Periodontal Bone Loss and Immune Response to Ovalbumin in ...

Vol. 32, No. 1

INFECTION AND ImMUNITY, Apr. 1981, p. 145-152 0019-9567/81/040145-08$02.00/0

Periodontal Bone Loss and Immune Response to Ovalbumin in Germfree Rats Fed Antigen-Free Diet with Ovalbumin MARTIN A. TAUBMAN,* JULIA M. BUCKELEW, JEFFREY L. EBERSOLE, AND DANIEL J. SMITH Department of Immunology, Forsyth Dental Center, Boston, Massachusetts 02115

A technique for the characterization of rat gingival lymphocytes has been described. The technique was used to obtain gingival cells from rats maintained on antigen-free diets or such diets with ovalbumin (OVA) added. Increases in gingival lymphocyte numbers in the antigen-fed (AF) animals occurred by 16 to 23 days of OVA feeding. The elevated gingival lymphocyte numbers were predominantly T lymphocytes at the initial intervals of the experiment (to 59 days of OVA feeding). At 128 days of OVA feeding T-lymphocyte numbers diminished but B-lymphocyte numbers increased, and AF animals had more than six times as many gingival B lymphocytes as animals not fed antigen. Also, AF animals showed immunoglobulin A antibody in intestinal perfusates (after 9 days of OVA feeding) and in saliva (within 23 days of OVA initiation). Plasma immunoglobulin G antibodies were not detected until 59 days of feeding. Spleen cells from AF rats showed in vitro blastogenic responses to OVA at 23 to 59 days of feeding. Periodontal bone loss was greater in AF animals after 59 and 128 days of OVA. Germfree animals fed only one antigen experienced more periodontal bone loss than animals fed the same diet not containing antigen. Therefore, immune phenomena can contribute to experimental bone loss in germfree rats. A consistently observed feature in human periodontal disease has been the presence in the periodontal tissues of a dense inflammatory infiltrate consisting almost totally of plasma cells and lymphocytes (30,36,37). This inflammatory component is also associated with the very early stages of gingival disease (32). There is substantial evidence that human B lymphocytes (14, 18) and T lymphocytes (19, 22) are sensitized to various products of oral microorganisms, suggesting that these products gain access to immunocompetent cells. Subsequent immune reactions may then contribute to the pathogenesis of periodontal disease (20). Germfree and gnotobiotic rodents have been used as model systems for periodontal disease (15, 23, 40, 42). When these animals are confronted with a massive antigenic challenge after infection with certain microorganisms, they demonstrate considerable increases in periodontal bone loss (9, 42). However, rats and mice

maintained in the gernfree state also demonstrate some bone loss (3, 4, 9, 42). Although such animals are not exposed to live bacteria, an antigenic challenge from diet and bedding still exists (47). The bedding challenge can be eliminated by maintaining animals on suspended caging. Furthernore, liquid diets are available which do not contain antigenic components (33, 34). 145

The experiments described in this paper were based on the premise that the introduction into rats of material (ovalbumin [OVA]) of known antigenic properties (35) and of approximate size to penetrate gingival tissues (29) could result in gingival cell sensitization and periodontal bone loss in rats. We demonstrate a gingival response and the presence of a spleen cell blastogenic response to the antigen (OVA). MATERALS AND METHODS Animals. Animals used in this experiment were germfree Sprague-Dawley rats (CD strain; Charles River Breeding Laboratories, Inc., Wilmington, Mass.). Animals were housed in plastic isolators (9) on suspended caging. Rats were fed liquid, antigen-free diet L-489E8a (GIBCO Laboratories, Grand Island, N.Y.), described by Pleasants et al. (34). Gamma globulin levels from rats maintained on this diet have been shown to be lowered by at least 80% (34). Experimental protocol. Twenty-six germfree weanling rats from three litters were randomly distributed into two separate groups in each of two plastic isolators. All animals were fed liquid, antigen-free diet L-489E8a from 23 days of age. One milligram of 5xrecrystallized egg albumin (OVA; Sigma Chemical Co., St. Louis, Mo.) per ml was added to the diet of one group of animals (antigen fed [AF]). The second group was not fed antigen (NAF). Two to four animals from each group were removed from the isolators 9, 16, 23, 59, and 128 days after antigen-supplemented diet was initiated. At these intervals, animals were exsanguin-

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ated and gingival tissues were removed for cell counts. Spleens were removed, and spleen cells were prepared individually for culture and determination of blastogenic responses. Preparation and specificity of anti-lymphocyte reagents: anti-thymocyte reagent (anti-T). Rabbit anti-rat thymocyte globulin (Microbiological Associates, Bethesda, Md.) was fluoresceinated as described by Cebra and Goldstein (7) to give a molar fluorescein/protein ratio of 3.3. This reagent was adsorbed with rat erythrocytes, bone marrow cells, peritoneal exudate cells (5, 16), and finally, bone marrow from neonatally thymectomized rats (11). Bone marrow cells were obtained by expressing plugs of marrow from the femurs of exsanguinated rats with Alsever solution. Peritoneal exudate cells, obtained from rats injected intraperitoneally with 3 ml of 12% sodium caseinate per 100 g of body weight, were harvested 48 h later after flooding the peritoneal cavity with 20 ml of phosphate-buffered saline (PBS). Cell preparation and immunofluorescence assay. Thymocytes, lymph node cells (mesenteric lymph node), splenocytes, and bone marrow cells were washed twice in cold Tyrodes solution and resuspended in PBS at a concentration of 2 x 107 cells per ml. Whole blood was collected into preservative-free heparin (GIBCO Laboratories), and the cells were centrifuged into a Ficoll-Hypaque gradient (6). Mononuclear cells were removed from the interface and washed. Living cells (approximately 106) were suspended in 0.05 ml of PBS with 0.05 ml of serially diluted anti-T in a U-shaped well of a microtiter tray (Linbro Scientific Co.) and incubated for 20 min at 40°C. The cells were washed in the tray three times with ice-cold PBS, and samples were examined by light-field fluorescence microscopy. The anti-T did not react with rat immunoglobulins when tested in gel diffusion analyses. The specificity of this reagent was further tested by determining the percentages of positively staining cells in suspensions from various rat tissues (Table 1). Percentages of positively stained cells obtained are comparable to the published values for these organs (16). Testing of the T-lymphocyte reagent with lymphoid cells from rats that were neonatally thymectomized (11) showed depletions in Tcell numbers in the spleen, peripheral blood, and bone marrow (Table 1). This reagent was also tested in two other ways. Rat spleen cells were applied to successive columns of glass wool and nylon wool according to the method of Julius et al. (24). Eighty-one to 94% of the spleen cells that passed through the column reacted with the anti-T. Spleen cells were also subjected to separation on a rabbit anti-rat L-chain adsorbent column prepared as described by Chess and co-workers (8). Eighty-eight to 96% of the cells that were not retained on this column reacted with the anti-T. Anti-B-lymphocyte reagent (anti-Fab). A reagent directed to the Fab fragment of rat immunoglobulin G (IgG) has been reported to optimally react with rat B cells (21, 45). Fab fragments of rat IgG were prepared by papain digestion, gel filtration (Sephadex G-100 [Pharmacia Fine Chemicals, Piscataway, N. J.]), and ion-exchange chromatography (diethylaminoethyl cellulose; 2). Rabbits were immunized with

INFECT. IMMUN.

TABLE 1. Characterization of fluorescein isothiocyanate-anti-T-Cell globulin % Positive staining cells

Mean ± standard error (n) Tissue

Thymus Lymph node Spleen Peripheral blood Bone marrow

From reference 16 (range)

Normal rats

99 79-85 -50 58-65

97 ± 2 (4) 80 ± 6 (5) 56 ± 9 (6) 64 ± 4 (9)

8-15

11 ± 1 (4)

Neonatal thymectomy _a

NTb 9 ± 1 (5) 13 ± 1 (2) 0 (2)

a_ Thymus removed. b NT, Not tested. the purified Fab fragments, and the antisera were adsorbed with the insolubilized Fc fragment of rat IgG. The sera react with rat serum immunoglobulins, but not with Fc fragments of rat IgG, in gel diffusion and immunoelectrophoretic analyses. Globulin (50% ammonium sulfate) from these sera was fluoresceinated and prepared (7) to give a fluorescein/protein ratio of 3.2. This reagent (tested by light-field fluorescence microscopy as described below; 25, 26) did not stain rat thymocytes but stained 16 to 18% of rat mesenteric lymph node cells (16). The anti-Fab also stained 75 to 88% of rat spleen cells that bound to an anti-rat lightchain adsorbent column and only 3 to 8% of cells that did not bind (8). Thirty-three percent of the spleen cells which bound to a nylon wool column (24) stained with the anti-Fab, and only 6 to 7% of the unbound cells stained with this reagent. Gingival tissue. Dissection was performed under a stereomicroscope (Bausch & Lomb, Inc., Rochester, N.Y.) equipped with a reticule eye piece (one grid square = 1 mm). The gingivae around the mandibular and maxillary molars were separated from the underlying tissue by cutting approximately 1 mm from the gingival crest after positioning the mandible or maxilla with the teeth facing upward. Then the gingival tissue segments were excised from attachment by cutting with a scalpel parallel to the tooth surface. Preliminary experiments indicated that the weights of these segments removed from AF or NAF animals (69, 99, 130 days of age) did not differ significantly at each day (mean weight at 69 days of age, 49.9 mg; at 130 days, 69.3 mg). After washing to remove all visible blood, these segments were expressed through 60-mesh stainless-steel screens and then through an 80-mesh copper sieve to obtain a single cell suspension in 2 ml of PBS. Less than 1% of the lymphocytes were retained on the screens. The viability of cells prepared in this manner was greater than 64% for all types of cells (see below) and more than 95% for lymphocyte-like cells. The gingival cells were counted in a hemacytometer, and the percentage of lymphocytes relative to total gingival cells was determined from Giemsa-stained smears (300 or more cells were counted). The percentage of lymphocytes in these experiments varied from 1.6 to 14.3. Typical preparations also contained: epithelial cells (56.3 to 73.8% of total cells), fibroblasts (13.9 to

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OVA-FED RATS EXHIBIT PERIODONTAL BONE LOSS

20.7%), erythrocytes (0 to 12.7%), and few if any other cell types (polymorphonuclear neutrophils, monocytemacrophage-like cells, and plasma cells, 0 to 7%). Since neutrophils constitute 18 to 36% ofthe total leukocytes of rats (17), the low neutrophil numbers suggested that the cell recoveries did not represent blood contamination. Also, the percentages of lymphocytes were higher by a factor of 10 or 100 than would be found relative to other cellular components in blood. Therefore, the cells recovered seem to be reflective of the gingival cell composition, and blood contamination could, at most, only account for 10% of the cells recovered. Portions of the initial cell suspension (50 pil) were then washed and directly stained with the anti-T and anti-Fab. The remaining cells were centrifuged into a Ficoll-Hypaque gradient (3 ml; Pharmacia). The mononuclear cells were removed from the interface and washed, and aliquots were stained with either the anti-T or the anti-Fab. Cells were stained for 20 min at 0 to 30C and washed three times with PBS containing 5% bovine serum albumin at 370C to eliminate cytophilic immunoglobulin and binding of the antiFab via Fc receptor-bearing cells (25, 26). Duplicate cell preparations were made for each reagent, using a cytocentrifuge (Shandon) followed by 95% ethanol fixation. Four slides were prepared for each gingival sample with each reagent and were observed with a Leitz fluorescence microscope equipped with a Ploem vertical illuminator, a high-pressure mercury vapor lamp, and excitation filters BG 38 and BG 12. Identification of small lymphocytes was made by using cell size and sharply delimited nuclear membrane as the primary positive criteria (46). All cells were thus identified as lymphocytes by morphological criteria, and those showing rim or more dffuse surface staining were considered to be positive T or B cells. These celLs were subsequently confirmed as lymphocytes after cover slip removal (in 95% ethanol) and Giemsa staining. The combined procedures enabled deternination of the number of total gingival cells, percentage of gingival lymphocytes, total gingival lymphocytes, gingival T lymphocytes, gingival B lymphocytes, and negative (nonstaining) lymphocytes. Lymphoid cell preparation and blastogenesis. Spleens were aspetically removed, and splenocytes were separately prepared for culture and determination of blastogenic responses by expression through stainless-steel mesh. Erythrocytes were lysed with 0.83% NH4Cl buffered to pH 7.5 with tris(hydroxymethyl)aminomethane and then washed in RPMI 1640 (GIBCO Laboratories). Lymphoid cells (2 x 105) from each individual spleen were resuspended in wells of microculture plates (COSTAR, Rochester, N.Y.) and cultured in RPMI 1640 with 10% heat-inactivated fetal calf serum (GIBCO Laboratories) or with 10% autologous plasma and antibiotics (100 mg of streptomycin per ml, 100 U of penicillin per ml). An optimal concentration (0.5 pig) of OVA was then added to experimental cultures, and plates were incubated at 37°C in a humidified 5% C02-95% air atmosphere. Tritiated thymidine (0.2 PCi; New England Nuclear Corp., Boston, Mass.; specific activity, 6.7 Ci/mmol) was added to each well 6 h before harvesting, with an automated collecting device (Brandel, Rockville, Md.),

147

on days 5, 6, and 7. The stimulation indices were determined relative to identical cultures not containing OVA. All cultures were performed at least in triplicate. The stimulation index was calculated according to the following equation: counts per minute in stimulated cultures/counts per minute in unstimulated cultures = stimulation index, where in each case the counts per minute are corrected for background radiation. Stimulation indices exceeding 2.5 were significant at the 95% confidence level as determined by normal distribution and a pooled estimate of variance (31). Antibody determinations. Antibody in saliva (collected and prepared as previously described [43]), plasma, and intestinal perfusates (10) was determined by a modification of the indirect enzyme-linked immunosorbent assay (13) that we have previously described (12). A 3-gug portion of OVA was attached to the wells of microtiter plates and reacted with antiOVA antibodies in the saliva, plasma, or intestinal perfusates. Reactions were developed with monospecific rabbit anti-rat a- or y-chain globulin (44) followed by goat anti-rabbit gamma globulins (CalbiochemBehring Corp., La Jolla, Calif.) conjugated to alkaline phosphatase (Sigma type VII). The potential existence of antibody to diet components was tested for in all rat sera by gel diffusion analyses against various concentrations of liquid diet. No precipitating antibody was ever detected. Bone loss. The distance from the cemento-enamel junction to the alveolar margin was measured under x25 magnification with a reticule eyepiece. Recordings were made in the long axis of the buccal and lingual root surfaces of all molar teeth with careful superimposition of buccal and lingual cusps to insure reproducibility, as previously described (9). The total horizontal bone loss (no vertical bone loss was detected) was determined without prior knowledge of the group designation of the animal.

RESULTS Gingival celIs. The sums of all gingival cell types recovered from animals that were AF or NAF were determined during a 128-day experimental interval. This comparison is shown in Fig. 1. Although there is a tendency for the AF animals to demonstrate somewhat higher gingival cell counts, the differences are only significant 128 days after feeding has been initiated. On the other hand, the gingival lymphocyte levels in the AF animals were consistently elevated by 16 to 23 days when compared with animals not fed OVA. These data suggested an influ2x of cellular components into the gingival tissues which appears to be related to antigenic stimulation with OVA. The characterization of these gingival lymphocytes, as determined by immunofluorescence, is shown in Fig. 2. After 9 days of antigen feeding no major differences between the means of the T- and B-cell numbers could be discerned in the gingival tissues of AF or NAF animals. However, after 16 or 23 days of antigen feeding,

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INFECT. IMMUN. 150 r

TOTAL GINGIVAL

(x

OCYTB

100 I

TOTAL GINGIVAIL CELLS

(x

106)

15

10

50

9

16

23

59

128

9

16

DAYS

59

2

128

DAYS

FIG. 1. Recovery of total gingival cells and gingival lymphocytes from AF rats (solid bars) and NAF rats (open bars). Bars indicate the means of the total cells or lymphocytes recovered from two to four animals from each group at each time interval after OVA supplementation ofthe diet. Brackets enclose two standard errors. Differences in total gingival cells and in gingival lymphocytes between groups were statistically significant as determined by analysis of variance (P < 0.01 and P < 0.05, respectively) 128 days after antigen initiation. Data are for all animals removed at each interval.

T

. _ _

100

B

GINGIVAL LYMPHOCYTE (x 10)

1

50

9

16

59

DAYS

128

9

16

59

128

DAYS FIG. 2. Characterization ofgingival lymphocytes from OVA-fed rats (AF, closed bars) and NAF rats (open bars) by immunofluorescence. Shown separately are the means of gingival T lymphocytes (surface thymus antigen positive) and B lymphocytes (surface immunoglobulin positive) recovered from two to four rats from each group. The abscissa, represents days of OVA feeding. Brackets enclose two standard errors. Differences between groups with respect to B lymphocytes were statistically significant, P < 0.02, 128 days after antigen initiation. Significant numbers of negative-staining lymphocytes were only observed after 59 and 128 days of feeding in the NAF group (1.1 0.8 x 104; day 128), and only on day 23 in the AF animals (6.4 ± 4.9 x 104). Data are from all animals removed at each interval.


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is diminished after 128 days of feeding, when immunoglobulin-positive (B) lymphocytes seem to predominate in the rat gingival tissues. Antibody response to OVA. Antibody responses to OVA were measured in plasma, intestinal perfusates, and saliva samples taken from the animals at various times after initiation of antigen feeding. A modification of the indirect enzyme-linked immunosorbent assay technique was used for these assays. Plasma IgG antibodies were not detected until 59 days of feeding (Table 2). These levels remained low throughout the duration of the experiment. Animals never exposed to OVA showed constant background values indicating the absence of serum or plasma antibody. On the other hand, antibody of the IgA class directed to OVA was detected in the intestinal perfusates of AF animals as early as 9 days after feeding was initiated. This response peaked after 23 days of feeding and was then present at similar levels at all subsequent test periods. Similarly, salivary IgA and IgG antibodies were detected on the first sampling day on which sufficient saliva could be obtained (Table 3). This was not until 23 days of the feeding regimen. The levels did not appear to increase appreciably during the time antibody was found. The presence of both salivary (at least by day 23) and intestinal (present by day 9) IgA antibody does not prevent gingival lymphocyte accumulation (presumably in response to antigen) by 16 to 23 days of feeding. Peripheral cells

slight increases in the numbers of both T and B cells occurred in the gingivae of OVA-fed rats. Differences between T-cell numbers became most pronounced after 59 days of feeding, when AF animals had more than four times as many T cells in their gingivae as animals not fed OVA. Differences in B cells are not apparent at 59 days. After feeding OVA for 128 days, differences between numbers of T cells in AF and NAF groups diminished, and the numbers of T cells did not change appreciably from the 59-day levels. In contrast, B cells are greatly increased at this time (128 days of feeding), and AF rats had more than six times as many B cells in their gingivae as NAF animals. Antigen-induced proliferation. We also monitored the cellular immune responses (as in vitro blastogenesis) as well as antibody to OVA during the course of antigen feeding. Spleen cells were cultured in microplates in 10% fetal calf serum (Fig. 3A) or 10% autologous plasma (Fig. 3B). The spleen cell blastogenic response was not evident until 23 to 59 days of OVA feeding. Similar results were found after feeding conventional rats OVA for 24 days (unpublished data). This measure of systemic immune response seems to occur slightly after the local gingival cellular response, which was detectable after 16 days of feeding (Fig. 1 and 2). These combined data suggest that gingival lymphocyte accumulation may precede peripheral lymphocyte sensitization. The spleen cell blastogenic response 10 r

SPLEEN CELL

A. STIMULATION I NDEX

5

I -

-

-

-

-

-

0

10 r SPLEEN CELL B.

STIMULATION 5 INDEX

0 9

16

23 DYS

59

128

FIG. 3. Spleen cel proliferation induced by OVA from OVA-fed rats (AF, closed bars) and from NAF rats (open bars). Shown are the means of stimulation indices for proliferation of rat spleen cells cultured for 5 to 7 days in (A) 10% fetal calf serum or (B) 10% autologous plasma. The abscissa represents days ofOVA feeding. Brackets enclose two standard errors. Data are from one to four animals removed at each interval. The dashed line indicates a stimulation index of 2.5.

150

TAUBMAN ET AL.

INFECT. IMMUN.

seemed to be sensitized to OVA after 23 days of tal bone loss and the distribution of bone loss in feeding, but a serum response was not seen until these animals. Mean bone loss in AF animals after 59 days of feeding. Therefore, in a situation was greater on both occasions (59 and 128 days where antigen-deprived germfree animals are of OVA) and was significantly greater at the 0.03 fed a defined protein antigen (OVA) the com- level on the latter occasion. Bone loss was also bined salivary and intestinal immune responses significantly greater on the buccal mandibular do not seem to be adequate to prevent systemic and on all buccal surfaces. Therefore, germfree sensitization. However, present sensitization animals fed a single antigen seem to experience may have been diminished by the extensive IgA more periodontal bone loss than animals fed the response that was detected. If these sensitized same diet not containing antigen. These data cells can contribute to periodontal bone loss, indicate that immune phenomena can contribthen OVA-fed animals should demonstrate ute to experimental bone loss in germfree rats. greater bone loss than unfed control rats. DISCUSSION Bone loss. Animals in the NAF and AF groups were evaluated with respect to bone loss In this report, genrfree antigen-deprived rats at 59 and 128 days after OVA feeding had begun. were sensitized by feeding OVA. OVA-fed aniTable 4 shows a comparison of the total horizon- mals demonstrated immunological sensitization as opposed to control rats, who showed no reTABLE 2. Antibody responses to OVA sponses. IgA antibody was detected in intestinal Optical density at 400 nm (mean ± standard error)' perfusates after 9 days of antigen exposure (Table 2). Salivary antibody and sensitized spleen FeedingInetnlpruas cells were present in OVA-fed rats within 23 duration Plasma (IgG) Intestinal pfut) (days) days of OVA-feeding (Table 3, Fig. 3). Therefore, feeding of OVA resulted in rapid stimulation of NAF AF NAF AF 9 0.07 ± 0.02 0.06 ± 0.01 0.06 ± 0.03 0.21 ± 0.17 at least two separate aspects of the secretory 16 0.05 + 0.01 0.05 ± 0.01 0.04 ± 0.04 0.12 ± 0.12 immunological system (intestinal and salivary; 23 0.07 ± 0.01 0.06 ± 0.01 0.08 ± 0.03 0.63 ± 0.14 Tables 2 and 3) and also in peripheral cell sen59 0.06 ± 0.01 0.12 ± 0.03 0.08 ± 0 0.23 ± 0.07 sitization. 128 0.09 + 0.01 0.19 ± 0.02 0±0 0.24 ± 0.03 We have described a technique for the detera No IgM antibody was detected in the plasiia samples. mination and characterization of gingival lymValues in IgA and IgG assays are not necessarily comparable since antisera with different potency were used. All plasma phocytes. We used this technique to characterize samples were tested at a 1/40 dilution, and all intestinal the gingival cells of rats maintained on antigenfree diets or such diets with OVA added. Inperfusates were tested at a 1/20 dilution. TABLE 3. Salivary antibody responses to OVA Optical density at 400 nm (mean ± standard error)a Feeding dura-

IgA

IgG

tion (days) NAF (n)

NAF

AF (n)

AF

23 NTb (0) 0.20 ± 0.06 (2) NT 0.13 ± 0.08 0.10 (1) 59 0.19 ± 0.11 (2) 0.09 0.12 ± 0.09 128 0.09 ± 0.01 (4) 0.25 ± 0.02 (2) 0.10 ± 0.01 0.18 ± 0.03 a Values in IgA and IgG assays are not necessarily comparable since antisera with different potency were used. All saliva samples were tested at a 1/10 dilution. b NT, not tested. Sufficient amounts of saliva for testing could not be obtained after 9 or 16 days of feeding.

Days of OVA

TABLE 4. Total bone loss in germfree rats fed OVA (AF) or in NAF animals Horizontal bone loss (mm)' Group (n) Total All lingual Lingual man- Lingual Buccal All buccal surfaces

59

NAF (3) AF (4)

18.0 ± 0.6 20.6 ± 1.7

NAF (4) 21.5 + 1.1b AF (3) 25.7 + 0.5b + standard error. 'Mean b P < 0.026.

128

maxilla

Buccal

14.7 + 0.2 16.0 ± 1.3

dible 10.7 ± 0.3 11.1 ± 0.5

4.0 + 0.5 5.0 + 0.8

surfaces 3.3 + 0.6 4.5 ± 0.5

17.3 ± 1.1 19.8 ± 0.9

11.9 + 0.6 12.7 + 0.6

5.4 + 0.6 7.1 ± 0.4

4.2 + 0.3b 2.2 + 0.3b 2.0 + 0.1 5.9 + 0.5b 3.2 ± 0.1b 2.7 ± 0.4

mandible

maxilla

1.3 + 0.4 2.4 ± 0.2

2.0 + 0.2 2.2 + 0.3

VOL. 32, 1981


creases in gingival lymphocytes occurred by 16 to 23 days of OVA feeding (Fig. 1) in AF rats. The elevated gingival lymphocyte numbers were predominantly T lymphocytes at the initial intervals of the experiment (to 59 days of OVA feeding). After 128 days of feeding, differences between numbers of gingival T cells in the OVAfed and control groups tended to diminish, and the numbers of T cells did not increase appreciably. However, at this time (128 days of feeding), B cells were greatly increased and AF animals had more than six times as many B cells in their gingivae as NAF animals. These findings generally agree with those of Mackler and coworkers (27, 28), who have examined human gingival lymphocytes from patients with different types of inflammatory periodontal lesions. They found that mild gingivitis was characterized by lymphocytes lacking surface immunoglobulin suggestive of thymus (T)-dependent cells. On the other hand, periodontitis lesions

151

palatal and lingual side of upper and lower second molars (1). The distance remains constant at all other sites. Such changes would not influence the results reported herein for the following reasons: (i) control animals should show the same bone loss attributed to passive eruption as do the OVA-fed animals to which they were compared; (ii) bone loss was assessed in animals that were more than 80 days old in our experiments. Therefore, the model described in these studies may be of considerable value in studying periodontal reactions in animals not infected with bacteria. In addition, this model has the advantage of eliminating direct toxic effects which may be attributed to bacteria. Although the present study does not permit precise determination of the immune mechanisms involved in tissue alteration, it is quite clear that these mechanisms can play an important role in periodontal bone loss.

ACKNOWLEDGMENTS contained predominantly immunoglobulin-posiThis research was performed persuant to Public Health tive lymphocytes and plasma cells. They sug- Service grant DE-03420 from the National Institute of Dental gested that the cellular infiltrate during the most Research. D.J.S. and J.L.E. are recipients of Public Health destructive stage of periodontal disease is pri- Service Research Career Development awards DE-00024 (to and DE-0075 (to J.L.E.) from the National Institute marily composed of bone marrow (B)-derived D.J.S.) Research. cells (28). This may also be the case in the of Dental We thank Linda Springs, James Cairns, and Michael Bialis germfree rat system. However, our data do not for expert technical assistance and Barbara Connolly for secpermit the establishment of a precise relation- retarial assistance. ship between cell composition and destruction. LITERATURE CITED Other investigators have found that a majority 1. Amatad-Jossi, M., and H. E. Schroeder. 1978. Ageof lymphoid cells in lesions of established related alterations of periodontal structures around the chronic inflammatory periodontal disease in hucemento-enamel junction and of the gingival connective mans were IgM-bearing B lymphocytes. Few T tissue composition in germ-free rats. J. Periodontal Res. 13:76-90. cells were also identified (38). On the basis of D. 1971. Two allotypic specificities of rat these findings it has been postulated that the 2. Armerding, immunoglobulin. Eur. J. Immunol. 1:39-45. pathogenesis of chronic inflammatory periodon- 3. Baer, P. N., and R. J. Fitzgerald. 1966. Periodontal tal disease involves conversion of a stable T-cell disease in the 18-month old germfree rat. J. Dent. Res. 45:406. lesion to a progressive B-cell lesion (39). Perhaps P. N., W. L Newton, and C. L White. 1964. the model proposed in this report can be utilized 4. Baer, Studies on periodontal disease in the mouse. VI. The to further determine the gingival lymphocyte older germ-free mouse and its conventional control. J. composition during active phases of disease. Periodontol. 35:388-396. Other investigators have found a preponder- 5. Balch, C. M., and J. D. Feldman. 1974. Thymus-dependent (T) lymphocytes in the rat. J. Immunol. 112: ance of T lymphocytes in surgical specimens of 79-6. chronically inflamed gingiva removed after a 6. Boyum, A. 1968. Isolation of mononuclear cells and granregime of scaling and plaque control for at least ulocytes from human blood. Scand. J. Clin. Lab. Invest. 2 months (41). However, the technique utilized 21(Suppl. 97):77-9. in those experiments may not have allowed re- 7. Cebra, J., and G. Goldstein. 1965. Chromatographic purification of tetramethylrhodamine-immune globulin covery of all gingival cells. Therefore, the burden conjugates and their use in the cellular localization of of evidence suggests that B lymphocytes can rabbit gamma-globulin polypeptide chains. J. Immunol. predominate in chronic periodontal conditions. 95:230-245. An important finding was that germfree ani- 8. Chess, L, R. P. MacDermott, and S. F. Schlossman. 1974. Immunologic functions of isolated human lymmals fed a protein antigen seemed to experience phocyte subpopulations. 1. Quantitative isolation of humore periodontal bone loss than animals fed the man T and B cells and response to mitogens. J. Immusame diet not containing antigen. It has been nol. 113:1113-1121. shown in germfree rats up to 80 days of age that 9. Crawford, J. M, ML A. Taubman, and D. J. Smith. 1978. The natural history of periodontal bone loss in the distance between the cemento-enamel juncgermfree and gnotobiotic rats infected with periodontion and the alveolar bone crest increased at the topathic microorganisms. J. Periodontal Res. 13:316lingual side of the lower first molar and the 325.

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