trchsowl B~ol.Vol. 21, pp. 749 to 753. Pergamon Press1976.Printed in GreatBritam.
IMMUNOPOTENTIATION BY DENTAL MICROBIAL PLAQUE AND ITS RELATIONSHIP TO ORAL DISEASE IN MAN T.
LEHNER, S. J. CHALLACOMEZE,J. M.
A. WILTON
and L.
IVANYI
Department of Oral Immunology and Microbiology, Guy’s Hospital Medical and Dental Schools, London SE1 9RT, England Summary-The relationship between cell-mediated immunity and paa caries experience, as well as experimental gingivitis, was investigated under the immunopotentiating conditions of dental plaque which had been allowed to accumulate for 28 days. The lymphoproliferative responses induced by Streptococcus mutans, plaque, Veillonella alcalescens and Actinomyces viscosus showed negative correlations with the DMF index, but positive correlations with the gingival index. No relationship with the DMF or gingival index was found with the unrelated purified protein derivative, or with the mitogen, phytohaemagglutinin. A second episode of plaque accumulation in the same subjects revealed that the lymphoproliferative response appeared earlier, was greater in magnitude and lasted longer in the second, as compared with the first plaque accumulation experiment. The results suggest that dental bacterial plaque has an immunopotentiating effect on cell-mediated immune responses to oral bacteria and facilitates recall of the immunological memory for bacterial antigens. A unified hypothesis is postulated for dental caries and gingival disease; immunopotentiation by dental microbial plaque has a protective immune effect on dental caries and a damaging, allergic effect on gingival disease.
INTRODUCIION
With the rapid progress towards the development of a vaccine against dental caries, the immune responses to cariogenic bacteria in man need urgent examination. Serum agglutinating (Kennedy et al., 1968), haemagglutinating (Lehner, Wilton and Ward, 1970; Challacombe, Lehner and Guggenheim, 1972) and complement-fixing (Challacombe et al., 1972; Challacombe, 1974) antibodies to Streptococcus mutans have been correlated with the prevalence of caries, using the decayed, missing and filled (DMF) teeth index. A sequential analysis of serum antibodies over a period of 32 months showed that the development of caries was associated with an increase and the removal of caries with a decrease in serum IgG and IgM antibody titres to Strep. mutans (Challacombe and Lehner, 1976). We did not find a relationship between caries experience and cellular immune responses when human peripheral blood lymphocytes were stimulated with Strep. mutans (unpublished). However, significant lymphocyte transformation and macrophage migration inhibition to a sonicate of Strep. mutans were found 7 to 14 days after allowing dental bacterial plaque to accumulate (Lehner et al., 1974a). It seems that an apparent failure to detect cellular responses was overcome by the potentiating effect of dental bacterial plaque on cell-mediated immune responses. These observations and the enhancing effect of dental plaque on the cellular responses to LPS (B cell mitogen) and on the mixed leucocyte culture reaction (T cell response) led to the hypothesis that dental bacterial plaque might act as an endogenous adjuvant, enhancing both T and B lymphocyte responses (Lehner er al., 1974a). 149
Dental bacterial plaque might be considered to have 3 functional components: (a) cariogenic organisms, of which Strep. mutans, Lactohacillus acidophilus and casei, and Actinomyces viscosus are of particular importance (Gibbons and van Houte, 1975), (b) organisms inducing periodontal disease, of which Veillonella alcalescens, Actinomyces viscosus, Bacteroides melaninogenicus and Fusobacterium ,fusiforme may be particularly relevant (Genco et al., 1969), and (c) an adjuvant component, the most potent being lipopolysaccharide, dextrans and levans (Lehner, 1976). As dental bacterial plaque can induce both caries (von der Fehr, Lije and Theilade, 1970) and gingivitis (Lije, Theilade and Jensen. 1965) and as there is a relationship between cell-mediated immunity (CMI) and gingivitis (Ivanyi and Lehner, 1970; Lehner rt al., 1974a), the relationship between CM1 and past caries experience was investigated.
MATERIALS AND METHODS
Subjects The investigation was carried out in 13 healthy subjects, 21-23 years old, of whom 9 were males and 4 females. Bacterial plaque was allowed to accumulate on the teeth for 28 days, by abstaining from any oral cleansing, as described (Lehner et al., 1974a). The DMF index, based on clinical and radiological assessment, the gingival index (Liie and Silness, 1963) and samples of dental bacterial plaque and of venous blood were taken at weekly intervals (Lehner rt ul., 1974a). The plaque accumulation experiment was repeated in 5 subjects, 210 days after plaque was removed and good oral hygiene reinstituted.
750
T. Lehner, S. J. Challacombe, J. M. A. Wilton and L. Ivanyi
Bacteria and antigens V. alcalescens, A. viscosus, B. melaninogenicus and L. acidophilus were grown from stock cultures as described (Ivanyi and Lehner, 1970). Strep. mutans (Ing-
britt) was grown in Todd-Hewitt broth for 48 h. These organisms and the dental plaque collected from each subject separately were disintegrated by ultrasonication and used as antigens in doubling dilutions (Ivanyi and Lehner, 1970). Attempts were also made to grow these organisms from samples of dental plaque from each subject (Lehner et al., 1974a), in order to find out if the presence of these organisms in the plaque might affect the immune response during plaque accumulation. Purified protein derivative (PPD; Weybridge) was used at a concentration of 101.18 per culture and phytohaemagglutinin (PHA; Wellcome Reagents) was used at a dilution of 1:lOO. Lymphocyte transformation
Leucocyte cultures were prepared from Ficoll-Triosil separated cells in triplicate or duplicate, cultured with each antigen in doubling dilutions and also without antigen, in the presence of 15 per cent autologous serum (Ivanyi and Lehner, 1970); the cultures were harvested and prepared for scintillation counting. The results were expressed in terms of the stimulation index (SI), as the ratio of [i4C]-thymidine uptake in antigen-stimulated and control cultures. The SI of lymphocytes to the antigens and mitogens were plotted against each subject’s DMF and GI before plaque accumulation. During plaque accumulation, maximum SI and GI were similarly analysed. In the DMF analysis, only those 10 subjects were utilized who were free from active caries and the index was based on the evidence of past caries experience, i.e. missing and filled teeth. The remaining
3 subjects had active caries and the number was inadequate to consider them in detail. The correlation between the SI of lymphocytes to each antigen and mitogen, and the DMF or the GI was calculated using the Spearman’s rank correlation method. During the second plaque accumulation study, the SI of lymphocytes stimulated by plaque, V. alcalescens and B. melaninogenicus were compared with those from the first study. RESULTS
All but two of the SI before plaque accumulation were negative (less than 2.0) and all the GI were less than 0.4, so that these were not correlated (Figs. 1 and 2). However, with plaque accumulation the SI of lymphocytes induced by all oral organisms increased and showed a negative correlation with the DMF and a positive correlation with the GI (Figs. 1 and 2, Table 1). Significant negative correlations between the SI and the DMF index were found with Strep. mutans (r = -0.824, p < OOOS), plaque (r = -0.739, p < 0.02), V. alcalescens (r = -0.842, p < 0.005) and A. uiscosus (r = -0.648, p < 0.05). In the analysis of SI against GI the positive correlation with V. alcalescens reaohed a significant level (r = f0.657, p < 0.02), whereas the positive correlations with Strep. mutans (r = +0.522), plaque (r = +0.478), L. acidophilus (r = +0.554), A. viscosus (r = +0.467) and B. melaninogenicus (r = +0.522) did
not reach the 5 per cent level of significance. The SI elicited by PPD and PHA showed no significant correlations with the DMF or GI (Table 1). During the second plaque accumulation study, significant lymphocyte transformation was found earlier, it was greater in magnitude and lasted longer in the second as compared with the first study (Fig. 3).
1.4 1.3
r=0.522 pco.05
1.2
r=-0.824 p
IMMUNOPOTENTIATION BY DENTAL MICROBIAL PLAQUE AND ITS RELATIONSHIP TO ORAL DISEASE IN MAN T.
LEHNER, S. J. CHALLACOMEZE,J. M.
A. WILTON
and L.
IVANYI
Department of Oral Immunology and Microbiology, Guy’s Hospital Medical and Dental Schools, London SE1 9RT, England Summary-The relationship between cell-mediated immunity and paa caries experience, as well as experimental gingivitis, was investigated under the immunopotentiating conditions of dental plaque which had been allowed to accumulate for 28 days. The lymphoproliferative responses induced by Streptococcus mutans, plaque, Veillonella alcalescens and Actinomyces viscosus showed negative correlations with the DMF index, but positive correlations with the gingival index. No relationship with the DMF or gingival index was found with the unrelated purified protein derivative, or with the mitogen, phytohaemagglutinin. A second episode of plaque accumulation in the same subjects revealed that the lymphoproliferative response appeared earlier, was greater in magnitude and lasted longer in the second, as compared with the first plaque accumulation experiment. The results suggest that dental bacterial plaque has an immunopotentiating effect on cell-mediated immune responses to oral bacteria and facilitates recall of the immunological memory for bacterial antigens. A unified hypothesis is postulated for dental caries and gingival disease; immunopotentiation by dental microbial plaque has a protective immune effect on dental caries and a damaging, allergic effect on gingival disease.
INTRODUCIION
With the rapid progress towards the development of a vaccine against dental caries, the immune responses to cariogenic bacteria in man need urgent examination. Serum agglutinating (Kennedy et al., 1968), haemagglutinating (Lehner, Wilton and Ward, 1970; Challacombe, Lehner and Guggenheim, 1972) and complement-fixing (Challacombe et al., 1972; Challacombe, 1974) antibodies to Streptococcus mutans have been correlated with the prevalence of caries, using the decayed, missing and filled (DMF) teeth index. A sequential analysis of serum antibodies over a period of 32 months showed that the development of caries was associated with an increase and the removal of caries with a decrease in serum IgG and IgM antibody titres to Strep. mutans (Challacombe and Lehner, 1976). We did not find a relationship between caries experience and cellular immune responses when human peripheral blood lymphocytes were stimulated with Strep. mutans (unpublished). However, significant lymphocyte transformation and macrophage migration inhibition to a sonicate of Strep. mutans were found 7 to 14 days after allowing dental bacterial plaque to accumulate (Lehner et al., 1974a). It seems that an apparent failure to detect cellular responses was overcome by the potentiating effect of dental bacterial plaque on cell-mediated immune responses. These observations and the enhancing effect of dental plaque on the cellular responses to LPS (B cell mitogen) and on the mixed leucocyte culture reaction (T cell response) led to the hypothesis that dental bacterial plaque might act as an endogenous adjuvant, enhancing both T and B lymphocyte responses (Lehner er al., 1974a). 149
Dental bacterial plaque might be considered to have 3 functional components: (a) cariogenic organisms, of which Strep. mutans, Lactohacillus acidophilus and casei, and Actinomyces viscosus are of particular importance (Gibbons and van Houte, 1975), (b) organisms inducing periodontal disease, of which Veillonella alcalescens, Actinomyces viscosus, Bacteroides melaninogenicus and Fusobacterium ,fusiforme may be particularly relevant (Genco et al., 1969), and (c) an adjuvant component, the most potent being lipopolysaccharide, dextrans and levans (Lehner, 1976). As dental bacterial plaque can induce both caries (von der Fehr, Lije and Theilade, 1970) and gingivitis (Lije, Theilade and Jensen. 1965) and as there is a relationship between cell-mediated immunity (CMI) and gingivitis (Ivanyi and Lehner, 1970; Lehner rt al., 1974a), the relationship between CM1 and past caries experience was investigated.
MATERIALS AND METHODS
Subjects The investigation was carried out in 13 healthy subjects, 21-23 years old, of whom 9 were males and 4 females. Bacterial plaque was allowed to accumulate on the teeth for 28 days, by abstaining from any oral cleansing, as described (Lehner et al., 1974a). The DMF index, based on clinical and radiological assessment, the gingival index (Liie and Silness, 1963) and samples of dental bacterial plaque and of venous blood were taken at weekly intervals (Lehner rt ul., 1974a). The plaque accumulation experiment was repeated in 5 subjects, 210 days after plaque was removed and good oral hygiene reinstituted.
750
T. Lehner, S. J. Challacombe, J. M. A. Wilton and L. Ivanyi
Bacteria and antigens V. alcalescens, A. viscosus, B. melaninogenicus and L. acidophilus were grown from stock cultures as described (Ivanyi and Lehner, 1970). Strep. mutans (Ing-
britt) was grown in Todd-Hewitt broth for 48 h. These organisms and the dental plaque collected from each subject separately were disintegrated by ultrasonication and used as antigens in doubling dilutions (Ivanyi and Lehner, 1970). Attempts were also made to grow these organisms from samples of dental plaque from each subject (Lehner et al., 1974a), in order to find out if the presence of these organisms in the plaque might affect the immune response during plaque accumulation. Purified protein derivative (PPD; Weybridge) was used at a concentration of 101.18 per culture and phytohaemagglutinin (PHA; Wellcome Reagents) was used at a dilution of 1:lOO. Lymphocyte transformation
Leucocyte cultures were prepared from Ficoll-Triosil separated cells in triplicate or duplicate, cultured with each antigen in doubling dilutions and also without antigen, in the presence of 15 per cent autologous serum (Ivanyi and Lehner, 1970); the cultures were harvested and prepared for scintillation counting. The results were expressed in terms of the stimulation index (SI), as the ratio of [i4C]-thymidine uptake in antigen-stimulated and control cultures. The SI of lymphocytes to the antigens and mitogens were plotted against each subject’s DMF and GI before plaque accumulation. During plaque accumulation, maximum SI and GI were similarly analysed. In the DMF analysis, only those 10 subjects were utilized who were free from active caries and the index was based on the evidence of past caries experience, i.e. missing and filled teeth. The remaining
3 subjects had active caries and the number was inadequate to consider them in detail. The correlation between the SI of lymphocytes to each antigen and mitogen, and the DMF or the GI was calculated using the Spearman’s rank correlation method. During the second plaque accumulation study, the SI of lymphocytes stimulated by plaque, V. alcalescens and B. melaninogenicus were compared with those from the first study. RESULTS
All but two of the SI before plaque accumulation were negative (less than 2.0) and all the GI were less than 0.4, so that these were not correlated (Figs. 1 and 2). However, with plaque accumulation the SI of lymphocytes induced by all oral organisms increased and showed a negative correlation with the DMF and a positive correlation with the GI (Figs. 1 and 2, Table 1). Significant negative correlations between the SI and the DMF index were found with Strep. mutans (r = -0.824, p < OOOS), plaque (r = -0.739, p < 0.02), V. alcalescens (r = -0.842, p < 0.005) and A. uiscosus (r = -0.648, p < 0.05). In the analysis of SI against GI the positive correlation with V. alcalescens reaohed a significant level (r = f0.657, p < 0.02), whereas the positive correlations with Strep. mutans (r = +0.522), plaque (r = +0.478), L. acidophilus (r = +0.554), A. viscosus (r = +0.467) and B. melaninogenicus (r = +0.522) did
not reach the 5 per cent level of significance. The SI elicited by PPD and PHA showed no significant correlations with the DMF or GI (Table 1). During the second plaque accumulation study, significant lymphocyte transformation was found earlier, it was greater in magnitude and lasted longer in the second as compared with the first study (Fig. 3).
1.4 1.3
r=0.522 pco.05
1.2
r=-0.824 p
