ANTIBODIES TO STREPTOCOCCUS MUTANS IMMUNOGLOBULIN LEVELS IN CHILDREN DENTAL CARIES
AND WITH
T. LEHNER,’ J. J. MURRAY,’ G. B. WINTER’ and JILL CALDWELL’ ‘Department
of Oral
‘Department
Immunology
of Children’s
and Microbiology. Guy’s Hospital London SEl. and Dentistry. Eastman Dental Hospital.
Medical
and Dental
Schools.
London
WCL. England
Summary-An immunological investigation of rampant and non-rampant caries was carried out in children aged 2.5-5.5 yr. Serum IgG, IgA and IgM classes of antibodies to Streptococcus mutuns were detected and significant negative correlations were found between the DMFS and the IgG:IgA or 1gG:IgM ratios. Immunological response against dental caries appears to be associated with the proportion of IgG to IgA and IgM classes of antibodies to Strep. muruns. Serum IgM. unlike IgG or IgA, concentrations showed a significant positive correlation with the DMFS and IgM concentration may be a measure of antigenic load in the child. The study emphasizes the importance of antibody class and age of evaluation of the immune responses in the mechanism of protection against dental caries.
SUBJECTS AND METHODS
INTRODUCTION Subjects
Twenty-seven
Streptococcus mutuns
is probably the most cariogenic organism and requires the tooth surface for large-scale colonization (Carlsson. 1967). Hence, the earliest time of colonization of Srrep. mutans is about 6 months, in contrast to Luctobucillus,
microorganisms in the mouth (Lehner, 1977). An into the comparative maternalfetal immune responses to Strep. mutuns (Ivanyi and Lehner, 1978) revealed maternal IgG but not IgM class of antibodies to Strep. mutuns in neonatal blood. Furthermore, of neonatal lymphocytes to Strep. mutans occurs in utero (Ivanyi and Lehner, 197X). Systemic humoral and cellular immune components enter the mouth through the gingival crevice (Attstram and Egelberg, 1970; Skapski and Lehner. 1976; Wilton, Renggli and Lehner, 1976, 1977). There may, therefore, be a critical timing between the eruption of deciduous teeth and the clearance of maternal antibodies (about 5 months after birth), as well as the duration of of lymphocytes which may influence the colonization of plaque bacteria. We have compared serum and salivary antibody titres and immunoglobulin concentrations in young children with rampant caries, with those having nonrampant caries of deciduous teeth.
children,
2.5-5.5 yr, were 1 consisted of 15 children with rampant caries, as defined by Winter, Hamilton and James (1966) and group 2 of 12 children with one or more untreated carious lesions (Table 1). Up to 10ml of blood was collected from each child under a general anaesthetic required for dental extraction. A specimen of whole saliva was collected from 24 children by aspiration from the floor of the mouth through a sterile plastic saliva ejector into a sterile 20-ml stoppered bottle. Dental plaque was collected with a sterile probe from the approximal surfaces of the deciduous molars and cervical aspects of the lower incisors. divided
Assay
into two groups.
aged
Group
qf immunoylobulins
Serum IgG, IgA and IgM and salivary IgA concentrations were estimated by the radial immunodiffusion method (Mancini, Carbonara and Heremans, 1965). The antisera used (Wellcome reagents) were checked by immunoelectrophoresis for specificity for y, t( and p chains. The corresponding immunoglobulin standards used were supplied by the Medical Research Council.
1061
Prepuration Strep.
of Strep. mutans for
untibody
ussuy
mutws
(serotype c) was grown in ToddHewitt broth for 18 h. The cells were washed 3 times with saline and resuspended to about 10’ organisms/ml. Immunojluorescent antibodies
ussuy for serum
IgG,
IgM
and lyA
Class-specific antibodies to Strep. mutans were assayed by the immunofluorescent method (Lehner,
15 12
Rampant Non-rampant t value I’
* Months. NS. not significant.
No.
Caries
47 (f3.4) 49.3 (13.4)
Mean age* (&SE)
Table
10 6
5 6
Sex M F 11.5 (kO.6) 5.1 (kO.6) 7.139 < 0.0001
DMF
in relation
DMFS
NS
1.899
9 4
Positive culture of Strep. tnutans
to Strup. rnutan.s and salivary
25.2 (k3.1) 8.3 (& 1.5) 4.512 < 0.001
Mean (*SE)
1. Age. sex and DMFS
13 11
No.
indices
6.3 (t0.7) 5.4 (+ 0.5) 1.074 NS
1.8 (iO.2) 1.9 (kO.2) 0.308 NS
Saliva (mean + SE) Agglutinating kA cont. antibody
Antibody
and immunoglobulin
1966). Doubling dilutions of sera prepared with saline (1:5, 1 : 10, etc.) were added to air-dried smears of cells of Strep. mutcms for 30 min. These were washed 3 times with phosphate-buffered saline (PBS; pH 7.2) and overlaid with anti-human IgG (F:P 3.1), IgM (F:P 4.1) or IgA (F:P 3.8) fluorescein-isothiocyanate conjugates (Wellcome reagents). The reagents were checked for class specificity by immunoelectrophoresis and double-diffusion precipitation tests. The specificity of the test was established by complete absorption of the antibody titre with the homologous and little or no absorption with heterologous organisms. The titres were expressed in doubling dilutions: log2 (I = 1:5). Sulitw),
agglutinating
unribodg
test
This was performed with serial dilutions of saliva (1:2. 1:4, etc.) with saline in microplates, using fresh, washed cells of the Strep. mutans (5~1 of lo9 colonyforming units per ml). The plates were read after overnight incubation at 4°C. Culture
of pluque ,fiw Strep.
mutans
The samples were dispersed by vigorous shaking with sterile, 3-mm dia glass beads in a vortex mixer (Fisons Ltd., Loughborough, Leics.) for 60s and cul-
levels in dental
caries
1063
tured on TYC medium as described by Caldwell, Challacombe and Lehner (1977). Colonies that produced extracellular polysaccharide (EPS) were differentiated according to their colonial morphology. Representative colonies were subcultured for positive identification. Strains that fermented mannitol and sorbitol were designated as Strep. mutans.
RESULTS
Age und decayed, missing und ,filled in rampant and non-rumpunt curies
surfuces
There was no difference in age (Table 1) between the group with rampant caries (47 i 3.4 months) and non-rampant caries (49.3 f. 3.4 months). However, a very significant difference in DMFS (p < 0.001) was present in children with rampant caries (25.2 + 3.1) as compared with non-rampant caries (8.3 k 1.5). Serum
immunoglobulin
concentrurions
A significant positive correlation (Fig. 1) was found between the DMFS and the IgM (r = 0.502; p < 0.01) but not IgA (r = 0.231; p > 0.05) or IgG concentrations (r = 0.348: p > 0.05; Table 2). Significant correlations were found between age and IgG
3000 2800 2400
.
.
2000
. .
.
.
IgG 1600 .
.
9.’
. ..
300
.
’
.
250 200
I@
150 100 50 0
’
300 l
250
.
.
.
IgM
0-m 0
5
10 15
20 25
30 45
60
DMFS *Rampant Fig. 1. lmmunoglobulin
I
I
0
10
I
I
I
I
1
117
20 30 40 50 60 70 100 Age in months
0 Non-rampant
concentrations
(DMFS)
(mg/lOOml)
in relation
to the DMFS
and age
1064
T. Lehner.
J. J. Murray,
G. B. Winter
and J. Caldwell
Antibody and immunoglobulin (r = 0.514; p < 0.01) and
but not IgM concentrations Serum
antibody
IgA (r = 0.536; p < 0.01) (Table 2).
titres
No significant difference in IgG, IgA or IgM antibody titres between sera from children with rampant or non-rampant caries was found (Table 2). No significant relationship was observed between the DMFS and IgG, IgA or IgM classes of antibodies. However, significantly decreased ratios of 1gG:IgA (t = 2.533; p < 0.02) or 1gG:IgM (t = 2.577; p < 0.02) were found in rampant as compared with non-rampant caries. Further analysis showed significant negative correlations between the DMFS and 1gG:IgA (r = 0.528; p < 0.01). 1gG:IgM (r = 0.4921 ; p < 0.01) and IgG:(IgA and IgM) (r = 0.484; p < 0.01). The age of the children was not related to the antibody ratios (Table 2), but showed a significant correlation with IgM antibodies to Strep. mutans (r = 0.518: p < 0.01). Sulicary
1gA concentration
and antibodies
No
difference was found between rampant and non-rampant caries in the salivary IgA concentration or agglutinating antibodies (Table 1). No correlation was found between salivary IgA concentration or agglutinating antibody and the DMFS. However, as whole saliva was used, some antibodies may have been lost by binding to Strep. mutans in the saliva. Culture
1065
levels in dental caries
entiate the two types of caries as a combined analysis of the two groups in terms of DMFS against the IgG:IgA, or 1gG:IgM ratios revealed significant negative correlations (p < 0.01; Fig. 2). These results are therefore consistent with the view that children with rampant caries are not immunologically compromised, but that the dietary conditions, such as are created by prolonged use of sweetened feeding bottles or sweetened comforters, are responsible for rampant caries (Winter et u/., 1971). Our investigation was based on the concept evolved from immunization of monkeys. that immunological protection against caries is dependent on the class of antibodies: a high ratio of IgG to IgA. and of IgG to IgM antibodies is found in macaque monkeys with a low caries incidence (Lehner et rrl., 1978). This concept also appears to apply to a population of young children (2.5-5.5 yr old) who also show the three classes of serum antibodies to Strep. mutans, and emphasizes the differences in the function of antibodies of the IgG, IgA and IgM classes. A significant correlation was not detectable with any of the three classes of fluorescent antibodies and the DMFS. but significant negative correlations were found between the DMFS and the ratios of IgG:IgA, 1gG:IgM and IgG:(IgA and IgM). These results argue in favour of a protective effect of IgG antibodies. if their titre and probably affinity
of Strep. mutans
Strep. mutans was cultured in 9 out of 15 samples of plaque from children with rampant caries and in 4 out of 12 children with non-rampant caries (Table 1). This difference was not significant (x2 = 1.894). Furthermore, the number of colony-forming units in the two groups was not significantly different.
4l 3 IgG-IgA
o
DO
2
0.
1
000
Log2
r 0
. . 0.
.
.0*
. .*
’
DISCUSSION A clinical differentiation of caries of deciduous teeth into rampant and non-rampant types (Winter et ul., 1971) showed a significant difference in the DMFS but not in the age of the two groups (Table 1). In our study. salivary IgA concentration or agglutinating antibodies were the same in the two groups investigated. However, non-immunoglobulin agglutinating factors have been described in saliva (Williams and Gibbons, 1975: Ericson, Pruitt and Wedel, 1975) so that these may have affected the results. Of the serum immunoglobulin concentrations, only IgM was significantly increased in rampant caries but, as there was a significant positive correlation between the DMFS and IgM concentration (p < O.Ol), irrespective of age, the difference in IgM might be due to the number of carious lesions and not to the type of caries. Immunofluorescent antibodies to Strep. mutans of the IgG, IgA and IgM classes were not significantly different, though decreased titres of IgG and increased titres of IgA and IgM antibodies were observed in rampant as compared with non-rampant caries (Table 2). This suggested that the ratios of IgG to IgA and IgG to IgM might be decreased in rampant caries, and indeed this was found (p < 0.02). However, these antibody ratios are unlikely to differ-
.
DMFS l
Rampant
o Non-rampant
Fig. 2. Relationship between DMFS and the ratios 01”IgG to IgA. IgM and IgA + IgM antibodies to .‘rrc,/~.IHL~~~I~I.L
1066
T. Lehner. J. J. Murray, G. B. Winter and J. Caldwell
are greater than those of IgA and IgM antibodies. The antibody ratios and titres in our study were independent of the age of the children, except for the IgM antibodies, unlike the summated agglutinating antibody titres to cell walls previously shown to four strains of Strep. mutuns and Strep. sunguis (Berkenbilt and Bahn, 1971). The method used by these workers, however, did not differentiate between the three antibody classes: it may have been biased in favour of the IgM class and may have assayed an increase in antibody load to a variety of oral streptococci associated with the age of the child. A comparison by Kennedy er al. (1968) in naval recruits, aged 17-19 yr, of serum-agglutinating antibodies to cell walls from caries-free subjects with those from rampant caries showed a higher titre in the caries-free subjects. However, haemagglutinating antibodies to sonicates of Strep. mutans showed that adults with high caries indices had a higher antibody titre than those with low caries indices (Lehner, Wilton and Ward, 1970). Further division of caries into a group with carious lesions and another without carious lesions, but some with past caries experience, has revealed a significant negative correlation between the DMF and IgG or IgM antibodies to cell walls or culture supernatant of Strep,. mutuns in the group free of carious lesions, but a positive correlation in the group with carious lesions (Challacombe, Lehner and Guggenheim, 1972: Challacombe. 1974: Challacombe and Lehner, 1976). In our present study, serum IgG and IgA concentrations showed an increase with DMFS and age of the children, though only the correlation with age was significant (p < 0.01: Table 2). However, IgM showed a significant positive correlation with DMFS but not with age, so that the IgM concentration may be a non-specific marker of increased antigenic load in rampant caries. These findings are consistent with the sequence of development of the immunoglobulins with age: IgM concentration increases rapidly in the first year, IgG after an initial fall increases over 2 years, whilst IgA increases slowly during infancy and childhood (Stiehm and Fudenberg, 1966). Our data suggest, therefore, that the younger the child the more likely it will develop IgM rather than IgG or IgA classes of antibodies in response to antigenic stimulation. The significance of IgG antibodies in protection against caries has been established in rice (Lehner et al., 1978). Passive transfer of whole immune serum with IgG, IgM and IgA antibodies to Strep. mutuns failed to protect against caries. but very significant protection was induced with the transfer of separated IgG (p < 0.001) though not with IgM (p > 0.05) or IgA (p > 0.05) antibodies. Passive transfer of IgA antibodies resulted in the highest index of caries (8.5) so that such transfer does not seem to have a protec-
tive function. The lack of protection in passive transfer of whole immune serum may be due to competition between IgG and IgA or 1gM antibodies. Indeed, IgA can inhibit IgG-mediated bacteriolysis (Hall, Manion and Zinneman, 1971: Griffis, 1975) and phagocytosis (Wilton, 1978) and it inhibits chemotaxis of polymorphonuclear leukocytes (Van Epps and Williams, 1976). IgM antibodies also failed to induce protection (caries index of 5.5) as compared with caries
found with transfer of IgG antibodies (2.0). There is evidence that only IgG is opsonic or that IgG is more efficient than IgM in opsonizing bacteria for phagocytosis (Rabinovitch, 1967; Laxdal et al., 1968; Bjornson and Michael, 1971). There is now direct evidence that IgG, unlike IgA, is involved in opsonization, phagocytosis and killing of Strep. mutans (Scully, Russell and Lehner, 1978). The opposing effects of different classes of antibodies has also been found with antibodies to glucosyltransferase (GTF) (Russell, Challacombe and Lehner, 1976). IgG class of antibodies showed GTFinhibitory activity, whereas IgA and IgM antibodies showed GTF-enhancing activity. IgM antibodies might enhance adherence of Strep. muruns to the tooth surface and thus increase the development of caries, as has been found on immunization with a preparation of culture supernatant of Sfrep. mur~ns (Russell et ul., 1976). Experimental work in rhesus monkeys kept on a human type of diet has clearly established that Strep. mutans, naturally colonizing teeth, is a poor immunogen (Lehner, Challacombe and Caldwell, 1976). A significant increase in IgG class of antibodies, assayed by precipitation and complement fixation tests, was not detected during a period of 2.5 years, throughout which Strep. muruns was isolated from the dental plaque sampled at monthly intervals. Only haemagglutinating antibodies, predominantly of the IgM class and directed to antigens in cell walls but not culture supernatant of Strep. mutuns, showed an increased antibody titre with natural colonization of Strep. murans. The IgM antibodies are unlikely to be protective as they may not be directed against essential antigens and they are inefficient in opsonizing bacteria for phagocytosis and killing. It is therefore not surprising that any naturally acquired immunity to dental caries in man is relatively ineffective. Ackno~,/etiyements-We thank Miss Christine A. Casswell and Mrs. Belinda J. Lund for their help in examining the patients
and collecting
the samples
for investigation.
REFERENCES Attstriim R. and Egelberg J. 1970. Emigration of blood neutrophils and monocytes into the gingival crevices. J. periodont.
Res. 5, 48-55.
Berger U., Kaparitis M. and Pfeifer G. 1959. Zur Besiedlung der klinichen Mundhiihle mit anaeroben Mikroorganismen. Z. Hyg. I$ Krunkh. 145, 564. Berkenbilt D. A. and Bahn A. N. 1971. Development of antibodies to cariogenic streptococci in children. J. Am. dent. Ass. 83, 332-331.
Bjornson A. B. and Michael J. G. 1971. Contribution of humoral and cellular factors to the resistance to experimental infection by Pseudomonas aeruginoscr in mice. Infect. Immun. 4, 462-467. Caldwell J., Challacombe S. J. and Lehner T. 1977. A sequential bacteriological and serological investigation of rhesus monkeys immunised against dental caries with Streptococcus
mutans.
.I. med. Microbial.
IO, 213-224.
Carlsson J. 1967. Presence of various types of non-haemolytic streptococci in dental plaque and in other sites of the oral cavity in man. O&n. Rery 18, 55-74. Challacombe S. J. 1974. Serum complement-fixing antibodies in human dental caries. Caries Res. 8, 84-95.
Antibody
and immunoglobulin
Challacombe S. J. and Lehner T. 1976. Serum and salivary antibodies to cariogenic bacteria in man. J. dent. Res. 55, Cl39-148. Challacombe S. J.. Lehwr T. and Guggenheim B. 1972. Serum and salivary antibodies to glucosyltransferase in dental caries in man. Nature 238, 219. Ericson T., Pruitt K. and Wedel M. 1975. The reaction of salivary substances with bacteria. J. oral Path. 4, 3077323. Griffis J. M. 1975. Bactericidal activity of meningococcal antisera. J. Itnmun. 114, 1779-1784. Hall W. H., Manion R. E. and Zinneman H. H. 1971. Blocking serum lysis of Bruce//a abortus by hyperimmune rabbit IgA. J. fmmun. 107, 41-46. lvanyi L. and Lehner T. 1978. The relationship between caries index and stimulation of lymphocytes by Srreproco(‘cu\ mutans in mothers and their neonates. Archs orul Biol. 23, 851 ~856. Kennedy A. E., Shklair I. L.. Hayashi J. A. and Bahn A. N. 1968. Antibodies to cariogenic streptococci in humans. Archs oral Biol. 13, 1275-1278. Laxdall T., Messner R. P., Williams R. C. Jr. and Quie P. G. 1968. Opsonic agglutinating and complementfixing antibodies in patients with sub-acute bacterial endocarditis. J. Lab. clin. Med. 71, 638-653. Lehner T. 1966. Immunofluorescence study of Candida ulbicws in candidiasis. carriers and controls. J. Puth. Btrct 91, 97m 104. Lehner T. 1977. Immunological responses to bacterial plaque in the mouth. In: Immunoloyp of the Gur. Vol. 46, pp. 135-154. Ciba Foundation Symposium. Lehner T., Wilton J. M. A. and Ward R. G. 1970. Serum antibodies in dental caries in man. Archs oral Biol. 15, 4x I 490. Lehner T.. Challacombe S. J. and Caldwell J. 1976. immunologic basis for vaccination against dental caries in rhesus monkeys. J. denf. Res. 55, C166180. Lchner T.. Russell M. W.. Challacombe S. J.. Scullv C. M. and Hawkes J. E. 1978. Passive immunisation -with strum and immunoglobulins against dental caries in rhesus monkeys. Luncrr i, 693. McCarthy C.. Snyder M. L. and Parker R. B. 1965. The indigenous oral flora of man. I. The newborn to the I-year old infant. Awhs owl Bioi. 10, 61-70. Mancini G.. Carbonara A. and Heremans J. P. 1965. Immunochemical quantitation of antigens by single radial
levels in dental
caries
1067
immunodiffusion. Immunochemistry 2, 235-239. Rabinovitch M. 1967. Studies of the immunoglobulins which stimulate the ingestion of glutaraldehyde-treated red cells attached to macrophages. 1. Immun. 99, 1115-1120. Russell M. W., Challacombe S. J. and Lehner T. 1976. Serum glucosyltransferase-inhibiting antibodies and dental caries in rhesus monkeys immunized against Streptococcus mutans. lmmunoloyy 30, 619.-627. Scully C. M. and Lehner T. 1978. Phagocytosis of Streptococcus mutuns in relation to protection of rhesus monkeys against dental caries. Proc. 7rk Itif. Srrnp. ou Srrrptoc’oc~ci cmd Strrptococcol Di.snr.st~.\. Oxford (in press). Skapski H. and Lehner T. 1976. A crevicular washing method for investigating immune components of crevicular fluid in man. J. periodontal Rex 11, 19-24. Stiehm E. R. and Fudenberg H. H. 1966. Serum levels of immune globulins in health and disease: A survey, Pediatrics 37, 715-727. Van Epps D. E. and Williams R. C. Jr. 1976. Suppression of leukocyte chemotaxis by human IgA myeloma components. J. exp. Med. 144, 1227-1231. Williams R. C. and Gibbons R. J. 1975. Inhibition of streptococcal attachment to receptors on human epithelial cells by antigenically similar salivary glycoproteins. Inject. Immun. 11, 71ll718. Wilton J. M. A. Polymeric and dimeric IgA inhibition of IgG mediated phagocytosis by human polymorphonuclear leucocytes. J. denr. Res. Abstract. in press. Wilton J. M. A.. Renggli H. H. and Lehner T. 1976. The isolation and identification of mononuclear cells from the gingival crevice in man. .I. prrioclorlr. Rcr. Il. 262-268. Wilton J. M. A.. Renggli H. H. and Lehner T. 1977. The role of Fc and C3b receptors in phagocytosis by inllammatory polymorphonuclear leucocytes in man. Immirnology 32, 955-961. Winter G. B., Hamilton M. C. and James P. M. C. 1966. Role of the comforter as an aetiological factor in rampant caries of the deciduous dentition. Art,hs Di\. Childh. 41, 207-212. Winter G. B.. Rule D. C., Mailer G. P., James P. M. C. and Gordon P. H. 1971. The prevalence of dental caries in pre-school children aged I to 4 years. Br tlen~. J. 130, 271-277.
AND WITH
T. LEHNER,’ J. J. MURRAY,’ G. B. WINTER’ and JILL CALDWELL’ ‘Department
of Oral
‘Department
Immunology
of Children’s
and Microbiology. Guy’s Hospital London SEl. and Dentistry. Eastman Dental Hospital.
Medical
and Dental
Schools.
London
WCL. England
Summary-An immunological investigation of rampant and non-rampant caries was carried out in children aged 2.5-5.5 yr. Serum IgG, IgA and IgM classes of antibodies to Streptococcus mutuns were detected and significant negative correlations were found between the DMFS and the IgG:IgA or 1gG:IgM ratios. Immunological response against dental caries appears to be associated with the proportion of IgG to IgA and IgM classes of antibodies to Strep. muruns. Serum IgM. unlike IgG or IgA, concentrations showed a significant positive correlation with the DMFS and IgM concentration may be a measure of antigenic load in the child. The study emphasizes the importance of antibody class and age of evaluation of the immune responses in the mechanism of protection against dental caries.
SUBJECTS AND METHODS
INTRODUCTION Subjects
Twenty-seven
Streptococcus mutuns
is probably the most cariogenic organism and requires the tooth surface for large-scale colonization (Carlsson. 1967). Hence, the earliest time of colonization of Srrep. mutans is about 6 months, in contrast to Luctobucillus,
microorganisms in the mouth (Lehner, 1977). An into the comparative maternalfetal immune responses to Strep. mutuns (Ivanyi and Lehner, 1978) revealed maternal IgG but not IgM class of antibodies to Strep. mutuns in neonatal blood. Furthermore, of neonatal lymphocytes to Strep. mutans occurs in utero (Ivanyi and Lehner, 197X). Systemic humoral and cellular immune components enter the mouth through the gingival crevice (Attstram and Egelberg, 1970; Skapski and Lehner. 1976; Wilton, Renggli and Lehner, 1976, 1977). There may, therefore, be a critical timing between the eruption of deciduous teeth and the clearance of maternal antibodies (about 5 months after birth), as well as the duration of of lymphocytes which may influence the colonization of plaque bacteria. We have compared serum and salivary antibody titres and immunoglobulin concentrations in young children with rampant caries, with those having nonrampant caries of deciduous teeth.
children,
2.5-5.5 yr, were 1 consisted of 15 children with rampant caries, as defined by Winter, Hamilton and James (1966) and group 2 of 12 children with one or more untreated carious lesions (Table 1). Up to 10ml of blood was collected from each child under a general anaesthetic required for dental extraction. A specimen of whole saliva was collected from 24 children by aspiration from the floor of the mouth through a sterile plastic saliva ejector into a sterile 20-ml stoppered bottle. Dental plaque was collected with a sterile probe from the approximal surfaces of the deciduous molars and cervical aspects of the lower incisors. divided
Assay
into two groups.
aged
Group
qf immunoylobulins
Serum IgG, IgA and IgM and salivary IgA concentrations were estimated by the radial immunodiffusion method (Mancini, Carbonara and Heremans, 1965). The antisera used (Wellcome reagents) were checked by immunoelectrophoresis for specificity for y, t( and p chains. The corresponding immunoglobulin standards used were supplied by the Medical Research Council.
1061
Prepuration Strep.
of Strep. mutans for
untibody
ussuy
mutws
(serotype c) was grown in ToddHewitt broth for 18 h. The cells were washed 3 times with saline and resuspended to about 10’ organisms/ml. Immunojluorescent antibodies
ussuy for serum
IgG,
IgM
and lyA
Class-specific antibodies to Strep. mutans were assayed by the immunofluorescent method (Lehner,
15 12
Rampant Non-rampant t value I’
* Months. NS. not significant.
No.
Caries
47 (f3.4) 49.3 (13.4)
Mean age* (&SE)
Table
10 6
5 6
Sex M F 11.5 (kO.6) 5.1 (kO.6) 7.139 < 0.0001
DMF
in relation
DMFS
NS
1.899
9 4
Positive culture of Strep. tnutans
to Strup. rnutan.s and salivary
25.2 (k3.1) 8.3 (& 1.5) 4.512 < 0.001
Mean (*SE)
1. Age. sex and DMFS
13 11
No.
indices
6.3 (t0.7) 5.4 (+ 0.5) 1.074 NS
1.8 (iO.2) 1.9 (kO.2) 0.308 NS
Saliva (mean + SE) Agglutinating kA cont. antibody
Antibody
and immunoglobulin
1966). Doubling dilutions of sera prepared with saline (1:5, 1 : 10, etc.) were added to air-dried smears of cells of Strep. mutcms for 30 min. These were washed 3 times with phosphate-buffered saline (PBS; pH 7.2) and overlaid with anti-human IgG (F:P 3.1), IgM (F:P 4.1) or IgA (F:P 3.8) fluorescein-isothiocyanate conjugates (Wellcome reagents). The reagents were checked for class specificity by immunoelectrophoresis and double-diffusion precipitation tests. The specificity of the test was established by complete absorption of the antibody titre with the homologous and little or no absorption with heterologous organisms. The titres were expressed in doubling dilutions: log2 (I = 1:5). Sulitw),
agglutinating
unribodg
test
This was performed with serial dilutions of saliva (1:2. 1:4, etc.) with saline in microplates, using fresh, washed cells of the Strep. mutans (5~1 of lo9 colonyforming units per ml). The plates were read after overnight incubation at 4°C. Culture
of pluque ,fiw Strep.
mutans
The samples were dispersed by vigorous shaking with sterile, 3-mm dia glass beads in a vortex mixer (Fisons Ltd., Loughborough, Leics.) for 60s and cul-
levels in dental
caries
1063
tured on TYC medium as described by Caldwell, Challacombe and Lehner (1977). Colonies that produced extracellular polysaccharide (EPS) were differentiated according to their colonial morphology. Representative colonies were subcultured for positive identification. Strains that fermented mannitol and sorbitol were designated as Strep. mutans.
RESULTS
Age und decayed, missing und ,filled in rampant and non-rumpunt curies
surfuces
There was no difference in age (Table 1) between the group with rampant caries (47 i 3.4 months) and non-rampant caries (49.3 f. 3.4 months). However, a very significant difference in DMFS (p < 0.001) was present in children with rampant caries (25.2 + 3.1) as compared with non-rampant caries (8.3 k 1.5). Serum
immunoglobulin
concentrurions
A significant positive correlation (Fig. 1) was found between the DMFS and the IgM (r = 0.502; p < 0.01) but not IgA (r = 0.231; p > 0.05) or IgG concentrations (r = 0.348: p > 0.05; Table 2). Significant correlations were found between age and IgG
3000 2800 2400
.
.
2000
. .
.
.
IgG 1600 .
.
9.’
. ..
300
.
’
.
250 200
I@
150 100 50 0
’
300 l
250
.
.
.
IgM
0-m 0
5
10 15
20 25
30 45
60
DMFS *Rampant Fig. 1. lmmunoglobulin
I
I
0
10
I
I
I
I
1
117
20 30 40 50 60 70 100 Age in months
0 Non-rampant
concentrations
(DMFS)
(mg/lOOml)
in relation
to the DMFS
and age
1064
T. Lehner.
J. J. Murray,
G. B. Winter
and J. Caldwell
Antibody and immunoglobulin (r = 0.514; p < 0.01) and
but not IgM concentrations Serum
antibody
IgA (r = 0.536; p < 0.01) (Table 2).
titres
No significant difference in IgG, IgA or IgM antibody titres between sera from children with rampant or non-rampant caries was found (Table 2). No significant relationship was observed between the DMFS and IgG, IgA or IgM classes of antibodies. However, significantly decreased ratios of 1gG:IgA (t = 2.533; p < 0.02) or 1gG:IgM (t = 2.577; p < 0.02) were found in rampant as compared with non-rampant caries. Further analysis showed significant negative correlations between the DMFS and 1gG:IgA (r = 0.528; p < 0.01). 1gG:IgM (r = 0.4921 ; p < 0.01) and IgG:(IgA and IgM) (r = 0.484; p < 0.01). The age of the children was not related to the antibody ratios (Table 2), but showed a significant correlation with IgM antibodies to Strep. mutans (r = 0.518: p < 0.01). Sulicary
1gA concentration
and antibodies
No
difference was found between rampant and non-rampant caries in the salivary IgA concentration or agglutinating antibodies (Table 1). No correlation was found between salivary IgA concentration or agglutinating antibody and the DMFS. However, as whole saliva was used, some antibodies may have been lost by binding to Strep. mutans in the saliva. Culture
1065
levels in dental caries
entiate the two types of caries as a combined analysis of the two groups in terms of DMFS against the IgG:IgA, or 1gG:IgM ratios revealed significant negative correlations (p < 0.01; Fig. 2). These results are therefore consistent with the view that children with rampant caries are not immunologically compromised, but that the dietary conditions, such as are created by prolonged use of sweetened feeding bottles or sweetened comforters, are responsible for rampant caries (Winter et u/., 1971). Our investigation was based on the concept evolved from immunization of monkeys. that immunological protection against caries is dependent on the class of antibodies: a high ratio of IgG to IgA. and of IgG to IgM antibodies is found in macaque monkeys with a low caries incidence (Lehner et rrl., 1978). This concept also appears to apply to a population of young children (2.5-5.5 yr old) who also show the three classes of serum antibodies to Strep. mutans, and emphasizes the differences in the function of antibodies of the IgG, IgA and IgM classes. A significant correlation was not detectable with any of the three classes of fluorescent antibodies and the DMFS. but significant negative correlations were found between the DMFS and the ratios of IgG:IgA, 1gG:IgM and IgG:(IgA and IgM). These results argue in favour of a protective effect of IgG antibodies. if their titre and probably affinity
of Strep. mutans
Strep. mutans was cultured in 9 out of 15 samples of plaque from children with rampant caries and in 4 out of 12 children with non-rampant caries (Table 1). This difference was not significant (x2 = 1.894). Furthermore, the number of colony-forming units in the two groups was not significantly different.
4l 3 IgG-IgA
o
DO
2
0.
1
000
Log2
r 0
. . 0.
.
.0*
. .*
’
DISCUSSION A clinical differentiation of caries of deciduous teeth into rampant and non-rampant types (Winter et ul., 1971) showed a significant difference in the DMFS but not in the age of the two groups (Table 1). In our study. salivary IgA concentration or agglutinating antibodies were the same in the two groups investigated. However, non-immunoglobulin agglutinating factors have been described in saliva (Williams and Gibbons, 1975: Ericson, Pruitt and Wedel, 1975) so that these may have affected the results. Of the serum immunoglobulin concentrations, only IgM was significantly increased in rampant caries but, as there was a significant positive correlation between the DMFS and IgM concentration (p < O.Ol), irrespective of age, the difference in IgM might be due to the number of carious lesions and not to the type of caries. Immunofluorescent antibodies to Strep. mutans of the IgG, IgA and IgM classes were not significantly different, though decreased titres of IgG and increased titres of IgA and IgM antibodies were observed in rampant as compared with non-rampant caries (Table 2). This suggested that the ratios of IgG to IgA and IgG to IgM might be decreased in rampant caries, and indeed this was found (p < 0.02). However, these antibody ratios are unlikely to differ-
.
DMFS l
Rampant
o Non-rampant
Fig. 2. Relationship between DMFS and the ratios 01”IgG to IgA. IgM and IgA + IgM antibodies to .‘rrc,/~.IHL~~~I~I.L
1066
T. Lehner. J. J. Murray, G. B. Winter and J. Caldwell
are greater than those of IgA and IgM antibodies. The antibody ratios and titres in our study were independent of the age of the children, except for the IgM antibodies, unlike the summated agglutinating antibody titres to cell walls previously shown to four strains of Strep. mutuns and Strep. sunguis (Berkenbilt and Bahn, 1971). The method used by these workers, however, did not differentiate between the three antibody classes: it may have been biased in favour of the IgM class and may have assayed an increase in antibody load to a variety of oral streptococci associated with the age of the child. A comparison by Kennedy er al. (1968) in naval recruits, aged 17-19 yr, of serum-agglutinating antibodies to cell walls from caries-free subjects with those from rampant caries showed a higher titre in the caries-free subjects. However, haemagglutinating antibodies to sonicates of Strep. mutans showed that adults with high caries indices had a higher antibody titre than those with low caries indices (Lehner, Wilton and Ward, 1970). Further division of caries into a group with carious lesions and another without carious lesions, but some with past caries experience, has revealed a significant negative correlation between the DMF and IgG or IgM antibodies to cell walls or culture supernatant of Strep,. mutuns in the group free of carious lesions, but a positive correlation in the group with carious lesions (Challacombe, Lehner and Guggenheim, 1972: Challacombe. 1974: Challacombe and Lehner, 1976). In our present study, serum IgG and IgA concentrations showed an increase with DMFS and age of the children, though only the correlation with age was significant (p < 0.01: Table 2). However, IgM showed a significant positive correlation with DMFS but not with age, so that the IgM concentration may be a non-specific marker of increased antigenic load in rampant caries. These findings are consistent with the sequence of development of the immunoglobulins with age: IgM concentration increases rapidly in the first year, IgG after an initial fall increases over 2 years, whilst IgA increases slowly during infancy and childhood (Stiehm and Fudenberg, 1966). Our data suggest, therefore, that the younger the child the more likely it will develop IgM rather than IgG or IgA classes of antibodies in response to antigenic stimulation. The significance of IgG antibodies in protection against caries has been established in rice (Lehner et al., 1978). Passive transfer of whole immune serum with IgG, IgM and IgA antibodies to Strep. mutuns failed to protect against caries. but very significant protection was induced with the transfer of separated IgG (p < 0.001) though not with IgM (p > 0.05) or IgA (p > 0.05) antibodies. Passive transfer of IgA antibodies resulted in the highest index of caries (8.5) so that such transfer does not seem to have a protec-
tive function. The lack of protection in passive transfer of whole immune serum may be due to competition between IgG and IgA or 1gM antibodies. Indeed, IgA can inhibit IgG-mediated bacteriolysis (Hall, Manion and Zinneman, 1971: Griffis, 1975) and phagocytosis (Wilton, 1978) and it inhibits chemotaxis of polymorphonuclear leukocytes (Van Epps and Williams, 1976). IgM antibodies also failed to induce protection (caries index of 5.5) as compared with caries
found with transfer of IgG antibodies (2.0). There is evidence that only IgG is opsonic or that IgG is more efficient than IgM in opsonizing bacteria for phagocytosis (Rabinovitch, 1967; Laxdal et al., 1968; Bjornson and Michael, 1971). There is now direct evidence that IgG, unlike IgA, is involved in opsonization, phagocytosis and killing of Strep. mutans (Scully, Russell and Lehner, 1978). The opposing effects of different classes of antibodies has also been found with antibodies to glucosyltransferase (GTF) (Russell, Challacombe and Lehner, 1976). IgG class of antibodies showed GTFinhibitory activity, whereas IgA and IgM antibodies showed GTF-enhancing activity. IgM antibodies might enhance adherence of Strep. muruns to the tooth surface and thus increase the development of caries, as has been found on immunization with a preparation of culture supernatant of Sfrep. mur~ns (Russell et ul., 1976). Experimental work in rhesus monkeys kept on a human type of diet has clearly established that Strep. mutans, naturally colonizing teeth, is a poor immunogen (Lehner, Challacombe and Caldwell, 1976). A significant increase in IgG class of antibodies, assayed by precipitation and complement fixation tests, was not detected during a period of 2.5 years, throughout which Strep. muruns was isolated from the dental plaque sampled at monthly intervals. Only haemagglutinating antibodies, predominantly of the IgM class and directed to antigens in cell walls but not culture supernatant of Strep. mutuns, showed an increased antibody titre with natural colonization of Strep. murans. The IgM antibodies are unlikely to be protective as they may not be directed against essential antigens and they are inefficient in opsonizing bacteria for phagocytosis and killing. It is therefore not surprising that any naturally acquired immunity to dental caries in man is relatively ineffective. Ackno~,/etiyements-We thank Miss Christine A. Casswell and Mrs. Belinda J. Lund for their help in examining the patients
and collecting
the samples
for investigation.
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1067
immunodiffusion. Immunochemistry 2, 235-239. Rabinovitch M. 1967. Studies of the immunoglobulins which stimulate the ingestion of glutaraldehyde-treated red cells attached to macrophages. 1. Immun. 99, 1115-1120. Russell M. W., Challacombe S. J. and Lehner T. 1976. Serum glucosyltransferase-inhibiting antibodies and dental caries in rhesus monkeys immunized against Streptococcus mutans. lmmunoloyy 30, 619.-627. Scully C. M. and Lehner T. 1978. Phagocytosis of Streptococcus mutuns in relation to protection of rhesus monkeys against dental caries. Proc. 7rk Itif. Srrnp. ou Srrrptoc’oc~ci cmd Strrptococcol Di.snr.st~.\. Oxford (in press). Skapski H. and Lehner T. 1976. A crevicular washing method for investigating immune components of crevicular fluid in man. J. periodontal Rex 11, 19-24. Stiehm E. R. and Fudenberg H. H. 1966. Serum levels of immune globulins in health and disease: A survey, Pediatrics 37, 715-727. Van Epps D. E. and Williams R. C. Jr. 1976. Suppression of leukocyte chemotaxis by human IgA myeloma components. J. exp. Med. 144, 1227-1231. Williams R. C. and Gibbons R. J. 1975. Inhibition of streptococcal attachment to receptors on human epithelial cells by antigenically similar salivary glycoproteins. Inject. Immun. 11, 71ll718. Wilton J. M. A. Polymeric and dimeric IgA inhibition of IgG mediated phagocytosis by human polymorphonuclear leucocytes. J. denr. Res. Abstract. in press. Wilton J. M. A.. Renggli H. H. and Lehner T. 1976. The isolation and identification of mononuclear cells from the gingival crevice in man. .I. prrioclorlr. Rcr. Il. 262-268. Wilton J. M. A.. Renggli H. H. and Lehner T. 1977. The role of Fc and C3b receptors in phagocytosis by inllammatory polymorphonuclear leucocytes in man. Immirnology 32, 955-961. Winter G. B., Hamilton M. C. and James P. M. C. 1966. Role of the comforter as an aetiological factor in rampant caries of the deciduous dentition. Art,hs Di\. Childh. 41, 207-212. Winter G. B.. Rule D. C., Mailer G. P., James P. M. C. and Gordon P. H. 1971. The prevalence of dental caries in pre-school children aged I to 4 years. Br tlen~. J. 130, 271-277.
