Vol. 13, No. 1
INFECTION AND IMMUNITY, Jan. 1976,l p. 108-113 Copyright © 1976 American Society for Microbiology
Printed in U.SA.
Lymphocyte Response to T-Cell Mitogen During Experimental Gingivitis in Humans NIKLAUS P. LANG'* AND FREDERIC N. SMITH Dental Research Institute and Department of Periodontics, The University of Michigan and Veterans Administration Hospital, Ann Arbor, Michigan 48104 Received for publication 10 June 1975
This study was conducted to evaluate the dose response relationships of peripheral blood lymphocytes (PBL) by stimulation of phytohemagglutinin (PHA) during the onset of oral inflammation. Eleven dental students underwent a 3-week experimental gingivitis program (Loe et al., 1965). At time zero, weeks 1, 2, and 3, and after 1 week of reinstituted oral hygiene (week 4), the plaque accumulations were evaluated, the degree of gingival inflammation was assessed, and a blood sample was taken. Quadruplicate microcultures each containing 2 x 105 PBL in 0.2 ml of tissue culture medium 199 and 10% fetal calf serum were stimulated with five concentrations of PHA (10 to 0.5 ,ug/ml) and incubated for 78 h at 37 C in 5% C02. [3H]thymidine was added to each culture for the final 8 h. The cultures were then harvested and counted by liquid scintillation, and stimulation indexes (SI) were determined. At time zero the maximum PBL response occurred at a PHA concentration of 5 ug/ml (SI = 100). During weeks 1, 2, and 3 the location of the maximum PBL response shifted to a lower PHA concentration (1.0 ,ug/ml) and increased to over SI = 400. The phenomenon of shifting of peak PHA responses to lower PHA concentrations could be observed after only 1 week of developing gingival inflammation. The PBL response returned to pre-experimental values after 1 week of reinstituted oral hygiene, which resolved the oral inflammation. The findings show that a dose response relationship exists between PHA concentrations and the PBL response. If these dose response changes seen during developing gingival inflammation are ignored, either a decrease, increase, or no change in PBL response can be shown depending upon the PHA concentration evaluated. Owing to the dose-dependent nature of this PBL response, it is advisable to routinely use dose response curves in order to properly evaluate the full responsiveness of PBL to mitogenic substances.
The response of peripheral blood lymphocytes gingival inflammation, upon the PBL response (PBL) to certain plant mitogens is often used as to PHA in humans. an in vitro correlate of their immunological MATERIALS AND METHODS responsiveness (2). Certain mitogens are spein Twelve dental students, aged 22 to 26 years, parcific their ability to induce proliferation of different lymphocyte subpopulations. Phytohe- ticipated in the study. They were divided into three magglutinin (PHA) and concanavalin A are groups of four to assure reproducibility of the systhorough prophylaxis and known to be T-cell specific (12) and are, there- tem. They each received abefore the beginning of the hygiene instruction fore, frequently used in assessing lymphocytes oral were and Clean teeth healthy experiment. involved in delayed hypersensitivity. However, required of each student at the start ofgingivae the research the responsiveness of T-lymphocytes to PHA period. They then abstained from all oral hygiene appears to be affected by both the experimental measures for 3 weeks, allowing dental plaque to method (4) and the status of the lymphocyte accumulate and gingivitis to develop (18). Thorough donor. Donor parameters including age (8, 25) oral hygiene measures were reinstituted after the and health status (13, 14, 19, 21) can influence 3rd week. At the beginning of the study, after 1, 2, T-cell responses. This study was undertaken to and 3 weeks of no oral hygiene, and 1 week after plaque was assessed determine the effect of a change in health sta- reinstitution of oral hygiene, to the criteria of the plaque index system tus, as demonstrated by the development of according (P1 I) (22). Gingival health was determined by the 1 Present Address: University of Berne, School of Dental criteria of the gingival index system (GI) (17). At the same time, 25 ml of blood was drawn from the ante-
Medicine, Freiburgstrasse 7, CH 3010, Berne, Switzerland.
108
PHA RESPONSE DURING EXPERIMENTAL GINGIVITIS
VOL. 13, 1976
109
cubital fossa. After sedimentation for 90 min, the lated to a P1 I of 1.46 (±0.2) after 1 week. A leukocyte-rich plasma was removed and the cells peak plaque load was reached after 3 weeks (P1 were centrifuged and washed three times in Hanks I = 1.78 ± 0.28). During this time the GI inbalanced salt solution (Difco). The number of viable creased in a linear fashion, reaching a peak cells was determined by the trypan blue dye exclu- after 3 weeks (GI = 1.11 ± 0.07). After reinstitusion test (6). Quadruplicate microcultures, each con- tion of oral hygiene procedures, the mean value taining 2 x 105 PBL in 0.2 ml of tissue culture PI I fell to the pre-experimental level of for the medium 199 (GIBCO) and 10% fetal calf serum (GIBCO), were stimulated with PHA-P (Difco) in 0.14 (±0.12) after 1 week. The mean values for concentrations of 0.5, 1.0, 2.5, 5.0, and 10.0 plg/ml. the GI also dropped significantly to 0.38 The cultures were incubated for 78 h at 37 C in a (±0.12). humid 95% air and 5% CO2 environment. For the Peak SI are shown in Fig. 2. At the start of final 8 h, [methyl-3H]thymidine (specific activity 6.7 the experiment the peak SI was 109 (±36). The Ci/mmol; New England Nuclear Corp.) was added to SI increased by 1 week to 249 (±32), rose to 403 each culture in order to measure deoxyribonucleic (±108) by the 2nd week, and decreased to 277 acid synthesis. The cells were harvested, precipiretated on filters with 10% trichloroacetic acid, and (±72) at the end of the 3rd week. The 1 SIweek prepared for liquid scintillation counting. The turned to pre-experimental levels after counts per minute were recorded and corrected for of reinstituted oral hygiene measures (SI = 109 chemical quenching. Stimulation indexes (SI) were ± 20). The weekly changes in SI all differed to a calculated by dividing the mean counts per minute statistically significant level (P < 0.01) except of the stimulated cultures by the mean counts per for the apparent decrease from weeks 2 to 3, minute of unstimulated saline controls. Dose re- which was not significant. sponse curves were obtained for each sample, and Initial and final peak SI occurred at a PHA the weekly results were compared with results ofthe concentration of 5 ,ug/ml. During the period of previous week by paired t tests. developing gingivitis, however, the peak SI responses occurred at 1 ,ug/ml, with a fivefold RESULTS decrease in PHA concentration (Fig. 2). The One of the subjects in group III was unable to total dose response relationships between the complete the entire 4 weeks required for the PBL stimulation and PHA concentration are experiment and was, therefore, excluded from demonstrated in Fig. 3. The peak SI generally increased, and this increase occurred at lower the data.
At the start of the experiment the subjects displayed a mean P1 I of 0.16 (±0.15) and a mean GI of 0. 12 (±0.07) (Fig. 1). After cessation of oral hygiene their plaque deposits accumu-
PHA concentrations during developing gingival inflammation. Although the percentage of lymphocytes in the cell-rich plasma was monitored, a strict P- i Gl DU' NG EXPEFIVETJA CMGNJI II IS N M.L:N
1 D-
FIG. 1. Mean plaque indexes (PI ) and gingival indexes (GI) ofthe 11 subjects during onset and recovery of experimental gingivitis. Oral hygiene was abolished after the clinical scoring of week 0 and was reinstituted after the clinical scoring of week 3.
110
LANG AND SMITH CPM
INFZCT. IMMUN.
Si
24000
500-
T
450-~
PEAK RESPONSE OF PBL TO PHA-P DURING EXPERIMENTAL GINGIVITIS IN MAN
350-
300T
250-
peak
150-
concentration pha-p * 5 ug/mI
100-
*pg/ml
200-
70W
: P
INFECTION AND IMMUNITY, Jan. 1976,l p. 108-113 Copyright © 1976 American Society for Microbiology
Printed in U.SA.
Lymphocyte Response to T-Cell Mitogen During Experimental Gingivitis in Humans NIKLAUS P. LANG'* AND FREDERIC N. SMITH Dental Research Institute and Department of Periodontics, The University of Michigan and Veterans Administration Hospital, Ann Arbor, Michigan 48104 Received for publication 10 June 1975
This study was conducted to evaluate the dose response relationships of peripheral blood lymphocytes (PBL) by stimulation of phytohemagglutinin (PHA) during the onset of oral inflammation. Eleven dental students underwent a 3-week experimental gingivitis program (Loe et al., 1965). At time zero, weeks 1, 2, and 3, and after 1 week of reinstituted oral hygiene (week 4), the plaque accumulations were evaluated, the degree of gingival inflammation was assessed, and a blood sample was taken. Quadruplicate microcultures each containing 2 x 105 PBL in 0.2 ml of tissue culture medium 199 and 10% fetal calf serum were stimulated with five concentrations of PHA (10 to 0.5 ,ug/ml) and incubated for 78 h at 37 C in 5% C02. [3H]thymidine was added to each culture for the final 8 h. The cultures were then harvested and counted by liquid scintillation, and stimulation indexes (SI) were determined. At time zero the maximum PBL response occurred at a PHA concentration of 5 ug/ml (SI = 100). During weeks 1, 2, and 3 the location of the maximum PBL response shifted to a lower PHA concentration (1.0 ,ug/ml) and increased to over SI = 400. The phenomenon of shifting of peak PHA responses to lower PHA concentrations could be observed after only 1 week of developing gingival inflammation. The PBL response returned to pre-experimental values after 1 week of reinstituted oral hygiene, which resolved the oral inflammation. The findings show that a dose response relationship exists between PHA concentrations and the PBL response. If these dose response changes seen during developing gingival inflammation are ignored, either a decrease, increase, or no change in PBL response can be shown depending upon the PHA concentration evaluated. Owing to the dose-dependent nature of this PBL response, it is advisable to routinely use dose response curves in order to properly evaluate the full responsiveness of PBL to mitogenic substances.
The response of peripheral blood lymphocytes gingival inflammation, upon the PBL response (PBL) to certain plant mitogens is often used as to PHA in humans. an in vitro correlate of their immunological MATERIALS AND METHODS responsiveness (2). Certain mitogens are spein Twelve dental students, aged 22 to 26 years, parcific their ability to induce proliferation of different lymphocyte subpopulations. Phytohe- ticipated in the study. They were divided into three magglutinin (PHA) and concanavalin A are groups of four to assure reproducibility of the systhorough prophylaxis and known to be T-cell specific (12) and are, there- tem. They each received abefore the beginning of the hygiene instruction fore, frequently used in assessing lymphocytes oral were and Clean teeth healthy experiment. involved in delayed hypersensitivity. However, required of each student at the start ofgingivae the research the responsiveness of T-lymphocytes to PHA period. They then abstained from all oral hygiene appears to be affected by both the experimental measures for 3 weeks, allowing dental plaque to method (4) and the status of the lymphocyte accumulate and gingivitis to develop (18). Thorough donor. Donor parameters including age (8, 25) oral hygiene measures were reinstituted after the and health status (13, 14, 19, 21) can influence 3rd week. At the beginning of the study, after 1, 2, T-cell responses. This study was undertaken to and 3 weeks of no oral hygiene, and 1 week after plaque was assessed determine the effect of a change in health sta- reinstitution of oral hygiene, to the criteria of the plaque index system tus, as demonstrated by the development of according (P1 I) (22). Gingival health was determined by the 1 Present Address: University of Berne, School of Dental criteria of the gingival index system (GI) (17). At the same time, 25 ml of blood was drawn from the ante-
Medicine, Freiburgstrasse 7, CH 3010, Berne, Switzerland.
108
PHA RESPONSE DURING EXPERIMENTAL GINGIVITIS
VOL. 13, 1976
109
cubital fossa. After sedimentation for 90 min, the lated to a P1 I of 1.46 (±0.2) after 1 week. A leukocyte-rich plasma was removed and the cells peak plaque load was reached after 3 weeks (P1 were centrifuged and washed three times in Hanks I = 1.78 ± 0.28). During this time the GI inbalanced salt solution (Difco). The number of viable creased in a linear fashion, reaching a peak cells was determined by the trypan blue dye exclu- after 3 weeks (GI = 1.11 ± 0.07). After reinstitusion test (6). Quadruplicate microcultures, each con- tion of oral hygiene procedures, the mean value taining 2 x 105 PBL in 0.2 ml of tissue culture PI I fell to the pre-experimental level of for the medium 199 (GIBCO) and 10% fetal calf serum (GIBCO), were stimulated with PHA-P (Difco) in 0.14 (±0.12) after 1 week. The mean values for concentrations of 0.5, 1.0, 2.5, 5.0, and 10.0 plg/ml. the GI also dropped significantly to 0.38 The cultures were incubated for 78 h at 37 C in a (±0.12). humid 95% air and 5% CO2 environment. For the Peak SI are shown in Fig. 2. At the start of final 8 h, [methyl-3H]thymidine (specific activity 6.7 the experiment the peak SI was 109 (±36). The Ci/mmol; New England Nuclear Corp.) was added to SI increased by 1 week to 249 (±32), rose to 403 each culture in order to measure deoxyribonucleic (±108) by the 2nd week, and decreased to 277 acid synthesis. The cells were harvested, precipiretated on filters with 10% trichloroacetic acid, and (±72) at the end of the 3rd week. The 1 SIweek prepared for liquid scintillation counting. The turned to pre-experimental levels after counts per minute were recorded and corrected for of reinstituted oral hygiene measures (SI = 109 chemical quenching. Stimulation indexes (SI) were ± 20). The weekly changes in SI all differed to a calculated by dividing the mean counts per minute statistically significant level (P < 0.01) except of the stimulated cultures by the mean counts per for the apparent decrease from weeks 2 to 3, minute of unstimulated saline controls. Dose re- which was not significant. sponse curves were obtained for each sample, and Initial and final peak SI occurred at a PHA the weekly results were compared with results ofthe concentration of 5 ,ug/ml. During the period of previous week by paired t tests. developing gingivitis, however, the peak SI responses occurred at 1 ,ug/ml, with a fivefold RESULTS decrease in PHA concentration (Fig. 2). The One of the subjects in group III was unable to total dose response relationships between the complete the entire 4 weeks required for the PBL stimulation and PHA concentration are experiment and was, therefore, excluded from demonstrated in Fig. 3. The peak SI generally increased, and this increase occurred at lower the data.
At the start of the experiment the subjects displayed a mean P1 I of 0.16 (±0.15) and a mean GI of 0. 12 (±0.07) (Fig. 1). After cessation of oral hygiene their plaque deposits accumu-
PHA concentrations during developing gingival inflammation. Although the percentage of lymphocytes in the cell-rich plasma was monitored, a strict P- i Gl DU' NG EXPEFIVETJA CMGNJI II IS N M.L:N
1 D-
FIG. 1. Mean plaque indexes (PI ) and gingival indexes (GI) ofthe 11 subjects during onset and recovery of experimental gingivitis. Oral hygiene was abolished after the clinical scoring of week 0 and was reinstituted after the clinical scoring of week 3.
110
LANG AND SMITH CPM
INFZCT. IMMUN.
Si
24000
500-
T
450-~
PEAK RESPONSE OF PBL TO PHA-P DURING EXPERIMENTAL GINGIVITIS IN MAN
350-
300T
250-
peak
150-
concentration pha-p * 5 ug/mI
100-
*pg/ml
200-
70W
: P
