Increased periodontal bone loss in temporarily B lymphocyte-deficient rats

Increased periodontal bone loss in temporarily B lymphocytedeficient rats

Bjarne Klausen, Hans Petter Hougen and Nils-Erik Fiehn Departments of Microbiology, Periodontology and Oral Diagnosis, Royal Dental College; Barthotin Institute, Kommunehospitaiet; University Institute of Forensic Medicine, Copenhagen, Denmark

Klausen B, Hougen HP, Fiehn N~E: Increased periodontal bone loss in temporarily B lymphocyte-deficient rats. J. Feriodont Res 1989; 24: 384-390. In order to study the role of T lymphocytes and B lymphocytes in the development of marginal periodontitis. experiments were performed on specificpathogen-free (SPF) rats with various immunologic profiles. The study comprised nude {congenitally T lymphocyte-deficient), thymus-grafted nude (T-!ymphocyte reconstituted), anti-/j treated (temporarily B lymphocyte-deficient), and normal rats. When they were 5 to 9 weeks old, some of the rats were inoculated with Actinomyces viscosus. Bacteroides gingivalis and a strain of oral spirochetes; 90 days later they were killed. A. viscosus and B. gingivalis colonized readily, but the spirochete strain could not be established permanently in the oral eavity of the rats. 95% of the inoculated rats raised serum IgG or IgM antibody against one or more of the microorganisms; nude rats tended to raise less antibody than the three other experimental groups. Inoculated rats had significantly less periodontal bone support than controls. Anti-/i treated inoculated rats had significantly less periodontal bone support than nude and normal rats, whereas no difference was found between normal, nude, and thymus-grafted rats. It is concluded that permanent T-lymphocyte deficiency did not interfere with the development of periodontal disease in this model, whereas a temporary and moderate reduction in B-lymphoeyte numbers seemed to predispose for aggravation of periodontal bone loss.

Introduction

In order to study the role of the immune system in the development of marginal periodontitis, several experiments have been performed on immune-deficient rats. General immune suppression has been achieved by whole-body-irradiation (1) or cyclophosphamide treatment (2, 3). Specific T-lymphocyte suppression has been studied in rats after cyclosporin A treatment (4), after neonatal thymectomy (5, 6), and in congenitally athymic nude rats (6, 7. 8). So far, periodontal reactions in specifically B lymphocyte-suppressed rats have not been studied; however, it is possible to suppress B lymphocytes in rats by treatment with anti-/i chain serum (9). Previous studies indicated that general immune suppression per se was not associated with periodonta] bone loss, but immunocompromised hosts seemed more susceptible to opportunistic infections causing rapid periodontal destruction (2, 3). Results of experimental periodontitis in T lymphocyte-deficient rats are somewhat conflicting. Some investigators found that nude and neonatally thymectomized rats developed more periodontal bone

Accepted for publication July 10, 1989

loss than normal (euthymic) rats and thymus cell reconstittited nude rats in response to their indigenous flora alone or superinfected with Actinobacillus actinomycetemcomitans (6, 7). However, in other studies normal and neonatally thymectomized rats reacted similarly to intragingival injections of streptococcal cell wall fragments (5), normal and cyclosporin A-treated rats developed similar bone destruction after monoinfection with Actinomyces viscosus (4), and normal and nude rats developed similar bone loss after monoinfection with Streptococcus mutans (8). One reason for this discrepancy could be that various bacteria affect various parts of the immune system differently; therefore further experiments on immune-deficient rats seem warranted. The purpose of the present investigation was to study periodontal bone loss and serum antibody reactions to a mixture of periodontal pathogens in specific-pathogen-free (SPF) normal ( + I + ) , nude (mu/mu), thymus reconstituted nude, and anti-^ treated +1+ rats. The pathogens were Actinomyces viscosus, Bacteriodes gingivalis, and a recently described strain of an oral spirochete with two endoflagella from each cell end. A. viscostis is as-

Bone loss in B cell-deficient rats sociated with human gingivitis development (10), induces periodontal bone loss in germfree and conventional rats (11, 12, 13), and enhances the adherence of ,8, gingivalis on tooth surfaces (14), B. gingivalis is associated with adult periodontitis and rapidly progressing adult periodontitis (15, 16), induces periodontal bone loss in germfree and conventional rats (13, 17), and produces a number of potential virulence factors (16, 18), Spirochetes constitute a considerable part of the flora in deep pockets (19), and high proportions of spirochetes in subgingival plaque seem positively correlated to active periodontal breakdown (20); the periodontally derived strain (21) used in this study synthesizes several tissue-damaging enzymes that may be important in the development of periodontal disease (22), Material and methods Animals

A total of 73 SPF male and female RTl' LEW/ Mol rats were used. Four -I-/-I- and 5 anti-// treated + / -I- rats were killed when 5 wk old without being inoculated. In Experiment 7, 6 rnu/rnu, 11 -I- / -t- and 6 anti-// treated -H / -I- rats were inoculated when they were 5 wk old and killed 90 d later; 8 rnu/rnu, 5 +/-I- and 4 anti-// treated + / + rats served as age-matched SPF controls. In Experiment 2, 4 rnu/rnu, 7 -I-/-I- and 8 thymus-grafted rnu/rnu rats were inoculated when they were 9 wk old and killed 90 d later; 5 rnu/rnu rats served as age-matched SPF controls, Anti-// treatment was administered by i,p, injections of 1;5 diluted sheep-anti-rat-IgM (ICN Immunobiologicals. Lisle, II 60532, USA) thrice a week from birth until the age of 5 wk. The doses were; 1, wk, 0,1 ml; 2,-3, wk, 0,2 ml; 4,-5, wk, 0,3 ml, Thymus grafting was performed on 4 wk old rnu/rnu rats by placing one neonatal isogeneic thymus gland subcutaneously on the left flank of the animals (23), At the termination of the experimental period, all animals were anesthetized with Brietal* and exsanguinated, Microbjology procedures

The indigenous oral flora of anesthetized 5 wk old SPF rats was assessed by examining dental plaque samples from the gingival margins (obtained by means of a curette) and oral swabs from the whole oral cavity. The samples were transferred to peptone yeast medium (PY) (24), 10-fold diluted in PY, and inoculated on enriched trypticase soy agar plates with 5% blood, 0,05 mg/ml menadion and

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0,5 mg/ml hemine. After 6-7 d, colonies from a selected dilution were examined morphologically on Gram-stained smears. For experimental periodontitis studies rats were inoculated with a mixture of Actinomyces viscosus ATCC 19246, Bacteriodes gingivalis ATCC 33277, and an oral spirochete strain with two endoflagella from each cell-end (21), A. viscosus and B. gingivalis were cultivated in fluid PY with hemine and menadione (24), and the spirochete isolate was cultivated in fluid brain heart infusion broth with rabbit serum and cocarboxylase (BHI-SC) (21), The cells of the cultures were harvested by centrifugation in the late logarithmic phase of growth, washed two times in PBS, and the cell density was adjusted to approximately 10^ cells/ml PBS. One ml of each strain was inoculated by peroral administration daily for 3 consecutive d. The spirochete strain was reinoculated by similar procedures 8 wk after the first inoculation. In order to verify the presence of the three bacterial strains, dental plaque samples and oral swabs were obtained 2 wk after the first inoculation and at the termination of the experiments. For detection of spirochetes the samples were examined in phase-contrast microscopy (xl200). For detection of ^, viscostis cultivation was carried out on selective medium containing the inhibitory components cadmium sulfate, sodium fluoride, neutral acriflavin and potassium tellurite (CFAT) (25); for detection of B. gingivalis cultivation was carried out on B. gingivalis agar (BGA), which contains bacitracin, colistin and nalidixic acid as selective agents (26); the identification was based on colony morphology and Gram-stained smears. Post-mortem measurements

Frozen sections of spleen and inguinal lymph nodes were stained with the monoclonal antibodies OX6 (B lymphocytes), OX-8 (T non-helper lymphocytes) (both Serotec, Blackthorn, Oxon, UK) and W3/25 (T-helper lymphocytes) (Sera-lab, Crawley Down, Sussex, UK) using an immunoperoxidase technique (27), OX-6* lymphocytes were counted in B lymphocyte compartments, OX-8* and W3/ 25+ in T-lymphocyte compartments of the organs. Serum IgG and IgM class antibodies against the three inoculated organisms were detected by indirect immunofluorescence microscopy (8), Periodontal bone support was assessed on magnified radiographs, Distaily to the first mandibular molar the ratio apex-deepest bony defect;apex-cusp top was measured with a slide gauge. In a previous study similar ratios were measured mesially and distaily to all mandibular molars (8), but a critical review of the data revealed that no essential infor-

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Table I. Effect of anti-/i treatment on B-lymphocyte numbers in spleen and inguinal lymph node % 0 X - 6 ^ cells in B cell areas Animals n spleen lymph node Anti'fi, 5 weeks old 5 82.0±2.7 a 62.4 + 7.9 a Control, 5 weeks old 4 9L0±2.0 90.8 + 3.8 Anti-fi, 4 months old 6 93,4 ± 2.6 ns 89.7 ± 1.6 ns Control, 4 months old 10 93,2 + 3.5 86.3±6.0 a: significantly different from age-matched control, p