12 % to 32 % of the cultivable bacteria. By the third week Actinomyces comprised 51 % of the isolated flora. The largest increase in Actittotnyces occurred with A. israelii, a species not tested in this study. However, an increase in relative amounts of A. viscosus correlated more closely with the increase in clinical gingivitis scores than did A. israelii (Loesche & Syed 1975). Relative amounts of A. ttaesluttdit also increased in the developing den-
tal plaque but could not be clinically related to gingivitis scores (Loesche & Syed 1975, Syed, Loesche & Loe 1975). It is apparent from the present situation that an increase in Actittotuyces species in dental plaque stimulates the immune mechanisms and that A. viscosus stimulates sensitized lymphocytes to a greater degree than the closely related A. naeslttttdii. This could indicate a difference in pathogenicity. Further work is needed to define the role of this species and to test A. israelii in particular. Cross-sectional studies in populations with varying degrees of periodontal inflammation have shown associations between several microorganisms and the severity of this disease. Ivanyi and Lehner (1970, 1971) reported that A. viscosus and Veillottella stimulated PBLs as did dental plaque extract. B. melatiinogetiicus seemed to stimulate less and F. fusifortue stimulated PBLs weakly. In an experimental gingivitis study, several other antigens, including Lactobacillus acidophihts, Proteus mirabilis and PPD as well as A. viscostts, Veillottella, Bacteroides attd Fusobacterium stimulated PBL (Lehner et al. 1974). A biphasic response with peaks occurring in weeks two and four with a drop in SI at about week three was suggested. The fact that such a biphasic pattern was not found in the current study, may have been due to differences in methodology preventing valid comparison. The transformation of PBL by Actinotnyces in patients with periodontal disease has been reported in several investigations (Horton et al. 1972, Baker et al. 1976, Lehner et al. 1974). A. ttaeslutulii and, to a greater extent, A. viscosus can cause PBL blastogenesis in subjects with periodontal disease, ranging from gingivitis to advanced periodontitis (Lang & Smith 1977). The response to A. viscostts seemed to correlate more closely to the Gingival Index and Plaque Index of an individual than to
pocket depth or loss of periodontal attachment (Smith & Lang 1977). The findings in the present study conlirm this relationship between A. viscosus and the gingival inflammatory response. The 78 hour incubation period used in this study lor cuituring PBLs was similar to that used in other studies (Lang & Smith J976, 1977, Ivanyi & Lehner 1970, 1971). This short incubation period most likely delines a peak in mitogenic activity from substances such as PHA, ConA and lipopolysaccharidos (LPS) from gram negative bacteria (Ling & Kay 1975). Purely antigenic PBL stimulation usually peaks in from 120 to 168 hours (Baker et al. 1976, Patters et al. 1976). The mitogenic potential of the bacterial extracts used in this study are unknown. However, the gram-negative organisms known to contain LPS, i. e. B. melaninogenicus, V. alccilescens and F. luicleattttn, did not yield significant mean peak SI at any time during the study. Although little is known about the mitogenic effects of Actinotnyces in humans, a potent mouse spleen B-cell mitogen has been described (Engel et al. 1977). Also in the germfree rat model, fractions of A. viscostts have been demonstrated to possess marked mitogenicity for spleen cells (Burckhardt & Guggenheim 1977). Since all subjects were repeatedly unresponsive to all antigens before or after experimental gingivitis the elevation in SI due to A. visco.stts most likely represents an antigenic response rather than a mitogenic. On the other hand, seven day cultures of human PBL have demonstrated Actinotnyces to be potent stimulators of blastogenesis (Baker et at. 1976). It has been reported that the PBL response to PHA increases during experimental gingivitis and that this occurs at lower PHA concentrations as well (Lang & Smith 1976, Gaumer, Holm-Pedersen & Folke 1976). The same phenomenon was seen in
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the present study. Increasing sensitivity to lower doses of an antigen after primary immunization has also been reported (Andersen, S0borg & S0rensen 1971). The elevated SI found at the four concentrations of A. viscostts used to stimulate PBL's in this study more closely resembles an antigenic response than the sharper peak more characteristic of mitogens (Moller 1970). No effect of bacterial lipopolysaccharide could be observed in this study even though the blastogenic response to LPS has been shown to increase during plaque accumulations (Gaumer et al. 1976, Lehner et al, 1973). None of the LPS containing bacterial extracts in the current study showed any significant changes in mean peak SI during the 28 days of experimental gingivitis. The sensitization of patients duritig plaque accumulations and the sensitivity of the gingival tissues to changes in the dental plaque are striking phenomenons. Although S. sanguis always constitutes a large percentage of plaque (Loesche 1975), the immune system does not seem to become sensitized to the antigenic preparation of this species (Lang & Smith 1977). Since only short incubation periods (78 hours) have been used in the present study, the assay system might not have been sensitive enough to detect blastogenesis with weaker antigens. Obviously, A. viscosus represents a very potent antigen (Baker et al. 1976). The uniqueness of Actinoinya's species in their ability to induce immunological sensitization of PBL during gingivitis development probably links them in some way to the pathogenesis of this disease. Further studies are underway to determine the mechanism of this sensitization. References
Andersen, V., S0borg, M. & S0rensen, S. F., 1971. Antigen induced lymphocyte transfor-
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mation //; vino during primary immunisation in man. I. Development and course. Acta Pathol. Microbiol. Scand. 79B: 489-494. Baker, J. J., Chan, S. P., Socransky, S. S., Oppenheim, J. H. & Mergenhagen, S. E., 1976. Importance of Actinomyces and certain gram-negative anaerobic organisms in the tran.storniation of lymphocytes from patients with periodontal disease. Infect, and Immunity J3: 1363-1368. Biirckhardt, J. J. & Guggenheim, B., 1977. Mitogenicity of fractions of Actinomyces viscosus. lADR Abstract No. 118, 55th General Session, Copenhagen. ./. Denl. Res. 56: Special issue A. 72. Engel, D., Clagett, J. Page, R. & Williams, B. 1977. Milogenic activity of Actinomyces viscosus. I. Effects on murine B- and T-Iymphocytes, and partial characterization. ./. Immunol. 118.- 1466-1471. Horton, J. E., Leiken, S. & Oppenheim, J. .1. 1972. Human proliferative reaction to saliva and dental plaque deposits: An in vitro correlation with periodontal disease. J. Pcriodontol. 43: 522-527. Ivanyi, L. & Lehner, T. 1970. Stimulalion of lymphocyte transformation by bacterial antigens in patients with periodontal disease. Arch. Oral Biol. 15: 1089-1096. Ivanyi, L. & Lehner, T. 1971. Lymphocyte transformation by sonicates of dental plaque in human periodontal disease. Arch. Oral Biol. 16: 1117-1121. Kiger, R, D., Wright, W. H. & Creamer, C. R., 1974. The significance of lymphocyte transformation responses to various miciobial stimulants. /. Periodontol. 45: 780-785. Lang, N. P. & Smith, E. N. 1976. Lymphocyte response to T-cell mitogen during experimental gingivitis in humans. Inject, and Immunity 13: 108-113. Lang, N. P. & Smith, F. N. 1977. Lymphocyte blastogenesis to plaque antigens in human periodontal disease. L Populations of vatying severity of disease. .'. Periodontal Res. 12: 298-309. Lehner, T., Challacombe, S. J., Ivanyi, L. & Wilton, J. M. A. 1973. The relationship between serum and salivary antibodies and cell mediated immunity in oral disease in man. Adv. Ex p. Med. Biol. 45: 485-496. Lehner, T,, Challacombe, S. J., Wilton, J. M. A. & Ivanyi, L. 1976, Immunopotentiation by dental microbial plaque and its relationship to oral disease in man. Arch. Oral Biol. 21: 749-753.
Lehner, T., Wilton, J. M. A., Challacombe, S. .(. & Ivanyi, L. 1974. Sequential cellmediated immune response in experimental gingivitis in man. CUn. E.\p. Imiiumol. 16: 481-492. Lehner, T., Wilton, J. M. A., Ivanyi, L. & Manson, .1. D. 1974. Immunological aspects of juvenile periodontitis (periodontosis). ./. Periodontal Res. 9: 261-272. Ling, N. R. & Kay, J. E. 1975. Lymphocyte stimulation. 2nd ed. P. 360-362. Amsterdam: North-Holland. Loe, H. 1971. Human research model for the production and prevention of gingivitis. ./. Dent. Ri's. 50: 256-264. Loe, H. & Silness, J. 1963. Periodontal disease in pregnancy. I. Prevalence and severity. Acta Odontol. Scand. 21: 533-551. Loe, H., Theiiade, E. & Jensen, S. B. 1965. E.xperimental gingivitis in man. ./. Periodontol. 36: 177-187. Loesche, W. .1. 1975. Bacterial sucsession in dental plaque: Role in dental disease. Microbiol. 2: 132-136. Loesche, W. J. & Syed, S. A. 1975. Bacteriology of dental plaque in experimental gingivitis. I. Relationship between gingivitis and flora. lADR Abst. No. 108. ./. Denl. Res. 54: Special Issue A. 71. Moller, G. 1970. Induction of DNA synthesis in human lymphocytes: Interaction between non-specific mitogens and antigens. Inmtttuology 19: 583-598. Silness, J. & Loe, H. 1964. Periodontal disease in pregnancy. II. Correlation between oral hygiene and periodontal condition. Ada Odontol. Scand. 22: 121-135. Smith, E. N. & Lang, N. P. 1977. Lymphocyte blaslogenesis to plaque antigens in human periodontal disease. 11. The relationship to clinical parameters. ./. Periodontal Res. 12: 310-317. Syed, S, A., Loesche, W. J. & Loe, H. 1975. Bacteriology of denial plaque in experimental gingivitis. 11, Relationship between time, plaque score and flora. lADR Abst. No. 109. ./. Dent. Res. 54: Special Issue A. 72. Williams, B. L., Pantalone, R. W. & Sherris, .1. C, 1976. Subgingival microflora and periodontitis. y. Periodontal Res. 11: 1-18. Address: The University of Michigan School of Dentistry Dept. of Periodontics Ann Arbor, Michigan 4HI09