Serum antibodies and loss of periodontal bone in patients with rheumatoid arthritis
KSre Tolo and Lars Jorkjend Department of Periodontoiogy, University of Oslo, Norway
Tolo K and Jorkjend L: Serum antibodies and loss of periodontal bone in patients with rheumatoid arthritis. J Clin Periodontol 1990; 17: 288-291. Abstract. The number of teeth, % of alveolar bone loss, serum IgG, and serum antibodies to Bacteroides gingivalis, Capnocytophaga ochracea and Eubacterium saburreum were recorded in 37 patients diagnosed with rheumatoid arthritis (RA) and in an age- and sex-matched control group of 37 individuals free from RA. The RA group had a significantly increased loss of teeth and loss of alveolar bone compared to the control group. The RA patients also had a significantly increased level of serum IgG. In the total material, 26% of the variation in loss Key words: periodontitis; rheumatoid arof alveolar bone was accounted for by age, diagnosis of rheumatoid arthritis, and levels of antibodies against B. gingivalis and E. saburreum. In the RA group, thritis; bone loss; tooth loss; serum antibodies; oral bacteria. 48% of this variation was accounted for by age, total serum IgG and IgG antibodies to B. gingivalis and E. saburreum. Accepted for publication 18 May 1989
Rheumatoid arthritis (RA) and related diseases afflict nearly 37 million people or 20% of the adult population in USA (Biennal Report of the National Institutes of Health 1985/86) and a similar prevalence has been found in northern Europe. The pattern of immune responses in rheumatoid arthritis is characterized by enhanced humoral and depressed cellular immunity (Talal 1985). Periodontitis is primarily a B-cell lesion (Seymour 1987) with a mechanism of tissue destruction analogous to that of rheumatoid arthritis (Snyderman & McCarthy 1982). The similarity between RA and periodontitis has prompted several studies of periodontal status in patients with rheumatoid arthritis. The observations are not concordant. Early studies from the Mayo Clinic in USA indicated similar frequencies of periodontitis in patients with or without RA. A later report from Europe (Liubomirova 1964) indicated that greater bone loss may occur in patients with RA. In a review of the literature, Helminen-Pakkala (1971) concluded that earlier studies supported her own observations in a large group of subjects in Finland, and stated that "periodontal disease does not seem to be a prominent feature in persons suffering from arthritis." Differences in disease criteria and methods for evaluation of the periodontal state form a
major problem in interpretation of the literature. A rough estimate of dental status is obtained by counting the number of teeth. Helminen-Pakkala (1971) reported a similar number of edentulous patients in RA patients as in controls, and up to the age of 50 years, the number of missing teeth was also similar in both groups. Malmstr0m & Calonius (1975) examined 126 RA patients and observed both an increased frequency of edentulousness and more missing teeth in dentate RA patients than in the control group. The questions asked in the present study were as follows: Is the loss of teeth and loss of periodontal bone higher in individuals with rheumatoid arthritis than in patients free from the disease? Is alveolar bone loss in RA correlated with specific antibodies to suspected periodontal pathogens or with the level of serum immunoglobulins? Material and Methods
'
Patients
37 patients diagnosed with RA according to the criteria set by Ropes et al. (1971) were studied. The patients were hospitalized at Bethanien Hospital, Skien, Norway for a period of 9 months. Edentulous patients and patients diagnosed with Sjogrens disease were excluded. AH other patients were included.
The mean duration of diagnosed RA was 13 years. All patients had received anti-inflammatory drugs for extended periods and 11 patients had also received cytostatica (Prednisone, Imuran, Methotrexate). A sex- and age-matched control group of 37 patients was drawn by paired matching from patients registered at the Dental Faculty Clinic, University of Oslo. Patients with rheumatoid arthritis and edentulous patients were excluded. Radiography
A Siemens Heliodent X-ray apparatus set at 60 kv and 7 mA, with a 1.6 mm AL-filter and Eggens film holder (Eggen, 1969). 10 intraoral, Kodak D-58 films were exposed per patient according to standard ej(posure times. The films were automatically processed at the Department of Radiology of the Faculty of Dentistry, University of Oslo. Loss of periodontal bone was recorded on the approximal surfaces of the front teeth in the upper and lower jaw. The distance from the cemento-enamel junction (CEJ) to the alveolar bone was measured and expressed as a % of the distance from the CEJ to the root apex. The measurements were made by an examiner who was not informed of the group assignment of the patient.
Periodontilis and rheumatoid arthritis phosphatase conjugated class specific antiserum (Orion Diagnostica, Helsin10 ml blood was obtained from each ki, Finland) and reaction with p-nitropatient and the serum was stored at phenol disodium phosphate (Sigma). — 20° C. Total serum immunoglobulins The test was accepted as valid if the were measured by single radial immuno- coefficient of variation did not exceed diffusion in Nor-partigen gels (Behring, 10%. 6 wells on each plate were incuMarburg, West Germany). The rheuma- bated with the reference serum and the toid factor was evaluated by a modified individual OD-readings were adjusted Waalers test (Waaler 1939) using rabbit for plate-to-plate variations according antiserum with human erythrocytes and to the formula: by latex agglutination (Behring). Specimean OD of individual serum - background fic activities of the serum immunoglobmean OD of reference serum - background ulins G, A and M against 3 suspected periodontal pathogens were measured The data were recorded as ELISA units, by enzyme-linked immunosorbent as- and the reference serum was assigned a says (Tolo and Schenck, 1985). Pure cul- value of 100 EU at an equal dilution tures of Bacteroides gingivalis, Capnocy- for each isotype. To obtain equality of tophaga ochracea and Euhacterium sa- variances, all immunological data 0') burreum were established from samples were transformed as y' = ln y before obtained from patients at the Depart- statistical analyses. Group differences ment of Periodontology, University of were analyzed by the Student /-test, 2Oslo as described by Tolo & Schenck tailed probability. The data were sub(1985). The cultures were grown to late jected to multiple regression analysis. exponential phase, centrifuged at 10,000 Loss of alveolar bone was selected as g^ for 10 min, the supernatants decanted, the dependent variable, and age, serum dialyzed for 3 days against running tap water and lyophilized. The powder was level of IgG, diagnosis of RA, antidissolved (100 mg/ml) in deionized bodies to B. gingivalis and E. saburreum water, applied on a Sephadex G 75 col- were entered stepwise into the equation. umn (100x2.5 cm) and eluted with The independent variables were entered water containing 0.2 g/1 sodium azide. according to the strength of bivariate The optical density of the eluate was correlation. The level of statistical sigmonitored at 280 nm and the fractions nificance was set at/I < 0.05. corresponding to the void volume were pooled, lyophilized and stored at —20° Results C. Dental status Immunological tests
The fractionated bacterial components were dissolved (1 /^g/ml) in 0.15 M phosphate buffered saline (PBS) pH 7.3, and 200 /il of the solution were applied per well in polystyrene microtiter plates (Dynatech MicroElisa, Greiner 129 B). A reference serum was prepared by pooling sera from 10 patients with periodontitis. The reference serum was positive for all 3 bacterial extracts (binding of IgG > mean + 2 SD obtained with serum from healthy individuals). Standard curves were established by testing serial dilutions of the reference serum. The binding of human Ig to the respective coatings was reduced by 70% to 90% by addition of homologous bacterial extract to the reference serum. The binding observed after maximal inhibition was considered equal to the background level of the assays. The serum samples were diluted 1:400 in phosphate buffered saline (PBS), pH 7.3 containing 5 mg/ml bovine serum albumin and tested in triplicate. The OD was measured after incubation with alkaline
The mean number of teeth was 17.9 in the RA-group and 22.6 in the control group. The mean loss of bone was 23.8% in the RA-group and 18.9% in the control group (Table 1). Serum Immunoglobulins
Serum IgG and IgA were significantly increased in the RA-patients compared to the control group. No group differences were observed with regard to serum IgM.
289
Table 2. Multiple regression analyses of data from 74 patients, 37 diagnosed with rheumatoid arthritis and 37 controls % of variation accounted for age age + Dia age + Dia-(-IgG B.g age-fDia + IgG B.g + IgG-E.s
14 20 24 26
Dependent variable = loss of alveolar bone. Variables entered stepwise into the equation: age, diagnosis (Dia), igG-antibody to B. gingivalis (IgG B.g) and E. saburreum (IgG E.s).
Multiple regression analyses
In the combined group of 74 individuals, age, diagnosis of RA, and IgGantibodies to B. gingivalis and E. saburreum accounted for 26% of the variation in loss of alveolar bone (Table 2). In the RA group, 48% of the variation in bone loss was accounted for by age, IgG antibodies to B. gingivalis, IgGantibodies to E. saburreum and total serum IgG (Table 3). Discussion
The study shows that patients diagnosed with rheumatoid arthritis had lost more teeth than patients with no RA. The mean number of teeth present in the control group was similar to that observed in a random sample of 194 individuals from the same district (Lervik et al. 1988), and this indicated that the control group was a representative sample of the population with regard to loss of teeth. Interestingly, the mean number of teeth present in the control group was also similar to that observed in employed, dentate persons in USA (NIH publication 87-2868). The RA group had a mean of 17.9 teeth present in dentate patients (mean age = 53 years). This is in accordance with Laurell (1985) who observed a mean of 18.1
Table 3. Multiple regression analyses of data from 37 patients diagnosed with rheumatoid arthritis Table I. Number of teeth and loss of alveolar bone
RA (n = 37) Ctrl (/i = 37)
Teeth
Bone loss
I7.9"'±8.9 22.6 ±5.8
23.8'"±12.2 18.9 + 7.9
"' Significantly decreased compared to controls. ''' Significantly increased compared to controls.
% of variation accounted for age + IgG B.g age-t-IgG B.g + SerIgG age + IgG B.g + SerIgG + IgG E.s
15 38 44 48
Dependent variable = loss of alveolar bone. Variables entered stepwise into the equation: Age, IgG-antibody to B. gingivalis (IgG B.g) IgG antibodies to E. saburreum (IgG E.s.) and serum concentration of IgG (SerIgG).
290
Kdre Tola & Lars Jorkjend
teeth present in a group of dentate RApatients with a mean age of 55 years. Accidents, caries, periodontitis and extractions associated with orthodontic treatment may have contributed to the loss of teeth. However, the observation of increased bone loss around the remaining teeth in the RA patients indicated that periodontitis may be an important cause of tooth loss in these patients. The multiple regression analyses indicated that age was the most important variable with regard to loss of alveolar bone, and accounted for about 14% of the variation. The second most important variable in the combined groups was diagnosis of RA. Further by taking into account the level^s of antibody against 2 suspected periodontal pathogens, the proportion of "explained variation" was increased to 26%. This proportion was not improved by the addition of the remaining variables into the multiple regression equation. In the RA group, the loss of alveolar bone was significantly correlated with age, IgG antibody against B. gingivalis and by the level of total serum IgG. The rheumatoid factor may have contributed to the increased proportion of "explained variation" in this group, since formation of IgG-complexes with rheumatoid factor may contribute both to increased ELISA readings and to the inflammatory activity in the periodontal lesion. 11 RA patients had high titers of rheumatoid factor (range 128-4096) and 19 patients had titers in the range 4-128. Data on the rheumatoid factor was lacking for 7 patients, and this factor was therefore not entered into the multiple regression analysis as an independent variable. Activity consistent with rheumatoid factor was observed in chronically inflamed gingiva by Kristoffersen & Tender (1973), and Malmstrom & Natvig (1975) observed that 25%-50% of the plasma cells in dental periapieal lesions in RA patients were positive for rheumatoid factor. Gargiulo et al. (1982) observed latex agglutination, indicating the presence of rheumatoid factor in dental plaque, saliva and extracts of gingiva in patients with periodontitis, and suggested that this faetor may contribute to chronic periodontal inflammation. Experimental data from mice (Lens & Beertsen, 1986) indicated that RAlesions can be influenced by injections of antigens into the oral mucosa. In periodontitis, the area of crevieular epi-
thelium exposed to the subgingival microbiota is substantially increased, and immune reactions will contribute to the permeability of the crevieular epithelium (Tolo et al. 1978). Penetration of bacterial products with a potential for B-cell activation in inflammatory infiltrates may therefore occur more frequently in periodontitis. The significant correlation between bone loss and IgGantibodies against 2 suspected periodontal pathogens in the RA patients is interesting, since it indicates the crevieular epithelium as a port of entry for Bcell activating agents. Among bacterial products reported to act as B-cell activators in RA are bacterial peptidoglycans (Pardo et al. 1984). Increased blast responses and polyclonal B-cell activation to extracts of dental plaque were observed in several studies of peripheral lymphocytes from patients with periodontitis (Patters 1976, Smith et al. 1979, Bick et al. 1981, Donaldson et al. 1982, Osterberg et al. 1983). The observations of the present study indicate that patients with RA may be regarded as a risk group with regard to periodontitis. Further studies are required to examine whether periodontitis may have a negative effect on RA. Zusammenfassung Scniimmlikdrper und parockmtaler Knochenschwund bci Patienlen mil rheumatischer Arihrill.s Bei 37 Palientcn mit rheumatischer Arthritis (RA) und einer, hinsiclitlich Alter- und Geschlechtszugehorigkeit vergleichbaren Kontrollgruppe, wurdc die Zahl vorhandener Zahne, der prozentuelle Knochenschwund, das Serum IgG und die Serumantikorper gegen Bacteroides gingivalis, Capnocytophaga ochracea und Eubacterium sahurreum registriert. Im Vergleich zu den KontroUprobanden kam bei der RA-Gruppe signifikant hoherer Zahnverlust und alveolarer Knochenschwund vor. Bei den RA-Patienten wurde ausserdem ein signifikant hoheres Serum IgG-Niveau festgestellt. In dem gesamten Probandengut wurden 26% des unterschiedlichen alveolaren Knochenschwunds dem Alter, der Diagnose "rheumatische Arthritis" und den Antikorperniveaus gegen B. gingivalis und E. saburreuni zugeschrieben. In der RA-Gruppe waren 48% dieser Variationen eine Folge des Alters, der totalen Serum IgG und der IgG Antikorper gegeniiber B. gingivalis und E. saburreuni
Resume Anticorps seriques el perte osseuse parodontale chez des patients souffrant d'arthrite rhuma-
lo'ide Lc nombre de dents, le pourcentage de perte osseuse alveolaire. Fig G et les anticorps seriques contre lc Bacteroides gingivalis, le Capnocytophaga ochracea et VEubaeterium saburreuni ont ete analyses chez 37 patients souffrant d'arthrite rhumatoide (RA) ainsi que chez 37 autres individus sans RA formant un groupe controle avec age et sexe as.sortis. Le groupe RA avait une perte dentaire et une perte osseuse significativetiient superieures a celles rencontrces dans le groupe controle. Les patients RA avaient egalement un taux d'lg G serique significativement plus important. Dans l'ensemble, 26% de la variation de perte d'os alveolaire otaient expliques par I'age, le diagnostic d'arthrite rhumatoide et les taux d'anticorps contre le B, gingivalis et VE. saburreuni, Dans le groupe RA, 48% de cette variation etaient expliques par I'age, l'lg G serique totale et les anticorps Ig G contre le B, gingivalis et VE, saburreuni.
References Bick, P H., Carpenter, A. B., Holdeman, L. V., Miller, G. A. Ranney, R. R., Palcanis, G. K. & Tew, J. G. (1981) Polyclonal Bcell activation induced by extracts of gramnegative bacteria isolated from periodontally diseased sites. Infection and Immunity 34, 4 3 ^ 9 . Biennial report of the Director, National Institutes of Health Volume II 1985-1986. NIH, Washington. Donaldson, S. L., Bick, P. H., Moore, W. E. C , Ranney, R. R. Burmeister, J. A. & Tew, J. G. (1982) Polyclonal B-cell activating capacities of gram-positive bacteria frequently isolated from periodontally diseased sites. Journal of Periodonlal Re,search 17, 569-575. Eggen, S. (1969) Standardiserad intraoral rontgenteknikunders0kning av felstorleken vid anvendanet av en forenklad innstallningsapparat. Tandlakartidningcn 61, 867. Gargiulo, A. V, Robinson, J., Toto, P. D. & Gargiulo, A. W. (1982) Identification of Rheumatoid Factor in Periodontal Disease. Journal of Periodontology, 53, 568-577. Helminen-Pakkala, E.- (1971) Periodontal conditions in rheumatoid arthritis. A clinical and r0ntgenological investigation. Part two. The study in rheumatoids. Proceedings of the Finnish Dental Societv Suppl. IV. Kristoffer.sen, T. & Tonder, O. (1973) Antiimmunoglobulin activity in inflamed human gingiva. Journal of Dental Research 52,991. Laurell, L. (1985) Oral status hos en grupp vuxna individer med rheumatoid artrit. Nordisk Forening for Handicaptandvdrd Bulletin 12, 3-16. Lens, J. W. & Beertsen, W. (1986) Injection of an antigen into the gingiva and its effect on an experimentally induced infiam-
Periodontitis and rheumatoid arthritis mation in the knee joint of the mouse. Journal of Perioiiontat Research 23, 1-6. Lervik, T., Haugejorden, O. & Holmbakken. N. (1988) Tannhelse hos 40 og 55 aringer i Skien i 1985. Den Norske Tannlegeforenings Tidende 98, 462-465. Liubomorova, I. M. (1964) State of parodontium in patients affected with rheumatism. Stomatologija (Moskva) 43, 33-37. Malmstr0m, M. & Calonius, P. E. B. (1975) Teeth loss and inflammalion of teeth supporting tissues in rheumatoid disease. Seandinavian Journal of Rheumatology 4, 49-55. Malmstr0m, M. & Natvig, J. (1975) IgG rheumatoid factor in dental periapical lesions of patients with rheumatoid disease. Scandinavian Journal of Rheumatology 4, 177-185. Pardo, I., Carafa, C , Dziarski, R. & Levinson, A. I. (1984) Analysis of in vitro polyclonal B-cell differentiation responses to bacterial peptidoglycan and pokeweed mitogen in rheumatoid arthritis. Clinical and Experimental Imnnmotogy 56, 253-262. Patters, M. R., Genco, R. J., Reed, M. J. & Mashimo, P. A. (1976) Blastogenic response of human lymphocytes to oral bacterial antigens: Comparison of individuals with periodontal disease to normal and
edentulous subjects. Infection and Immunity 14, 1213-1220. Ropes, M. W., Bennet, G. A., Cobbs, S., Jacox, R. & Jessar, R. A. 1958 revision of diagnostic criteria for rheumatoid arthritis. Bultelin of Rheumatoid Diseases 9. 175-176. Seymour, G. J. (1987) Possible mechanisms involved in the immunoregulation of ehronic periodontal disease. Journat of Dental Research 66, 2-9. Smith, F. N. & Lang, N. P. (1977) Lymphocyte blastogenesis to plaque antigens in human periodontal disease. 11. The relationship to clinical parameters. Journal of Periodontal Research 12, 310-317. Snyderman, R. & McCarty, G. A. (1982) Analogous mechanisms of tissue destruction in rheumatoid arthritis and periodontal research. In Hosi-parasite interactions in periodonlat disease (ed. R. J. Genco & S. E. Mergenhagen) pp. 354-362. American Society for Microbiology, Washington D. C. Talal, N. (1985) Interleukins, Interferon and Rheumatic Disease. Clinics in Rheumatic Diseases II, 633-644. Tolo, K., Brandtzaeg, P & Jonsen, J. (1978) Mucosal penetration of antigen in the presence or absence of serum-derived antibody.
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An in vitro study of rabbit oral and intestinal mucosa. Immunology 33, 733-743. Tolo, K. & Schenck, K. (1985) Activity of scrum immunoglobulins G, A, and M to six oral bacteria in diagnosis of periodontitis. Journal of Periodonlal Re.search 20, 113-121. Waaler, E. (1939) A factor in human serum activating the specific agglutination of sheep blood corpuscles. Third International Congress for Microbiology, New York 1939. Abstract of communication p. 351. 0sterberg, S. K., Page, R. C , Sims, T. & Wilde, G. (1983) Blastogenic responsivetiess of peripheral blood mononuclear cells from individuals with various forms of periodontitis and effects of treatments. Jourtial of Clinical Periodontologv 10, 72-88. Address: Kdre Tolo Ktinikk for Periodonti Det Odonlologiske Fakuliet Posthoks 1109 Blindern Oslo 3 Norge
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KSre Tolo and Lars Jorkjend Department of Periodontoiogy, University of Oslo, Norway
Tolo K and Jorkjend L: Serum antibodies and loss of periodontal bone in patients with rheumatoid arthritis. J Clin Periodontol 1990; 17: 288-291. Abstract. The number of teeth, % of alveolar bone loss, serum IgG, and serum antibodies to Bacteroides gingivalis, Capnocytophaga ochracea and Eubacterium saburreum were recorded in 37 patients diagnosed with rheumatoid arthritis (RA) and in an age- and sex-matched control group of 37 individuals free from RA. The RA group had a significantly increased loss of teeth and loss of alveolar bone compared to the control group. The RA patients also had a significantly increased level of serum IgG. In the total material, 26% of the variation in loss Key words: periodontitis; rheumatoid arof alveolar bone was accounted for by age, diagnosis of rheumatoid arthritis, and levels of antibodies against B. gingivalis and E. saburreum. In the RA group, thritis; bone loss; tooth loss; serum antibodies; oral bacteria. 48% of this variation was accounted for by age, total serum IgG and IgG antibodies to B. gingivalis and E. saburreum. Accepted for publication 18 May 1989
Rheumatoid arthritis (RA) and related diseases afflict nearly 37 million people or 20% of the adult population in USA (Biennal Report of the National Institutes of Health 1985/86) and a similar prevalence has been found in northern Europe. The pattern of immune responses in rheumatoid arthritis is characterized by enhanced humoral and depressed cellular immunity (Talal 1985). Periodontitis is primarily a B-cell lesion (Seymour 1987) with a mechanism of tissue destruction analogous to that of rheumatoid arthritis (Snyderman & McCarthy 1982). The similarity between RA and periodontitis has prompted several studies of periodontal status in patients with rheumatoid arthritis. The observations are not concordant. Early studies from the Mayo Clinic in USA indicated similar frequencies of periodontitis in patients with or without RA. A later report from Europe (Liubomirova 1964) indicated that greater bone loss may occur in patients with RA. In a review of the literature, Helminen-Pakkala (1971) concluded that earlier studies supported her own observations in a large group of subjects in Finland, and stated that "periodontal disease does not seem to be a prominent feature in persons suffering from arthritis." Differences in disease criteria and methods for evaluation of the periodontal state form a
major problem in interpretation of the literature. A rough estimate of dental status is obtained by counting the number of teeth. Helminen-Pakkala (1971) reported a similar number of edentulous patients in RA patients as in controls, and up to the age of 50 years, the number of missing teeth was also similar in both groups. Malmstr0m & Calonius (1975) examined 126 RA patients and observed both an increased frequency of edentulousness and more missing teeth in dentate RA patients than in the control group. The questions asked in the present study were as follows: Is the loss of teeth and loss of periodontal bone higher in individuals with rheumatoid arthritis than in patients free from the disease? Is alveolar bone loss in RA correlated with specific antibodies to suspected periodontal pathogens or with the level of serum immunoglobulins? Material and Methods
'
Patients
37 patients diagnosed with RA according to the criteria set by Ropes et al. (1971) were studied. The patients were hospitalized at Bethanien Hospital, Skien, Norway for a period of 9 months. Edentulous patients and patients diagnosed with Sjogrens disease were excluded. AH other patients were included.
The mean duration of diagnosed RA was 13 years. All patients had received anti-inflammatory drugs for extended periods and 11 patients had also received cytostatica (Prednisone, Imuran, Methotrexate). A sex- and age-matched control group of 37 patients was drawn by paired matching from patients registered at the Dental Faculty Clinic, University of Oslo. Patients with rheumatoid arthritis and edentulous patients were excluded. Radiography
A Siemens Heliodent X-ray apparatus set at 60 kv and 7 mA, with a 1.6 mm AL-filter and Eggens film holder (Eggen, 1969). 10 intraoral, Kodak D-58 films were exposed per patient according to standard ej(posure times. The films were automatically processed at the Department of Radiology of the Faculty of Dentistry, University of Oslo. Loss of periodontal bone was recorded on the approximal surfaces of the front teeth in the upper and lower jaw. The distance from the cemento-enamel junction (CEJ) to the alveolar bone was measured and expressed as a % of the distance from the CEJ to the root apex. The measurements were made by an examiner who was not informed of the group assignment of the patient.
Periodontilis and rheumatoid arthritis phosphatase conjugated class specific antiserum (Orion Diagnostica, Helsin10 ml blood was obtained from each ki, Finland) and reaction with p-nitropatient and the serum was stored at phenol disodium phosphate (Sigma). — 20° C. Total serum immunoglobulins The test was accepted as valid if the were measured by single radial immuno- coefficient of variation did not exceed diffusion in Nor-partigen gels (Behring, 10%. 6 wells on each plate were incuMarburg, West Germany). The rheuma- bated with the reference serum and the toid factor was evaluated by a modified individual OD-readings were adjusted Waalers test (Waaler 1939) using rabbit for plate-to-plate variations according antiserum with human erythrocytes and to the formula: by latex agglutination (Behring). Specimean OD of individual serum - background fic activities of the serum immunoglobmean OD of reference serum - background ulins G, A and M against 3 suspected periodontal pathogens were measured The data were recorded as ELISA units, by enzyme-linked immunosorbent as- and the reference serum was assigned a says (Tolo and Schenck, 1985). Pure cul- value of 100 EU at an equal dilution tures of Bacteroides gingivalis, Capnocy- for each isotype. To obtain equality of tophaga ochracea and Euhacterium sa- variances, all immunological data 0') burreum were established from samples were transformed as y' = ln y before obtained from patients at the Depart- statistical analyses. Group differences ment of Periodontology, University of were analyzed by the Student /-test, 2Oslo as described by Tolo & Schenck tailed probability. The data were sub(1985). The cultures were grown to late jected to multiple regression analysis. exponential phase, centrifuged at 10,000 Loss of alveolar bone was selected as g^ for 10 min, the supernatants decanted, the dependent variable, and age, serum dialyzed for 3 days against running tap water and lyophilized. The powder was level of IgG, diagnosis of RA, antidissolved (100 mg/ml) in deionized bodies to B. gingivalis and E. saburreum water, applied on a Sephadex G 75 col- were entered stepwise into the equation. umn (100x2.5 cm) and eluted with The independent variables were entered water containing 0.2 g/1 sodium azide. according to the strength of bivariate The optical density of the eluate was correlation. The level of statistical sigmonitored at 280 nm and the fractions nificance was set at/I < 0.05. corresponding to the void volume were pooled, lyophilized and stored at —20° Results C. Dental status Immunological tests
The fractionated bacterial components were dissolved (1 /^g/ml) in 0.15 M phosphate buffered saline (PBS) pH 7.3, and 200 /il of the solution were applied per well in polystyrene microtiter plates (Dynatech MicroElisa, Greiner 129 B). A reference serum was prepared by pooling sera from 10 patients with periodontitis. The reference serum was positive for all 3 bacterial extracts (binding of IgG > mean + 2 SD obtained with serum from healthy individuals). Standard curves were established by testing serial dilutions of the reference serum. The binding of human Ig to the respective coatings was reduced by 70% to 90% by addition of homologous bacterial extract to the reference serum. The binding observed after maximal inhibition was considered equal to the background level of the assays. The serum samples were diluted 1:400 in phosphate buffered saline (PBS), pH 7.3 containing 5 mg/ml bovine serum albumin and tested in triplicate. The OD was measured after incubation with alkaline
The mean number of teeth was 17.9 in the RA-group and 22.6 in the control group. The mean loss of bone was 23.8% in the RA-group and 18.9% in the control group (Table 1). Serum Immunoglobulins
Serum IgG and IgA were significantly increased in the RA-patients compared to the control group. No group differences were observed with regard to serum IgM.
289
Table 2. Multiple regression analyses of data from 74 patients, 37 diagnosed with rheumatoid arthritis and 37 controls % of variation accounted for age age + Dia age + Dia-(-IgG B.g age-fDia + IgG B.g + IgG-E.s
14 20 24 26
Dependent variable = loss of alveolar bone. Variables entered stepwise into the equation: age, diagnosis (Dia), igG-antibody to B. gingivalis (IgG B.g) and E. saburreum (IgG E.s).
Multiple regression analyses
In the combined group of 74 individuals, age, diagnosis of RA, and IgGantibodies to B. gingivalis and E. saburreum accounted for 26% of the variation in loss of alveolar bone (Table 2). In the RA group, 48% of the variation in bone loss was accounted for by age, IgG antibodies to B. gingivalis, IgGantibodies to E. saburreum and total serum IgG (Table 3). Discussion
The study shows that patients diagnosed with rheumatoid arthritis had lost more teeth than patients with no RA. The mean number of teeth present in the control group was similar to that observed in a random sample of 194 individuals from the same district (Lervik et al. 1988), and this indicated that the control group was a representative sample of the population with regard to loss of teeth. Interestingly, the mean number of teeth present in the control group was also similar to that observed in employed, dentate persons in USA (NIH publication 87-2868). The RA group had a mean of 17.9 teeth present in dentate patients (mean age = 53 years). This is in accordance with Laurell (1985) who observed a mean of 18.1
Table 3. Multiple regression analyses of data from 37 patients diagnosed with rheumatoid arthritis Table I. Number of teeth and loss of alveolar bone
RA (n = 37) Ctrl (/i = 37)
Teeth
Bone loss
I7.9"'±8.9 22.6 ±5.8
23.8'"±12.2 18.9 + 7.9
"' Significantly decreased compared to controls. ''' Significantly increased compared to controls.
% of variation accounted for age + IgG B.g age-t-IgG B.g + SerIgG age + IgG B.g + SerIgG + IgG E.s
15 38 44 48
Dependent variable = loss of alveolar bone. Variables entered stepwise into the equation: Age, IgG-antibody to B. gingivalis (IgG B.g) IgG antibodies to E. saburreum (IgG E.s.) and serum concentration of IgG (SerIgG).
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Kdre Tola & Lars Jorkjend
teeth present in a group of dentate RApatients with a mean age of 55 years. Accidents, caries, periodontitis and extractions associated with orthodontic treatment may have contributed to the loss of teeth. However, the observation of increased bone loss around the remaining teeth in the RA patients indicated that periodontitis may be an important cause of tooth loss in these patients. The multiple regression analyses indicated that age was the most important variable with regard to loss of alveolar bone, and accounted for about 14% of the variation. The second most important variable in the combined groups was diagnosis of RA. Further by taking into account the level^s of antibody against 2 suspected periodontal pathogens, the proportion of "explained variation" was increased to 26%. This proportion was not improved by the addition of the remaining variables into the multiple regression equation. In the RA group, the loss of alveolar bone was significantly correlated with age, IgG antibody against B. gingivalis and by the level of total serum IgG. The rheumatoid factor may have contributed to the increased proportion of "explained variation" in this group, since formation of IgG-complexes with rheumatoid factor may contribute both to increased ELISA readings and to the inflammatory activity in the periodontal lesion. 11 RA patients had high titers of rheumatoid factor (range 128-4096) and 19 patients had titers in the range 4-128. Data on the rheumatoid factor was lacking for 7 patients, and this factor was therefore not entered into the multiple regression analysis as an independent variable. Activity consistent with rheumatoid factor was observed in chronically inflamed gingiva by Kristoffersen & Tender (1973), and Malmstrom & Natvig (1975) observed that 25%-50% of the plasma cells in dental periapieal lesions in RA patients were positive for rheumatoid factor. Gargiulo et al. (1982) observed latex agglutination, indicating the presence of rheumatoid factor in dental plaque, saliva and extracts of gingiva in patients with periodontitis, and suggested that this faetor may contribute to chronic periodontal inflammation. Experimental data from mice (Lens & Beertsen, 1986) indicated that RAlesions can be influenced by injections of antigens into the oral mucosa. In periodontitis, the area of crevieular epi-
thelium exposed to the subgingival microbiota is substantially increased, and immune reactions will contribute to the permeability of the crevieular epithelium (Tolo et al. 1978). Penetration of bacterial products with a potential for B-cell activation in inflammatory infiltrates may therefore occur more frequently in periodontitis. The significant correlation between bone loss and IgGantibodies against 2 suspected periodontal pathogens in the RA patients is interesting, since it indicates the crevieular epithelium as a port of entry for Bcell activating agents. Among bacterial products reported to act as B-cell activators in RA are bacterial peptidoglycans (Pardo et al. 1984). Increased blast responses and polyclonal B-cell activation to extracts of dental plaque were observed in several studies of peripheral lymphocytes from patients with periodontitis (Patters 1976, Smith et al. 1979, Bick et al. 1981, Donaldson et al. 1982, Osterberg et al. 1983). The observations of the present study indicate that patients with RA may be regarded as a risk group with regard to periodontitis. Further studies are required to examine whether periodontitis may have a negative effect on RA. Zusammenfassung Scniimmlikdrper und parockmtaler Knochenschwund bci Patienlen mil rheumatischer Arihrill.s Bei 37 Palientcn mit rheumatischer Arthritis (RA) und einer, hinsiclitlich Alter- und Geschlechtszugehorigkeit vergleichbaren Kontrollgruppe, wurdc die Zahl vorhandener Zahne, der prozentuelle Knochenschwund, das Serum IgG und die Serumantikorper gegen Bacteroides gingivalis, Capnocytophaga ochracea und Eubacterium sahurreum registriert. Im Vergleich zu den KontroUprobanden kam bei der RA-Gruppe signifikant hoherer Zahnverlust und alveolarer Knochenschwund vor. Bei den RA-Patienten wurde ausserdem ein signifikant hoheres Serum IgG-Niveau festgestellt. In dem gesamten Probandengut wurden 26% des unterschiedlichen alveolaren Knochenschwunds dem Alter, der Diagnose "rheumatische Arthritis" und den Antikorperniveaus gegen B. gingivalis und E. saburreuni zugeschrieben. In der RA-Gruppe waren 48% dieser Variationen eine Folge des Alters, der totalen Serum IgG und der IgG Antikorper gegeniiber B. gingivalis und E. saburreuni
Resume Anticorps seriques el perte osseuse parodontale chez des patients souffrant d'arthrite rhuma-
lo'ide Lc nombre de dents, le pourcentage de perte osseuse alveolaire. Fig G et les anticorps seriques contre lc Bacteroides gingivalis, le Capnocytophaga ochracea et VEubaeterium saburreuni ont ete analyses chez 37 patients souffrant d'arthrite rhumatoide (RA) ainsi que chez 37 autres individus sans RA formant un groupe controle avec age et sexe as.sortis. Le groupe RA avait une perte dentaire et une perte osseuse significativetiient superieures a celles rencontrces dans le groupe controle. Les patients RA avaient egalement un taux d'lg G serique significativement plus important. Dans l'ensemble, 26% de la variation de perte d'os alveolaire otaient expliques par I'age, le diagnostic d'arthrite rhumatoide et les taux d'anticorps contre le B, gingivalis et VE. saburreuni, Dans le groupe RA, 48% de cette variation etaient expliques par I'age, l'lg G serique totale et les anticorps Ig G contre le B, gingivalis et VE, saburreuni.
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edentulous subjects. Infection and Immunity 14, 1213-1220. Ropes, M. W., Bennet, G. A., Cobbs, S., Jacox, R. & Jessar, R. A. 1958 revision of diagnostic criteria for rheumatoid arthritis. Bultelin of Rheumatoid Diseases 9. 175-176. Seymour, G. J. (1987) Possible mechanisms involved in the immunoregulation of ehronic periodontal disease. Journat of Dental Research 66, 2-9. Smith, F. N. & Lang, N. P. (1977) Lymphocyte blastogenesis to plaque antigens in human periodontal disease. 11. The relationship to clinical parameters. Journal of Periodontal Research 12, 310-317. Snyderman, R. & McCarty, G. A. (1982) Analogous mechanisms of tissue destruction in rheumatoid arthritis and periodontal research. In Hosi-parasite interactions in periodonlat disease (ed. R. J. Genco & S. E. Mergenhagen) pp. 354-362. American Society for Microbiology, Washington D. C. Talal, N. (1985) Interleukins, Interferon and Rheumatic Disease. Clinics in Rheumatic Diseases II, 633-644. Tolo, K., Brandtzaeg, P & Jonsen, J. (1978) Mucosal penetration of antigen in the presence or absence of serum-derived antibody.
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An in vitro study of rabbit oral and intestinal mucosa. Immunology 33, 733-743. Tolo, K. & Schenck, K. (1985) Activity of scrum immunoglobulins G, A, and M to six oral bacteria in diagnosis of periodontitis. Journal of Periodonlal Re.search 20, 113-121. Waaler, E. (1939) A factor in human serum activating the specific agglutination of sheep blood corpuscles. Third International Congress for Microbiology, New York 1939. Abstract of communication p. 351. 0sterberg, S. K., Page, R. C , Sims, T. & Wilde, G. (1983) Blastogenic responsivetiess of peripheral blood mononuclear cells from individuals with various forms of periodontitis and effects of treatments. Jourtial of Clinical Periodontologv 10, 72-88. Address: Kdre Tolo Ktinikk for Periodonti Det Odonlologiske Fakuliet Posthoks 1109 Blindern Oslo 3 Norge
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