Vol. 13, No. 5 Printed in U.S A.
INFzcTION AND IMMUNITY, May 1976, P. 1347-1353 Copyright © 1976 American Society for Microbiology
Indirect Blastogenesis of Peripheral Blood Leukocytes in Experimental Gingivitis H. RICHARD GAUMER,* POUL HOLM-PEDERSEN,'
AND
LARS E. A. FOLKE
Division of Periodontology, University of Minnesota School of Dentistry, Minneapolis, Minnesota 55455
Received for publication 11 November 1975
The blastogenic response of peripheral blood leukocytes to lipopolysaccharide (LPS) was followed over a short course of experimental gingivitis, developed in human volunteers who strictly avoided oral hygiene procedures for periods up to 9 days. Eleven young males initially received thorough dental prophylaxes and supervised oral hygiene until they acquired optimal gingival health. At this point, leukocytes (5 x 105) incubated with 1.5 to 25 ,ug of LPS in serum-free media showed no response as measured by tritiated thymidine uptake. Coincubation of cells with LPS and phytohemagglutinin (PHA), however, caused synergistic enhancement of blastogenesis in every LPS-PHA dose combination tried. With progressive accumulation of dental plaque and the concomitant development of gingival inflammation, this synergistic response was lost and replaced, proportionately, by a direct response to LPS. The leukocyte response to PHA was marginally enhanced with gingivitis.
The mononuclear cells that infiltrate crevicular epithelium in developing gingivitis in man are predominantly lymphocytes (15, 35) that may have become sensitized to ubiquitous antigens of dental plaque and contribute to immunopathogenesis of gingival inflammation. Sensitization to plaque antigens is expressed systemically as transformation in peripheral blood leukocytes (PBL) cultured with plaque antigen (16-18, 20, 22, 26). Many of the cited studies have shown, however, that blastogenesis of leukocytes with plaque antigen was absent or reduced in subjects who had minimum inflammation induced by plaque (17-19, 26). Horton et al. (17) have reported that plaquestimulated blastogenesis is directly proportional to severity of periodontal disease. Similarly, Lehner et al. (26) found that expression of in vitro cellular immune response to plaque antigens, particularly lipopolysaccharides (LPS), became progressively stronger as experimental gingivitis developed during oral hygiene abstention. The response subsided precipitously when accumulated plaque was removed from the teeth. However, significant titer of serum antibody specific for indigenous oral bacteria does not vary in experimental gingivitis or chronic periodontal disease, showing that immunocompetence towards these antigens is maintained independently of clinically overt dental disease (3, 12). Specific immune tolerance for environmental antigens may normally ' Present address: Royal Dental College, DK-8000 Aarhus C, Denmark.
prevent cellular expression of this immunocompetence, e.g., in vitro blastogenesis, unless overwhelmed by abnormal antigenic exposure as there may be in periodontal inflammation. Direct blastogenic response of PBL to LPS increases in individuals subjected to experimental gingivitis during controlled bacterial perturbation (26; Gaumer et al., J. Dent. Res. 54:101, 1975). An indirect expression of LPS blastogenesis, synergy between LPS and coincubated phytohemagglutinin (PHA), has been described in generally healthy human subjects whose oral status was undetermined (34). The investigation reported here follows the mode of LPS response (i.e., direct or indirect) of PBL over a longitudinal course of progressive dental plaque accumulation and concomitant gingivitis. MATERIALS AND METHODS Experimental plan. A panel of 11 healthy male volunteers, 20 to 24 years old, having at least 28 teeth with no subgingival restorations or any gingival crevices extending beyond 3 mm was selected for the study. After a thorough dental prophylaxis and several weeks of supervised oral hygiene to ensure optimal gingival health, each subject was asked to abstain from all oral hygiene practices for successive 4-day (period I) and 9-day (period II) intervals. Supervised oral hygiene and plaque control was reinstated for 1 week between these periods. Thirty-five milliliters of venous blood was collected from each individual at the beginning and end of each experimental period. In addition, supragingival plaque accumulation was removed from all tooth surfaces at the end of each period and pooled.
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GAUMER, HOLM-PEDERSON, AND FOLKE
INFECT. IMMUN.
stention periods were 0.11 and 0.14, respectively. After 4 days of plaque accumulation, the gingival index increased to 0.44, and after 9 days it increased to 1.05. Calculation of the percentage of the maximum gingival index possible for each individual (number of tooth surfaces examined x 3, the maximum score assigned any surface) (27) showed that percentage maximum scores at the start of the 4- and 9-day plaque accumulation periods were 3.5 and 4.6% of maximum, respectively. Corresponding values at the termination of the two periods were 14.8 and 34.5% (Table 1). Before plaque accumulation in periods I and II (Table 2), the leukocytes responded well to PHA, with maximum responses of greater than 40-fold. The Y. PHA represented a nine- to sevenfold stimulation over the mean unstimulated response. This maximum response varied among individual subjects from 3,317 counts/ min (subject 4) induced with 10 ug to 30,073 counts/min (subject 8) induced with 5 ug. At the end of period I (Table 2) the mean maximum PHA response was 9,321 counts/min lower, but not significantly different from the initial expression. After period II the mean maximum response rose from 6,596 to 13,613 counts/min. In addition to the increased maximum responses for every subject after 9 days of oral hygiene abstention, the dose that induced the maximum response shifted down from 5 ,ug at the start of period I to 1.5 ug at the end of period II. Even though increases or decreases in the maximum response of PBL measured were irregular for each subject at different intervals of oral hygiene abstention, they reflect a transition associated with gingivitis. Expressed as E RESULTS PHA (Fig. 2, Table 4), the increased response is The mean gingival indexes of all subjects at emphasized. the start of the 4- and 9-day oral hygiene abThe changes in PBL responsiveness to LPS
Experimental gingivitis. Gingival inflammation induced by dental plaque accumulating over 4- and 9-day periods was evaluated at the beginning and the end of each period. The gingival tissues abutting buccal, lingual, mesial, and distal surfaces of every tooth were individually evaluated for clinical signs of inflammation according to previously published criteria (27, 28). Culture protocol. Leukocytes were recovered by differential sedimentation of erythrocytes with 5% dextran as previously described by Gothier et al. (13) and washed two times with Hanks balanced salt solution. Leukocytes were made up to a concentration of 2.5 x 10" per ml in medium (RPMI 1640, Grand Island Biological Co.) containing NaHCO3, 6.4 AM 2-mercaptoethanol, streptomycin, and penicillin, but devoid of serum supplement. Microcultures (0.2 ml, Falcon Plastics) were set up to accommodate a full range of mitogens: 1, 3.125, 6.25, 12.5, 25.0, and 50.0 ±g of LPS per culture (Escherichia coli, Difco) and 0, 1.25, 2.5, 5.0, and 10.0 ,ug of PHA per culture. The leukocytes were incubated for 72 h and were pulse-labeled for the final 6 h with 0.5 ,uCi of ['H]thymidine per culture. Thymidine incorporated was measured as previously described (9), and the data are reported as the difference in counts per minute between stimulated and unstimulated cultures. Evaluation of data. Synergistic blastogenesis developing in cultures containing both LPS and PHA constituted that response (counts per minute) in excess of the sum of the individual responses (counts per minute) to PHA and LPS (Fig. 1). The sum of the positive synergistic responses to all LPS-PHA dose combinations used is referred to as E synergism. Similarly, the sums of independent developed responses to LPS and PHA in each trail are referred to as Y. LPS and Y. PHA, respectively. The paired t test was used for the statistical analyses.
LIPOPOLYSACCHARIDE (LPS) (At / culture )
4
az z
0 a
734., -J
o
3.1
6.2 12.5
25
Synergistic CPM LPS PHA
50
11 hWl LPS CPM
L)
IL
( (0 ;) Pt
4 I 0 I 0
cr
15523
_
( v:
3 It /
PHA CPM O0t
)
a.
FIG. 1. Leukocyte blastogenesis in experimental gingivitis. Culture conditions were as follows: 5 x 105 cells per 0.2 ml of RPMI 1640, serum free, 3-day cultures, two to three replicate cultures for each dose.
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BLASTOGENESIS OF PBL IN GINGIVITIS
VOL. 13, 1976
TABLE 1. Gingival inflammation in experimental gingivitis Period II
Period I
Subject
0 days 7/112a 11/116 12/111 27/120
1
2 3 4 5 6 7 8 9 10 11
4 days 60/112
0 days 12/112
54/116
13/116 1/112 35/120 6/112 8/112 10/120 16/112 26/112 16/104 26/104
24/111 30/120
43/112
14/112 4/112 6/120 24/112 7/112 8/104 10/104
46/112 65/120 64/112 41/112 50/104 70/104
9 days 106/112 126/116 88/112 105/120 83/112 125/112 117/120 115/112 131/112 89/104 154/104
Accumulated total scores 131 547 169 1,282 Surfaces examined 1,235 1,235 1,237 1,237 Gingival index (mean 0.106 0.443 0.137 1.036 score/surface) % Maximum scoreb 14.8 4.6 34.5 3.5 a Score accumulated per total surfaces examined. b Maximum score = surfaces examined x3, the maximum score assigned any surface by criteria of LOe and Silness (27). TABLE 2. Maximum blastogenic response to PHA in experimental gingivitis Counts/min Period Ta
Subject OC
1
2 3 4 5 6 7 8 9 10 11
Mean
12,591" (5)'
Period IIb 4
0
9
2,033 (5)
7,864 (5)
11,784 (0.1)
6,765 5,156 3,317 11,431
(2.5) (5) (10) (5)
12,844 2,745 3,397 8,392
(1.25) (2.5) (5) (2.5)
10,070 30,074 10,661 10,539 8,210
(5) (5) (2.5) (2.5) (5)
2,706 12,635 13,300 25,865
(5) (2.5) (2.5) (2.5)
10,060
9,321
16,334 6,440 1,342 1,517 11,222 341 11,037 9,468
404 (10)
6,596
P = 0.4 Changes within periods I and II Changes between days 4 and 9 a10, 5, 2.5, and 1.25 ug of PHA/culture. b10, 5, 1, and 0.1 ,ug of PHA/culture. ' Days without oral hygiene. d Mean of selected maxima for 11 subjects. e Mean dose at which maximum response occurred is shown in parentheses.
were clearly observed (Table 3). Immediately before period I, the mean maximum response to LPS was only 518 counts/min below a twofold stimulation index recognized as the minimal positive response. After period I (4 days), moderate elevation in mean maximum response (1,034 counts/min) and markedly increased response in five of nine subjects (1, 2, 4, 5, and 11)
(2.5) (5) (0.1) (5) (1) (10) (2.5) (2.5)
14,878 8,100 7,864 17,516 22,131 4,586 19,428 11,414 5,571 26,476
(1) (1) (1) (1) (1) (1) (0.1) (0.1) (10) (1)
13,613
P = 0.01 P = 0.1
indicated a trend that was emphasized at the end of period II (9 days), when the maximum response to LPS had increased to 20,542 counts/ min (Table 3). Mean L value for all LPS doses included rose in period II from 501 counts/min at day 0 to 81,146 counts/min at day 9. Median LPS response was 23,176 (Table 4). A correlation of LPS responses (Table 3) with individual
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INFECT. IMMUN.
GAUMER, HOLM-PEDERSON, AND FOLKE 110 .
Synergistic CPM
100
Dl
o 90 X w F
v
( 2
L PS
-
PHA
mixledJ
PHA CPM
80
(2 125 U) LPS CPS
70
(
50
t)
z
60
E
a. 50 X 40 z D 30 0 o 20
I0
0
........
L
4 DAYS
START 00
---
PLAQUE
9 DAYS
ACCUMULATION
FIG. 2. Blastogenesis in experimental gingivitis (11 young subjects). TABLE 3. Maximum blastogenic response to LPS in experimental gingivitis Counts/min
Subject
Period Ta eY
1 2
3 4
5 6 7 8 9 10 11
716" 282 340 216 76
(12.5)' (50) (12.5) (6.25) (12.5)
404 (50) 2,506 (50) 404 (50) 368 (50) 52 (25)
Period IIb 4
(12.5) (2.5) (50) (3) 2,331 (12.5) 982 1,676 234 928
163 (12) 303 (50) 247 2,348 (12.5)
0
(25) (25) (12.5) (12.5) 349 (6.2) 368 (25) 516 (6.2)
643 378 295 239
0 10 (6.2) 50 (6.2)
9
13,772 12,754 12,654 19,737 44,252 22,467 3,152 21,018 19,332 19,920 36,854
(6.2) (3.1) (3.1) (6.2) (6.2) (6.2) (6.2) (6.2) (6.2) (6.2) (3.1)
20,542
Mean 518 Changes within periods I and II Changes between days 4 and 9 a 50, 25, 12.5, 6.25, and 3.12 ,g of LPS/culture. b 25, 12.5,6.25, 3.12, and 1.56 ,ug of LPS/culture. ' See Table 2.
1,034 P = 0.2
gingivitis scores (Table 1) was anticipated. Correlation analysis failed to prove any significant relationship between these parameters by rank, although this was not surprising since a qualitative measure (gingival index) was being compared with quantitative in vitro data. A subsequent paper shows that elderly subjects (>60 years old) have a significantly different in vitro PBL response (Holm-Pedersen, Gaumer, and Folke, in preparation) than the young panel of subjects presented in this investigation. Synergistic responses were observed consistently in all subjects just before periods I and II
of oral hygiene abstention. Maximum synergism occurred with 12.5 to 50 ,ug of PHA. Synergism deteriorated with increasing gingivitis scores and was absent by day 9 of period II. Before development of gingivitis, the positive synergistic response of all 20 LPS-PHA dose combinations tested yielded a I value of 85,787 counts/min (Table 4). This mean represents data pooled from day 0 of both periods I and II. DISCUSSION This report documents modulation of immunocompetence of PBL expressed in vitro accompanying progressive dental plaque accumula-
284
P = 0.001 P = 0.001
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BLASTOGENESIS OF PBL IN GINGIVITIS
VOL. 13, 1976
tion and gingivitis. The most prominent change in leukocyte response was the emergence, at the end of period II (9 days), of LPS-induced blastogenesis. This direct response to LPS (E LPS) increased over 100 times from day 0 to day 9 of period II (Table 4), as the clinical assessment of gingivitis (the gingival index) changed from 0.137 to 1.036 (Table 1). The PBL response to PHA rose too, but not as dramatically. Increased antigenic load at the gingival crevice (28) is implicated, since removal of plaque led to a decrease of the gingival inflammatory response. Modulation of immunocompetence after in vivo bacterial (4) or viral (21, 36) infection is probably analogous to this observation. Horton et al. (17) have reported that plaquestimulated blastogenesis is directly proportional to the severity of periodontal disease. Similarly, Lehner et al. (26) found that expression of in vitro cellular immune response to endotoxin of Veillonella alcalescens, indigenous to the dental plaque flora, became progressively stronger as experimental gingivitis developed, but fell precipitously when accumulated plaque was removed from the teeth. The increase noted in their study developed after 21 days of experimental gingivitis, but was modest compared with the marked elevation in direct LPS response observed in the present study of a 9-day development of experimental gingivitis (Table 3). Serum antibody specific for dental plaque antigen, including LPS, has been demonstrated in homologous sera (3, 5, 12). Serum-supplemented tissue culture media may, therefore, depress or enhance blastogenic response (10, 20). Consequently, we elected to exclude serum in this initial study since we have observed that serum is not necessary to achieve blastogenic
stimulation comparable with that observed in other studies (16, 25) that have used serum. Serum-free conditions, on the other hand, are not prerequisites for synergism or direct LPS response. Factors in sera effecting PBL response are the subject of current investigations in this laboratory. Both quality and quantity of mitogen and antigen determine in vitro transformation of lymphocytes. Numerous subpopulations of variably differentiated T and B lymphocytes respond only if presented with their own optimal dose (6, 7, 14). Pure T lymphocytes can be functionally subdivided into T, and T. cells, responsive to high and low concentrations of antigen, respectively, the latter being predominant in a more mature immune response (6). Likewise, exposing mixed lymphocytes in vitro to a single antigen dose will functionally reveal just one of the several subpopulations that may be potentially responsive in vivo, i.e., as long as the influence of factors secreted by immunocompetent cells exposed to antigen may be disregarded (2). The technique of summation of data from multiple doses of mitogen or antigen (Fig. 1) permits representation of every response obtained. Sigma values (Table 4) reflect overall immunocompetence and may be more relevant to an in vivo situation. The sum of responses, however, is only an approximation of overall immunocompetence, which is the integral approached as a limit, as the difference between PHA doses applied, over a reasonable range (1 to 10 ,ug per culture), is reduced: A[PHA], counts/min
Using a range of mitogen doses also secures the chances of measuring maximum response
TABLE 4. Immunocompetence of PBL in experimental gingivitis oral hygiene abstention Period II
Period I Mitogen
PHA
Determination
Ia Mean Median
LPS
lb
Mean Median LPS-PHA
0 days
4 days
0 days
9 days
15,919 3,979
23,032 5,758
12,084 3,021
28,488 7,122
3,608
4,870
2,902
8,219
300 60 0
2,158 432 336
501 31 0
81,146 16,229
23,176
722 85,787 32,342 36 Mean 4,289 1,617 (Incomplete) 4 0 Median 863 a Response (counts per minute) accumulated from PHA concentration doses of 10, 5, 2.5, and 1.25 tg. b Response (counts) accumulated from LPS doses of 50, 25, 12.5, 6.25, and 3.125 yg. Synergistic response accumulated from 20 combinations of PHA and LPS.
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GAUMER, HOLM-PEDERSON, AND FOLKE
throughout the course of gingival infection. Maximum PBL response observed at day 9 of period II was elicited by a lower dose of mitogen than what had been required to produce maxima at day 0 of period I (Tables 2 and 3). Note that total PHA response (E) increased after 9 days as well (Table 4). Lang and Smith (25) have also described a shift in mitogen dose optima with progressive experimental gingivitis. We concur with their interpretation that varying reports (22, 26) of changing PHA response with gingivitis may have resulted because of this dose response transition. Synergism (Table 4) occurred with all combinations of PHA and LPS used (Fig. 1), elevating the blastogenic response to PHA by 30 to 80%. Maximum synergism occurred with combinations of the highest doses of mitogen used (lower right quadrant, Fig. 1). The minimal response, developed with the lowest doses used (upper left quadrant, Fig. 1), accounts for the skewed distribution indicated by disparate mean and median synergistic response per dose (Table 4). After period II, the synergistic expression is no longer detectable. The magnitude of E values (Table 4) suggests that it has been replaced with direct response to LPS (Fig. 2). Note that whereas direct response to LPS developed with five selected doses, synergism occurred with twenty different dose combinations of LPS and PHA. Schmidke and Najarian (34) previously reported significant synergistic response in mononuclear cells of PBL co-incubated with LPS and PHA or concanavalin A. In an analogous study, Ozato and co-workers (31) measured LPS activation of mouse thymocytes unresponsive to LPS alone by showing synergistic enhancement by combining LPS with Concanavalin A. The latter observations were interpreted as showing that LPS, a "B cell mitogen" (30, 32), participates in triggering T cells, although it may not operate directly. "Helper" and "suppressor" functions of T lymphocytes are likewise considered to be influenced by exposure to LPS (1, 8, 24). Thus, the different expression of immunocompetence of PBL in vitro before and after plaque accumulation may be related to maturation of T lymphocytes. Before gingivitis, lowaffinity receptors of T, cells require higher concentrations of PHA for activation. LPS may react with T, cells, as described for mouse cells (1, 8, 31), and expand the proportion of cells responding to mitogen in a culture with both LPS and PHA, i.e., synergism. Plaque antigen, which includes LPS, may produce similar effects in vivo, causing maturation of T, to T, cells (6) that are responsive to lower concentrations of mitogen.
INFECT. IMMUN.
During gingivitis, synergism decreased with emergence of PBL response to LPS directly. These cells, perhaps B cells, may be newly mobilized by enhanced antigen stimulation, or they may represent a population whose competence for direct LPS response is normally suppressed in immune tolerance. That there was no increase in yields of PBL with early gingivitis does not favor the former alternative. More direct quantification of B and T cells is still required. The absence of PBL response to LPS before period I is interesting considering the subject's recurrent exposure to the gram-negative flora of the gastrointestinal tract, including the oral cavity (11, 23, 29). Specific antibodies and plasma factors (33, 37) as well as the indirect (synergism) and direct in vitro PBL response to LPS attests to the immunocompetence of humans for LPS. Ubiquitous dental plaque in general, and LPS in particular, may represent antigenic "noise" to the immune system, and a tolerance mechanism to prevent irrelevant response may be required. Such a mechanism may be overwhelmed by abnormal buildup of plaque during experimental gingivitis. Whether subjects whose normal oral hygiene is neglected respond to a sudden plaque buildup as did the subjects studied in this investigation remains to be determined. ACKNOWLEDGMENTS We thank Harriet Kasprick and Gerald Sherek for technical assistance and Eileen Jenkins for preparing the manuscript. This work was supported by Public Health Service grant 5R01-DE-03174-03 from the National Institute of Dental Research and a research grant from the Graduate School of the University of Minnesota. LITERATURE CITED 1. Amerding, D., and D. H. Katz. 1972. Activation of T and B lymphocytes in vitro. I. Regulatory influence of bacterial lipopolysaccharide (LPS) on specific T-cell helper function. J. Exp. Med. 139:24-43. 2. Anderson, J., G. Moller, and 0. Sjoberg. 1972. B lymphocytes can be stimulated by concanavalin A in the presence of humoral factors released by T cells. Eur. J. Immunol. 2:99-101. 3. Berkenbilt, D. A., and A. N. Bahn. 1971. Development of antibodies to cariogenic streptococci in children. J. Am. Dent. Ass. 83:332-337. 4. Blackman, U., S. R. Graboff, G. E. Hagg, E. Gottfeld, and M. J. Pickett. 1974. Experimental cholera in chinchillas: the immune response in serum and intestinal secretions to Vibrio cholerae and choleratoxin. Infect. Immun. 10:1094-1104. 5. Bratthall, D., and R. J. Gibbons. 1975. Changing agglutination activities of salivary immunoglobulin A preparations against oral streptococci. Infect. Immun. 11:603-606. 6. Cohen, J. J., and H. N. Claman. 1971. Thymus-marrow immunocompetence. V. Hydrocortisone-resistant cells and processes in the hemolytic antibody response of mice. J. Exp. Med. 133:1026-1034.
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7. Douglas, S. D., R. M. Kamin, and H. H. Fudenberg. 1969. Human lymphocyte response to phytomitogens in vitro: normal, agammaglobulinemic and paraproteinemic individuals. J. Immunol. 103:1185-1195. 8. Forbes, J. T., Y. Nako, and R. Smith. 1975. T mitogens trigger LPS responsiveness in mouse thymus cells. J. Immunol. 114:1004-1007. 9. Gaumer, H. R., and J. H. Schwab. 1971. Differential susceptibility of mouse lymphocytes to an immunosuppressant from group A streptococci. Cell. Immunol. 4:394-406. 10. Genco, R. J., P. A. Mashimo, G. Krygier, and S. A. Ellison. 1974. Antibody mediated effects on the periodontium. J. Periodontol. 45:330-337. 11. Gibbons, R. J., S. S. Socransky, S. Sawyer, B. Kapsimalis, and J. B. McDonald. 1963. The microbiota of the gingival crevice area of man. II. The predominant cultivatable organisms. Arch. Oral Biol. 8:281-289. 12. Gilmour, M., and R. J. Nisengard. 1974. Interactions between serum titers to filamentous bacteria and their relationship to human periodontal disease. Arch. Oral Biol. 19:959-968. 13. Gothier, D., H. R. Gaumer, B. Pihlstrom, and L. E. A. Folke. 1975. Elevation of a serum component in periodontal disease capable of modulating chemotactic infiltration. J. Periodont. Res. 10:65-72. 14. Gronowicz, E., A. Coutinko, and G. Moller. 1974. Differentiation of B cells: sequential appearance of responsiveness to polydorsal activators. Scand. J. Immunol. 3:413-421. 15. Guggenheim, B., and H. E. Schroeder. 1974. Reactions in the periodontium to continuous antigenic stimulation in sensitized gnotobiotic rats. Infect. Immun. 10:565-577. 16. Hayri, P., and V. Defendi. 1970. Cultivation conditions and mixed lymphocyte interaction of mouse peripheral lymphocytes. Clin. Exp. Immunol. 6:345-346. 17. Horton, J. E., S. Leiken, and J. J. Oppenheim. 1972. Human lymphoproliferative reaction to saliva and dental plaque deposits: an in vitro correlation with periodontal disease. J. Periodontol. 43:522-527. 18. Ivanyi, L., and T. Lehner. 1970. Stimulation of lymphocytes by bacterial antigens in patients with periodontal disease. Arch. Oral Biol. 15:1089-1096. 19. Ivanyi, L., and T. Lehner. 1971. Lymphocyte transformation by sonicates of dental plaque in human periodontal disease. Arch. Oral Biol. 16:1117-1121. 20. Ivanyi, L., and T. Lehner. 1971. The significance of serum factors in stimulation of lymphocytes from patients with periodontal disease by Veillonella alkalescens. Int. Arch. Allergy 41:620-627. 21. Kauffman, C., J. Phair, C. Linnemann, and G. Schiff. 1974. Cell-mediated immunity in humans during viral infection. I. Effect of rubella on hypersensitivity, phytohemagglutinin response, and T lymphocyte numbers. Infect. Immun. 10:212-215.
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22. Kiger, R. D., W. H. Wright, and H. R. Creamer. 1974. The significance of lymphocyte transformation responses to various microbial stimulants. J. Periodontol. 45:780-785. 23. Knox, D. W. 1970. Antigens of oral bacteria. Adv. Oral Biol. 4:92-103. 24. Lagrange, P. H., G. B. Mackaness, T. E. Miller, and P. Pardon. 1975. Effects of bacterial lipopolysaccharide on the induction and expression of cell mediated immunity. I. Depression of the afferant arc. J. Immunol. 114:442-446. 25. Lang, N. P., and F. N. Smith. 1976. Lymphocyte response to T-cell mitogen during experimental gingivitis. Infect. Immun. 13:108-113. 26. Lehner, T., S. J. Challacombe, L. Ivanyi, and J. M. A. Wilton. 1973. The relationship between serum and salivary antibodies and cell mediated immunity in oral disease in man. Adv. Exp. Med. Biol. 45:485-496. 27. Loe, H., and J. Silness. 1963. Periodontal disease in pregnancy. I. Prevalence and severity. Acta Odontol. Scand. 21:533-551. 28. Loe, H., E. Thielade, and S. B. Jensen. Experimental gingivitis in man. J. Periodont. Res. 36:177-187. 29. Loesche, W. J., and R. J. Gibbons. 1968. Amino acid fermentation by Fusobacterium nucleatum. Arch. Oral Biol. 13:191-201. 30. Moller, G., 0. Sjoberg, and J. Anderson. 1973. Immunogenicity, tolerogenicity, and mitogenicity of lipopolysaccharides. J. Infect. Dis. 128:552-556. 31. Ozato, K., W. H. Adler, and J. D. Ebert. 1975. Synergism of bacterial lipopolysaccharides and concanavalin A in the activation of thymic lymphocytes. Cell. Immunol. 17:532-541. 32. Peavy, D. L., J. W. Shands, W. Adler, and R. T. Smith. 1973. Selective effects of bacterial endotoxins on various subpopulations of lymphoreticular cells. J. Infect. Dis. 128:583-591. 33. Rudbach, J. A., and M. K. Luoma. 1974. Endotoxinaltering activity of plasma does not affect antigenicity of native protoplasmic polysaccharide. Infect. Immun. 10:1183-1184. 34. Schmidke, J. R., and J. S. Najarian. 1975. Synergistic effects on DNA synthesis of phytohemagglutinin or concanavalin A and lipopolysaccharide on human peripheral blood lymphocytes. J. Immunol. 114:742-746. 35. Schroeder, H. E., S. Munzel-Pedrazzoli, and R. Page. 1973. Correlated morphometric and biochemical analysis of gingival tissue in early chronic gingivitis in man. Arch. Oral Biol. 18:899-923. 36. Vesikari, T., and E. Buimovici-Klein. 1975. Lymphocyte responses to rubella antigens and phytohemagglutinin after administration of the RA27/3 strain of live, attenuated rubella vaccine. Infect. Immun. 11:748-753. 37. Wolff, S. M. 1973. Biological effects of bacterial endotoxins in man. J. Infect. Dis. 128:5251-5256.
INFzcTION AND IMMUNITY, May 1976, P. 1347-1353 Copyright © 1976 American Society for Microbiology
Indirect Blastogenesis of Peripheral Blood Leukocytes in Experimental Gingivitis H. RICHARD GAUMER,* POUL HOLM-PEDERSEN,'
AND
LARS E. A. FOLKE
Division of Periodontology, University of Minnesota School of Dentistry, Minneapolis, Minnesota 55455
Received for publication 11 November 1975
The blastogenic response of peripheral blood leukocytes to lipopolysaccharide (LPS) was followed over a short course of experimental gingivitis, developed in human volunteers who strictly avoided oral hygiene procedures for periods up to 9 days. Eleven young males initially received thorough dental prophylaxes and supervised oral hygiene until they acquired optimal gingival health. At this point, leukocytes (5 x 105) incubated with 1.5 to 25 ,ug of LPS in serum-free media showed no response as measured by tritiated thymidine uptake. Coincubation of cells with LPS and phytohemagglutinin (PHA), however, caused synergistic enhancement of blastogenesis in every LPS-PHA dose combination tried. With progressive accumulation of dental plaque and the concomitant development of gingival inflammation, this synergistic response was lost and replaced, proportionately, by a direct response to LPS. The leukocyte response to PHA was marginally enhanced with gingivitis.
The mononuclear cells that infiltrate crevicular epithelium in developing gingivitis in man are predominantly lymphocytes (15, 35) that may have become sensitized to ubiquitous antigens of dental plaque and contribute to immunopathogenesis of gingival inflammation. Sensitization to plaque antigens is expressed systemically as transformation in peripheral blood leukocytes (PBL) cultured with plaque antigen (16-18, 20, 22, 26). Many of the cited studies have shown, however, that blastogenesis of leukocytes with plaque antigen was absent or reduced in subjects who had minimum inflammation induced by plaque (17-19, 26). Horton et al. (17) have reported that plaquestimulated blastogenesis is directly proportional to severity of periodontal disease. Similarly, Lehner et al. (26) found that expression of in vitro cellular immune response to plaque antigens, particularly lipopolysaccharides (LPS), became progressively stronger as experimental gingivitis developed during oral hygiene abstention. The response subsided precipitously when accumulated plaque was removed from the teeth. However, significant titer of serum antibody specific for indigenous oral bacteria does not vary in experimental gingivitis or chronic periodontal disease, showing that immunocompetence towards these antigens is maintained independently of clinically overt dental disease (3, 12). Specific immune tolerance for environmental antigens may normally ' Present address: Royal Dental College, DK-8000 Aarhus C, Denmark.
prevent cellular expression of this immunocompetence, e.g., in vitro blastogenesis, unless overwhelmed by abnormal antigenic exposure as there may be in periodontal inflammation. Direct blastogenic response of PBL to LPS increases in individuals subjected to experimental gingivitis during controlled bacterial perturbation (26; Gaumer et al., J. Dent. Res. 54:101, 1975). An indirect expression of LPS blastogenesis, synergy between LPS and coincubated phytohemagglutinin (PHA), has been described in generally healthy human subjects whose oral status was undetermined (34). The investigation reported here follows the mode of LPS response (i.e., direct or indirect) of PBL over a longitudinal course of progressive dental plaque accumulation and concomitant gingivitis. MATERIALS AND METHODS Experimental plan. A panel of 11 healthy male volunteers, 20 to 24 years old, having at least 28 teeth with no subgingival restorations or any gingival crevices extending beyond 3 mm was selected for the study. After a thorough dental prophylaxis and several weeks of supervised oral hygiene to ensure optimal gingival health, each subject was asked to abstain from all oral hygiene practices for successive 4-day (period I) and 9-day (period II) intervals. Supervised oral hygiene and plaque control was reinstated for 1 week between these periods. Thirty-five milliliters of venous blood was collected from each individual at the beginning and end of each experimental period. In addition, supragingival plaque accumulation was removed from all tooth surfaces at the end of each period and pooled.
1347
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GAUMER, HOLM-PEDERSON, AND FOLKE
INFECT. IMMUN.
stention periods were 0.11 and 0.14, respectively. After 4 days of plaque accumulation, the gingival index increased to 0.44, and after 9 days it increased to 1.05. Calculation of the percentage of the maximum gingival index possible for each individual (number of tooth surfaces examined x 3, the maximum score assigned any surface) (27) showed that percentage maximum scores at the start of the 4- and 9-day plaque accumulation periods were 3.5 and 4.6% of maximum, respectively. Corresponding values at the termination of the two periods were 14.8 and 34.5% (Table 1). Before plaque accumulation in periods I and II (Table 2), the leukocytes responded well to PHA, with maximum responses of greater than 40-fold. The Y. PHA represented a nine- to sevenfold stimulation over the mean unstimulated response. This maximum response varied among individual subjects from 3,317 counts/ min (subject 4) induced with 10 ug to 30,073 counts/min (subject 8) induced with 5 ug. At the end of period I (Table 2) the mean maximum PHA response was 9,321 counts/min lower, but not significantly different from the initial expression. After period II the mean maximum response rose from 6,596 to 13,613 counts/min. In addition to the increased maximum responses for every subject after 9 days of oral hygiene abstention, the dose that induced the maximum response shifted down from 5 ,ug at the start of period I to 1.5 ug at the end of period II. Even though increases or decreases in the maximum response of PBL measured were irregular for each subject at different intervals of oral hygiene abstention, they reflect a transition associated with gingivitis. Expressed as E RESULTS PHA (Fig. 2, Table 4), the increased response is The mean gingival indexes of all subjects at emphasized. the start of the 4- and 9-day oral hygiene abThe changes in PBL responsiveness to LPS
Experimental gingivitis. Gingival inflammation induced by dental plaque accumulating over 4- and 9-day periods was evaluated at the beginning and the end of each period. The gingival tissues abutting buccal, lingual, mesial, and distal surfaces of every tooth were individually evaluated for clinical signs of inflammation according to previously published criteria (27, 28). Culture protocol. Leukocytes were recovered by differential sedimentation of erythrocytes with 5% dextran as previously described by Gothier et al. (13) and washed two times with Hanks balanced salt solution. Leukocytes were made up to a concentration of 2.5 x 10" per ml in medium (RPMI 1640, Grand Island Biological Co.) containing NaHCO3, 6.4 AM 2-mercaptoethanol, streptomycin, and penicillin, but devoid of serum supplement. Microcultures (0.2 ml, Falcon Plastics) were set up to accommodate a full range of mitogens: 1, 3.125, 6.25, 12.5, 25.0, and 50.0 ±g of LPS per culture (Escherichia coli, Difco) and 0, 1.25, 2.5, 5.0, and 10.0 ,ug of PHA per culture. The leukocytes were incubated for 72 h and were pulse-labeled for the final 6 h with 0.5 ,uCi of ['H]thymidine per culture. Thymidine incorporated was measured as previously described (9), and the data are reported as the difference in counts per minute between stimulated and unstimulated cultures. Evaluation of data. Synergistic blastogenesis developing in cultures containing both LPS and PHA constituted that response (counts per minute) in excess of the sum of the individual responses (counts per minute) to PHA and LPS (Fig. 1). The sum of the positive synergistic responses to all LPS-PHA dose combinations used is referred to as E synergism. Similarly, the sums of independent developed responses to LPS and PHA in each trail are referred to as Y. LPS and Y. PHA, respectively. The paired t test was used for the statistical analyses.
LIPOPOLYSACCHARIDE (LPS) (At / culture )
4
az z
0 a
734., -J
o
3.1
6.2 12.5
25
Synergistic CPM LPS PHA
50
11 hWl LPS CPM
L)
IL
( (0 ;) Pt
4 I 0 I 0
cr
15523
_
( v:
3 It /
PHA CPM O0t
)
a.
FIG. 1. Leukocyte blastogenesis in experimental gingivitis. Culture conditions were as follows: 5 x 105 cells per 0.2 ml of RPMI 1640, serum free, 3-day cultures, two to three replicate cultures for each dose.
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BLASTOGENESIS OF PBL IN GINGIVITIS
VOL. 13, 1976
TABLE 1. Gingival inflammation in experimental gingivitis Period II
Period I
Subject
0 days 7/112a 11/116 12/111 27/120
1
2 3 4 5 6 7 8 9 10 11
4 days 60/112
0 days 12/112
54/116
13/116 1/112 35/120 6/112 8/112 10/120 16/112 26/112 16/104 26/104
24/111 30/120
43/112
14/112 4/112 6/120 24/112 7/112 8/104 10/104
46/112 65/120 64/112 41/112 50/104 70/104
9 days 106/112 126/116 88/112 105/120 83/112 125/112 117/120 115/112 131/112 89/104 154/104
Accumulated total scores 131 547 169 1,282 Surfaces examined 1,235 1,235 1,237 1,237 Gingival index (mean 0.106 0.443 0.137 1.036 score/surface) % Maximum scoreb 14.8 4.6 34.5 3.5 a Score accumulated per total surfaces examined. b Maximum score = surfaces examined x3, the maximum score assigned any surface by criteria of LOe and Silness (27). TABLE 2. Maximum blastogenic response to PHA in experimental gingivitis Counts/min Period Ta
Subject OC
1
2 3 4 5 6 7 8 9 10 11
Mean
12,591" (5)'
Period IIb 4
0
9
2,033 (5)
7,864 (5)
11,784 (0.1)
6,765 5,156 3,317 11,431
(2.5) (5) (10) (5)
12,844 2,745 3,397 8,392
(1.25) (2.5) (5) (2.5)
10,070 30,074 10,661 10,539 8,210
(5) (5) (2.5) (2.5) (5)
2,706 12,635 13,300 25,865
(5) (2.5) (2.5) (2.5)
10,060
9,321
16,334 6,440 1,342 1,517 11,222 341 11,037 9,468
404 (10)
6,596
P = 0.4 Changes within periods I and II Changes between days 4 and 9 a10, 5, 2.5, and 1.25 ug of PHA/culture. b10, 5, 1, and 0.1 ,ug of PHA/culture. ' Days without oral hygiene. d Mean of selected maxima for 11 subjects. e Mean dose at which maximum response occurred is shown in parentheses.
were clearly observed (Table 3). Immediately before period I, the mean maximum response to LPS was only 518 counts/min below a twofold stimulation index recognized as the minimal positive response. After period I (4 days), moderate elevation in mean maximum response (1,034 counts/min) and markedly increased response in five of nine subjects (1, 2, 4, 5, and 11)
(2.5) (5) (0.1) (5) (1) (10) (2.5) (2.5)
14,878 8,100 7,864 17,516 22,131 4,586 19,428 11,414 5,571 26,476
(1) (1) (1) (1) (1) (1) (0.1) (0.1) (10) (1)
13,613
P = 0.01 P = 0.1
indicated a trend that was emphasized at the end of period II (9 days), when the maximum response to LPS had increased to 20,542 counts/ min (Table 3). Mean L value for all LPS doses included rose in period II from 501 counts/min at day 0 to 81,146 counts/min at day 9. Median LPS response was 23,176 (Table 4). A correlation of LPS responses (Table 3) with individual
1350
INFECT. IMMUN.
GAUMER, HOLM-PEDERSON, AND FOLKE 110 .
Synergistic CPM
100
Dl
o 90 X w F
v
( 2
L PS
-
PHA
mixledJ
PHA CPM
80
(2 125 U) LPS CPS
70
(
50
t)
z
60
E
a. 50 X 40 z D 30 0 o 20
I0
0
........
L
4 DAYS
START 00
---
PLAQUE
9 DAYS
ACCUMULATION
FIG. 2. Blastogenesis in experimental gingivitis (11 young subjects). TABLE 3. Maximum blastogenic response to LPS in experimental gingivitis Counts/min
Subject
Period Ta eY
1 2
3 4
5 6 7 8 9 10 11
716" 282 340 216 76
(12.5)' (50) (12.5) (6.25) (12.5)
404 (50) 2,506 (50) 404 (50) 368 (50) 52 (25)
Period IIb 4
(12.5) (2.5) (50) (3) 2,331 (12.5) 982 1,676 234 928
163 (12) 303 (50) 247 2,348 (12.5)
0
(25) (25) (12.5) (12.5) 349 (6.2) 368 (25) 516 (6.2)
643 378 295 239
0 10 (6.2) 50 (6.2)
9
13,772 12,754 12,654 19,737 44,252 22,467 3,152 21,018 19,332 19,920 36,854
(6.2) (3.1) (3.1) (6.2) (6.2) (6.2) (6.2) (6.2) (6.2) (6.2) (3.1)
20,542
Mean 518 Changes within periods I and II Changes between days 4 and 9 a 50, 25, 12.5, 6.25, and 3.12 ,g of LPS/culture. b 25, 12.5,6.25, 3.12, and 1.56 ,ug of LPS/culture. ' See Table 2.
1,034 P = 0.2
gingivitis scores (Table 1) was anticipated. Correlation analysis failed to prove any significant relationship between these parameters by rank, although this was not surprising since a qualitative measure (gingival index) was being compared with quantitative in vitro data. A subsequent paper shows that elderly subjects (>60 years old) have a significantly different in vitro PBL response (Holm-Pedersen, Gaumer, and Folke, in preparation) than the young panel of subjects presented in this investigation. Synergistic responses were observed consistently in all subjects just before periods I and II
of oral hygiene abstention. Maximum synergism occurred with 12.5 to 50 ,ug of PHA. Synergism deteriorated with increasing gingivitis scores and was absent by day 9 of period II. Before development of gingivitis, the positive synergistic response of all 20 LPS-PHA dose combinations tested yielded a I value of 85,787 counts/min (Table 4). This mean represents data pooled from day 0 of both periods I and II. DISCUSSION This report documents modulation of immunocompetence of PBL expressed in vitro accompanying progressive dental plaque accumula-
284
P = 0.001 P = 0.001
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BLASTOGENESIS OF PBL IN GINGIVITIS
VOL. 13, 1976
tion and gingivitis. The most prominent change in leukocyte response was the emergence, at the end of period II (9 days), of LPS-induced blastogenesis. This direct response to LPS (E LPS) increased over 100 times from day 0 to day 9 of period II (Table 4), as the clinical assessment of gingivitis (the gingival index) changed from 0.137 to 1.036 (Table 1). The PBL response to PHA rose too, but not as dramatically. Increased antigenic load at the gingival crevice (28) is implicated, since removal of plaque led to a decrease of the gingival inflammatory response. Modulation of immunocompetence after in vivo bacterial (4) or viral (21, 36) infection is probably analogous to this observation. Horton et al. (17) have reported that plaquestimulated blastogenesis is directly proportional to the severity of periodontal disease. Similarly, Lehner et al. (26) found that expression of in vitro cellular immune response to endotoxin of Veillonella alcalescens, indigenous to the dental plaque flora, became progressively stronger as experimental gingivitis developed, but fell precipitously when accumulated plaque was removed from the teeth. The increase noted in their study developed after 21 days of experimental gingivitis, but was modest compared with the marked elevation in direct LPS response observed in the present study of a 9-day development of experimental gingivitis (Table 3). Serum antibody specific for dental plaque antigen, including LPS, has been demonstrated in homologous sera (3, 5, 12). Serum-supplemented tissue culture media may, therefore, depress or enhance blastogenic response (10, 20). Consequently, we elected to exclude serum in this initial study since we have observed that serum is not necessary to achieve blastogenic
stimulation comparable with that observed in other studies (16, 25) that have used serum. Serum-free conditions, on the other hand, are not prerequisites for synergism or direct LPS response. Factors in sera effecting PBL response are the subject of current investigations in this laboratory. Both quality and quantity of mitogen and antigen determine in vitro transformation of lymphocytes. Numerous subpopulations of variably differentiated T and B lymphocytes respond only if presented with their own optimal dose (6, 7, 14). Pure T lymphocytes can be functionally subdivided into T, and T. cells, responsive to high and low concentrations of antigen, respectively, the latter being predominant in a more mature immune response (6). Likewise, exposing mixed lymphocytes in vitro to a single antigen dose will functionally reveal just one of the several subpopulations that may be potentially responsive in vivo, i.e., as long as the influence of factors secreted by immunocompetent cells exposed to antigen may be disregarded (2). The technique of summation of data from multiple doses of mitogen or antigen (Fig. 1) permits representation of every response obtained. Sigma values (Table 4) reflect overall immunocompetence and may be more relevant to an in vivo situation. The sum of responses, however, is only an approximation of overall immunocompetence, which is the integral approached as a limit, as the difference between PHA doses applied, over a reasonable range (1 to 10 ,ug per culture), is reduced: A[PHA], counts/min
Using a range of mitogen doses also secures the chances of measuring maximum response
TABLE 4. Immunocompetence of PBL in experimental gingivitis oral hygiene abstention Period II
Period I Mitogen
PHA
Determination
Ia Mean Median
LPS
lb
Mean Median LPS-PHA
0 days
4 days
0 days
9 days
15,919 3,979
23,032 5,758
12,084 3,021
28,488 7,122
3,608
4,870
2,902
8,219
300 60 0
2,158 432 336
501 31 0
81,146 16,229
23,176
722 85,787 32,342 36 Mean 4,289 1,617 (Incomplete) 4 0 Median 863 a Response (counts per minute) accumulated from PHA concentration doses of 10, 5, 2.5, and 1.25 tg. b Response (counts) accumulated from LPS doses of 50, 25, 12.5, 6.25, and 3.125 yg. Synergistic response accumulated from 20 combinations of PHA and LPS.
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GAUMER, HOLM-PEDERSON, AND FOLKE
throughout the course of gingival infection. Maximum PBL response observed at day 9 of period II was elicited by a lower dose of mitogen than what had been required to produce maxima at day 0 of period I (Tables 2 and 3). Note that total PHA response (E) increased after 9 days as well (Table 4). Lang and Smith (25) have also described a shift in mitogen dose optima with progressive experimental gingivitis. We concur with their interpretation that varying reports (22, 26) of changing PHA response with gingivitis may have resulted because of this dose response transition. Synergism (Table 4) occurred with all combinations of PHA and LPS used (Fig. 1), elevating the blastogenic response to PHA by 30 to 80%. Maximum synergism occurred with combinations of the highest doses of mitogen used (lower right quadrant, Fig. 1). The minimal response, developed with the lowest doses used (upper left quadrant, Fig. 1), accounts for the skewed distribution indicated by disparate mean and median synergistic response per dose (Table 4). After period II, the synergistic expression is no longer detectable. The magnitude of E values (Table 4) suggests that it has been replaced with direct response to LPS (Fig. 2). Note that whereas direct response to LPS developed with five selected doses, synergism occurred with twenty different dose combinations of LPS and PHA. Schmidke and Najarian (34) previously reported significant synergistic response in mononuclear cells of PBL co-incubated with LPS and PHA or concanavalin A. In an analogous study, Ozato and co-workers (31) measured LPS activation of mouse thymocytes unresponsive to LPS alone by showing synergistic enhancement by combining LPS with Concanavalin A. The latter observations were interpreted as showing that LPS, a "B cell mitogen" (30, 32), participates in triggering T cells, although it may not operate directly. "Helper" and "suppressor" functions of T lymphocytes are likewise considered to be influenced by exposure to LPS (1, 8, 24). Thus, the different expression of immunocompetence of PBL in vitro before and after plaque accumulation may be related to maturation of T lymphocytes. Before gingivitis, lowaffinity receptors of T, cells require higher concentrations of PHA for activation. LPS may react with T, cells, as described for mouse cells (1, 8, 31), and expand the proportion of cells responding to mitogen in a culture with both LPS and PHA, i.e., synergism. Plaque antigen, which includes LPS, may produce similar effects in vivo, causing maturation of T, to T, cells (6) that are responsive to lower concentrations of mitogen.
INFECT. IMMUN.
During gingivitis, synergism decreased with emergence of PBL response to LPS directly. These cells, perhaps B cells, may be newly mobilized by enhanced antigen stimulation, or they may represent a population whose competence for direct LPS response is normally suppressed in immune tolerance. That there was no increase in yields of PBL with early gingivitis does not favor the former alternative. More direct quantification of B and T cells is still required. The absence of PBL response to LPS before period I is interesting considering the subject's recurrent exposure to the gram-negative flora of the gastrointestinal tract, including the oral cavity (11, 23, 29). Specific antibodies and plasma factors (33, 37) as well as the indirect (synergism) and direct in vitro PBL response to LPS attests to the immunocompetence of humans for LPS. Ubiquitous dental plaque in general, and LPS in particular, may represent antigenic "noise" to the immune system, and a tolerance mechanism to prevent irrelevant response may be required. Such a mechanism may be overwhelmed by abnormal buildup of plaque during experimental gingivitis. Whether subjects whose normal oral hygiene is neglected respond to a sudden plaque buildup as did the subjects studied in this investigation remains to be determined. ACKNOWLEDGMENTS We thank Harriet Kasprick and Gerald Sherek for technical assistance and Eileen Jenkins for preparing the manuscript. This work was supported by Public Health Service grant 5R01-DE-03174-03 from the National Institute of Dental Research and a research grant from the Graduate School of the University of Minnesota. LITERATURE CITED 1. Amerding, D., and D. H. Katz. 1972. Activation of T and B lymphocytes in vitro. I. Regulatory influence of bacterial lipopolysaccharide (LPS) on specific T-cell helper function. J. Exp. Med. 139:24-43. 2. Anderson, J., G. Moller, and 0. Sjoberg. 1972. B lymphocytes can be stimulated by concanavalin A in the presence of humoral factors released by T cells. Eur. J. Immunol. 2:99-101. 3. Berkenbilt, D. A., and A. N. Bahn. 1971. Development of antibodies to cariogenic streptococci in children. J. Am. Dent. Ass. 83:332-337. 4. Blackman, U., S. R. Graboff, G. E. Hagg, E. Gottfeld, and M. J. Pickett. 1974. Experimental cholera in chinchillas: the immune response in serum and intestinal secretions to Vibrio cholerae and choleratoxin. Infect. Immun. 10:1094-1104. 5. Bratthall, D., and R. J. Gibbons. 1975. Changing agglutination activities of salivary immunoglobulin A preparations against oral streptococci. Infect. Immun. 11:603-606. 6. Cohen, J. J., and H. N. Claman. 1971. Thymus-marrow immunocompetence. V. Hydrocortisone-resistant cells and processes in the hemolytic antibody response of mice. J. Exp. Med. 133:1026-1034.
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7. Douglas, S. D., R. M. Kamin, and H. H. Fudenberg. 1969. Human lymphocyte response to phytomitogens in vitro: normal, agammaglobulinemic and paraproteinemic individuals. J. Immunol. 103:1185-1195. 8. Forbes, J. T., Y. Nako, and R. Smith. 1975. T mitogens trigger LPS responsiveness in mouse thymus cells. J. Immunol. 114:1004-1007. 9. Gaumer, H. R., and J. H. Schwab. 1971. Differential susceptibility of mouse lymphocytes to an immunosuppressant from group A streptococci. Cell. Immunol. 4:394-406. 10. Genco, R. J., P. A. Mashimo, G. Krygier, and S. A. Ellison. 1974. Antibody mediated effects on the periodontium. J. Periodontol. 45:330-337. 11. Gibbons, R. J., S. S. Socransky, S. Sawyer, B. Kapsimalis, and J. B. McDonald. 1963. The microbiota of the gingival crevice area of man. II. The predominant cultivatable organisms. Arch. Oral Biol. 8:281-289. 12. Gilmour, M., and R. J. Nisengard. 1974. Interactions between serum titers to filamentous bacteria and their relationship to human periodontal disease. Arch. Oral Biol. 19:959-968. 13. Gothier, D., H. R. Gaumer, B. Pihlstrom, and L. E. A. Folke. 1975. Elevation of a serum component in periodontal disease capable of modulating chemotactic infiltration. J. Periodont. Res. 10:65-72. 14. Gronowicz, E., A. Coutinko, and G. Moller. 1974. Differentiation of B cells: sequential appearance of responsiveness to polydorsal activators. Scand. J. Immunol. 3:413-421. 15. Guggenheim, B., and H. E. Schroeder. 1974. Reactions in the periodontium to continuous antigenic stimulation in sensitized gnotobiotic rats. Infect. Immun. 10:565-577. 16. Hayri, P., and V. Defendi. 1970. Cultivation conditions and mixed lymphocyte interaction of mouse peripheral lymphocytes. Clin. Exp. Immunol. 6:345-346. 17. Horton, J. E., S. Leiken, and J. J. Oppenheim. 1972. Human lymphoproliferative reaction to saliva and dental plaque deposits: an in vitro correlation with periodontal disease. J. Periodontol. 43:522-527. 18. Ivanyi, L., and T. Lehner. 1970. Stimulation of lymphocytes by bacterial antigens in patients with periodontal disease. Arch. Oral Biol. 15:1089-1096. 19. Ivanyi, L., and T. Lehner. 1971. Lymphocyte transformation by sonicates of dental plaque in human periodontal disease. Arch. Oral Biol. 16:1117-1121. 20. Ivanyi, L., and T. Lehner. 1971. The significance of serum factors in stimulation of lymphocytes from patients with periodontal disease by Veillonella alkalescens. Int. Arch. Allergy 41:620-627. 21. Kauffman, C., J. Phair, C. Linnemann, and G. Schiff. 1974. Cell-mediated immunity in humans during viral infection. I. Effect of rubella on hypersensitivity, phytohemagglutinin response, and T lymphocyte numbers. Infect. Immun. 10:212-215.
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22. Kiger, R. D., W. H. Wright, and H. R. Creamer. 1974. The significance of lymphocyte transformation responses to various microbial stimulants. J. Periodontol. 45:780-785. 23. Knox, D. W. 1970. Antigens of oral bacteria. Adv. Oral Biol. 4:92-103. 24. Lagrange, P. H., G. B. Mackaness, T. E. Miller, and P. Pardon. 1975. Effects of bacterial lipopolysaccharide on the induction and expression of cell mediated immunity. I. Depression of the afferant arc. J. Immunol. 114:442-446. 25. Lang, N. P., and F. N. Smith. 1976. Lymphocyte response to T-cell mitogen during experimental gingivitis. Infect. Immun. 13:108-113. 26. Lehner, T., S. J. Challacombe, L. Ivanyi, and J. M. A. Wilton. 1973. The relationship between serum and salivary antibodies and cell mediated immunity in oral disease in man. Adv. Exp. Med. Biol. 45:485-496. 27. Loe, H., and J. Silness. 1963. Periodontal disease in pregnancy. I. Prevalence and severity. Acta Odontol. Scand. 21:533-551. 28. Loe, H., E. Thielade, and S. B. Jensen. Experimental gingivitis in man. J. Periodont. Res. 36:177-187. 29. Loesche, W. J., and R. J. Gibbons. 1968. Amino acid fermentation by Fusobacterium nucleatum. Arch. Oral Biol. 13:191-201. 30. Moller, G., 0. Sjoberg, and J. Anderson. 1973. Immunogenicity, tolerogenicity, and mitogenicity of lipopolysaccharides. J. Infect. Dis. 128:552-556. 31. Ozato, K., W. H. Adler, and J. D. Ebert. 1975. Synergism of bacterial lipopolysaccharides and concanavalin A in the activation of thymic lymphocytes. Cell. Immunol. 17:532-541. 32. Peavy, D. L., J. W. Shands, W. Adler, and R. T. Smith. 1973. Selective effects of bacterial endotoxins on various subpopulations of lymphoreticular cells. J. Infect. Dis. 128:583-591. 33. Rudbach, J. A., and M. K. Luoma. 1974. Endotoxinaltering activity of plasma does not affect antigenicity of native protoplasmic polysaccharide. Infect. Immun. 10:1183-1184. 34. Schmidke, J. R., and J. S. Najarian. 1975. Synergistic effects on DNA synthesis of phytohemagglutinin or concanavalin A and lipopolysaccharide on human peripheral blood lymphocytes. J. Immunol. 114:742-746. 35. Schroeder, H. E., S. Munzel-Pedrazzoli, and R. Page. 1973. Correlated morphometric and biochemical analysis of gingival tissue in early chronic gingivitis in man. Arch. Oral Biol. 18:899-923. 36. Vesikari, T., and E. Buimovici-Klein. 1975. Lymphocyte responses to rubella antigens and phytohemagglutinin after administration of the RA27/3 strain of live, attenuated rubella vaccine. Infect. Immun. 11:748-753. 37. Wolff, S. M. 1973. Biological effects of bacterial endotoxins in man. J. Infect. Dis. 128:5251-5256.
