663
CD4 to CD8 Ratio and In Vitro
Lymphoproliferative Responses During Experimental Gingivitis in Pregnancy and Post-Partum
J.E.
Raber-Durlacher,"
W.P.
Zeijlemaker,* A.A. P. Meinesz,' and L. Abraham-Inpijn
The absolute numbers and percentages of peripheral , B, and NK cells were assessed in 7 women, both during the second trimester of pregnancy and 6 months Postpartum. Furthermore, the in vitro responses of peripheral blood lymphocytes (PBL) to several mitogens and a preparation of Prevotella intermedia were compared in a period of experimentally-induced gingivitis during pregnancy and post-partum. Clinically, the periodontal pocket bleeding index (PPBI) was found to be higher during pregnancy than post-partum. The absolute numbers of CD3, CD4, and CD19 positive cells appeared to be decreased during pregnancy as compared to post-partum. However, the results did not indicate any evidence for a reduced in vitro PBL response to several mitogens and a preparation of P. intermedia during pregnancy. J Periodontol 1991; 62:663-667.
Key Words: Lymphocytes; pregnancy; gingivitis; Prevetolla intermedia.
Pregnancy gingivitis may be partly due to changes in maternal immunity. The existence of such changes that may
result in an overall increased susceptibility to infection1 can be inferred from diminished in vitro proliferative responses of peripheral blood lymphocytes (PBL) to preparations of Prevotella intermedia.2 This black-pigmented anaerobic microorganism is associated with pregnancy gingivitis,3 acute necrotizing gingivitis,4 and experimental gingivitis.5 In addition, a decrease in the ratio of peripheral helper cells to suppressor-cytotoxic cells (CD4/CD8 ratio) has been observed during pregnancy.6 It has been suggested that this phenomenon may contribute to increased gingival inflammation during pregnancy, as helper cells are thought to play a primarily protective role in the pathogenesis of periodontal diseases.7 Recently, we found evidence for the contention that the in vitro stimulation of lymphocytes by preparations of P. intermedia is antigen-specific, although a secondary cell-dependent polyclonal cell activation may occur.8 The present study forms a part of an investigation in which the development of experimentally induced gingival inflammation9 was studied during pregnancy, and 6 months post-partum. The aims of this study were to determine whether: 1) An alteration in the levels of peripheral and cells, and a decrease in the numbers of helper
'Department of General Pathology and Internal Medicine, Academic Cenfor Dentistry Amsterdam, (ACTA) Amsterdam, The Netherlands. Department of Clinical Immunobiology and Hybridoma Laboratory, Central Laboratory of the Netherlands Red Cross Blood Transfusion Service, ter
Amsterdam, The Netherlands.
resulting in lower CD4/CD8 ratios could be observed during the second trimester of pregnancy as compared to the levels of these cell populations post-partum; and 2) if any evidence for immunosuppression during pregnancy could be found when the in vitro proliferation of PBL to mitogens and preparations of P. intermedia was assessed during the various phases of the study. cells
MATERIALS AND METHODS Human Subjects and Study Design Nine gravidae (mean age 25.3 years, range 20 to 34) were selected to participate in this study before their 12th week of pregnancy (initial evaluation). The outline of the investigation is presented in Figure 1. All participants were in good physical health and no medication was taken that could be expected to influence the immune system. No features of periodontal diseases were present. Furthermore, there were no inadequate restorations interfering with the removal of dental plaque. After informed consent was secured, all patients were subjected to a carefully controlled oral hygiene program in order to reduce pre-existent inflammation as much as possible. The amount of dental plaque and gingival health were evaluated at least every 3 weeks, until experimental gingivitis was induced. At day 0, in their 25th week of pregnancy, the subjects were instructed to abstain from all oral hygiene procedures for a period of 14 days. Clinical examinations were performed at the initial evaluation and at day 0, 4, 8, and 14. These examinations in-
664
J Periodontol November 1991
LYMPHOCYTES AND PREGNANCY
POSTPARTUM
PREGNANT EXPERIMENTAL
EXPERIMENTAL
GINGIVITIS
GINGIVITIS
OPTIMAL
CESSATION OF
OPTIMAL
ORAL HYGIENE
ORAL HYGIENE
ORAL HYGIENE
CESSATION OF ORAL HYGIENE
-M-
Figure
1. Outline
of the investigation.
eluded the assessment of the
Plaque Index, (PI)10 and the index periodontal pocket bleeding (PPBI)11 at the mesial and distal sites of the teeth. At day 0 and day 14, peripheral venous blood was obtained to isolate mononuclear cells. At the end of the oral hygiene abstention period, a thorough dental prophylaxis was performed, and oral hygiene measures were reinstituted. About 12 weeks post-partum the participants in this study were seen again and the procedures as described above were repeated. Unfortunately, 1 subject was unable to participate in the investigation after parturition.
of the bicinchoninic acid protein assay.15 The protein concentration was 0.85 mg/ml in a dilution of 1:64. In addition, PBL were cultured in the presence of the following cell mitogens: PHA (purified phytohaemagglutinin HA 16)11 in a final (optimal) concentration of 12 pg/ml, ALS1 (Horse anti-human lymphocyte serum), in a final dilution of 1:32, Con A,# (Concanavalin A no. C-2010 type IV) in a final concentration of 60 µg/ ll. Furthermore, cells CD3 (subclass IgG2a)1 were cultured in the presence of in a final dilution of 1:10.
Isolation of Mononuclear Cells Sixty ml of blood was collected by venipuncture of the median cubital vein, between 9:00 and 9:30 a.m. Peripheral blood mononuclear cells (PBL) were isolated from the heparinized blood by Ficoll-Isopaque density layer centrifugádon and preserved in liquid nitrogen.12
Culture Procedures All cultures were performed in round-bottom microtiter plates (200 µ per well).** Lymphocytes were cultured in Iscove's modified Dulbecco's medium (IMDM) supplemented with 20% heat-inactivated pooled human serum and standard antibiotics at 37°C in a humidified 95% air, 5% C02 atmosphere. Cultures stimulated by PHA, ALS, Con A, and 104 lymphocytes per well aCD3 were performed with 4 and harvested after 4 days. On the basis of earlier findings for measurement of antigen-specific lymphocyte stimulation, a higher cell number (20 105 per well), and a longer culture period (8 days) were used for cultures stimulated by P. intermedia? Proliferative responses were determined by measuring the incorporation of 3H-thymidine (0.4 µ . : specific activity 200 Ci/mol) added 24 hours before harvesting. The cells were harvested onto glass fiber filters and the radioactivity was measured with a liquid scintillation counter. All cultures were performed in triplicate. Variability among triplicate values was always less than 10%. In unstimulated cultures, 3H-thymidine incorporation was nearly always less than 100 cpm; therefore, no standard deviation is given. Lymphocyte cultures of 1 subject were performed in one experiment on the same day, thus avoiding day-to-day variations.
Determination of Lymphocyte Subsets Lymphocyte subsets were determined by FACS cytofluorimetry with monoclonal antibodies against the following surface markers: CD3, CD4, CD8, CDllb,* and CD19.§ Peripheral blood mononuclear cells were incubated with 30 µ of these monoclonal antibodies. Subsequently, the cells were washed twice with PBS containing 0.5% bovine serum albumin and 0.01% sodium azide, incubated on ice for a further 30 minutes with fluorescein-conjugated-goat antimouse IgG. After 2 washings, the number of positive cells was counted in a flow cytometer system. The absolute numbers of cell subsets were calculated from the total number of leukocytes, the percentage of lymphocytes, and the distribution of lymphocyte subpopulations.
Lymphocyte Stimulants A preparation of P. intermedia strain H6 110 (ATCC 25611) was kindly provided by Dr. T.J.M. van Steenbergen, Academic Center for Dentistry Amsterdam (ACTA). Bacteria were cultured in liquid BM medium,13 centrifuged, and collected in phosphate buffered saline. An extract was prepared by freeze-thawing, sonication, and centrifugádon of cell wall fragments as described by Baker and Tondreau.14 Protein concentration of bacterial sonicate was measured by *Becton-Dickinson, Mountain View, CA. 'Central Laboratory of The Netherlands Service, Amsterdam.
Red Cross Blood Transfusion
means
Statistical Procedures The student r-test (double tailed) for matched samples and the Wilcoxon signed rank test were used for the statistical evaluation of the different parameters. Values of < 0.05 were
accepted as statistically significant.
Wellcome Diagnostics, Amsterdam, The Netherlands. "CLB, Amsterdam, The Netherlands. 'Sigma Chemical Co., St. Louis, MO. "Greiner, Langenthal, Switzerland.
Volume 62 Number 11
RABER-DURLACHER, ZEULEMAKER, MEINESZ, ABRAHAM-INPUN
PPBI
665
As seen in Figure 2, all subjects had gingivitis the initial evaluation in the first trimester of pregnancy which decreased after a period of optimal oral hygiene. During the 14-day period of cessation of all oral hygiene measures, the PPBI increased in all subjects tested. On day 0 as well as on day 14 of experimental gingivitis the mean PPBI was higher in the group during pregnancy than in the post-partum group. The amount of dental plaque that accumulated during both experimental phases of the study was rather similar (data not shown).
gingivitis. at
day
day
0
day
day ß
4
during
14
post partum
pregnancy
Figure 2. The mean periodontal pocket bleeding index (PPBI) + SD for the interproximal sites of the investigated subjects (n 8) during preg=
nancy and post-partum.
RESULTS Induction of Experimental Gingivitis In 8 individuals, some parameters of immunocompetence were studied in relation to the development of experimental gingivitis during pregnancy and post-partum (see Fig. 1 for an outline of the study). The periodontal pocket bleeding index (PPBI) was taken as a measure for the severity of
Leukocyte Subsets Leukocyte subsets in peripheral blood, as assessed in the experiment outlined above, are shown in Table 1. However,
in one donor we were not able to determine the distribution of these subsets, due to a lack of cells. In the pregnant group the absolute numbers of CD3-positive cells seemed to be slightly decreased during pregnancy as compared to the numbers of these cells assessed post-partum (marginally significant 0.05). The same was true for the mean absolute numbers of CD4-positive cells (P 0.07). Howthe number of CD4 cells decreased was not ever, (absolute) in each pregnant individual. The mean numbers of CD8positive suppressor/cytotoxic cells were found to be not =
=
Phenotypic Characterization of Cells Isolated From Peripheral Blood in 7 Subjects During Pregnancy and Post-Partum in Percentages and Absolute Values Expressed in lO'/Liter
Table 1.
During Pregnancy
CD4
CD3 abs
CD8 abs.
CD19 abs
CD4/CD8
Patient
%
A
79.2 85.2 86.2 74.3 89.0 93.7 89.7
1.13 1.98 1.99 1.04 0.77 1.47 0.76
52.5 48.2 52.6 50.7 65.0 61.4 65.9
0.73 1.09 1.21 0.71 0.56 0.96 0.65
32.5 37.3 33.3 26.1 23.0 33.6 22.1
0.82 0.77 0.37 0.20 0.53 0.22
ratio 1.59 1.29 1.57 1.92 2.80 1.81 2.98
85.30 6.62
1.31* 0.52
56.60 7.28
0.84* 0.24
29.70 5.91
0.48 0.25
1.99 0.65
1.23 2.78 3.01 0.74 1.13 1.70 1.69
52.7 69.5 77.1 49.9 59.0 55.0 55.1
0.76 2.13 1.96 0.51 0.80 1.10 1.08
39.4 23.3 33.8 26.5 17.0 33.3 31.3
0.57 0.72 1.15 0.27 0.23 0.67 0.62
1.33 2.96 1.70 1.88 3.48 1.64 1.74
1.75 0.85
59.76 9.89
1.19 0.24
29.23 7.50
0.60 0.31
2.10 0.79
C D E F G mean
SD
±
no.
Post-Partum 84.7 A 90.6 C 88.5 D 72.1 E 83.0 F 85.0 G 86.0 mean
SD
±
84.30 5.94
%
no.
%
no.
0.46
abs
4.0 9.0
0.26 0.14 0.21 0.11 0.07 0.06 0.12
CDllb % 17.6 9.4 11.5 9.3 29.0 11.1 12.0
8.84 4.87
0.14t 0.07
14.27 7.06
31.3 5.2 7.7 24.0 13.0 5.6 8.1
0.45 0.16 0.26 0.20 0.18 0.11 0.19
12.5 19.5 9.3 12.1 28.0 20.4 14.3
13.56 10.18
0.22 0.11
16.59 6.43
%
no.
18.8 4.6 9.4 8.1 8.0
Normal Values
CD3
CD4
CD8
CD4/CD8
abs
abs
abs
ratio
CD19 abs
0.77-2.84
0.10-0.72
no.
no.
no.
0.87-2.38
0.50-1.56
0.31-1.11
'Marginally significant. fVery significant.
no.
CD3: total number of T-cells; CD4: helper cells; CD8: suppressor cells, cytototoxic cells; CDllb: natural killer cells, monocytes, polymorphonuclear cells; and CD19: cells. The normal values were derived from over 1,000 healthy individuals.
J Periodontol November 1991
LYMPHOCYTES AND PREGNANCY
666
significantly
different during pregnancy and post-partum. individual level, however, there was some variation present in the numbers of these cells during pregnancy and 6 months after parturition. As a result the CD4/CD8 ratio was decreased only in subjects and E during pregnancy as compared to the CD4/CD8 ratio found in these individuals post-partum. The mean CD4/CD8 ratio calculated for the pregnant and the post-partum groups did not differ significantly. With regard to the CD19 positive cells, the numbers were found to be decreased during pregnancy as compared to the numbers of these cells post-partum (P < 0.01). In addition, no significant differences were observed during pregnancy and post-partum with respect to CDllb positive cells. The values of lymphocyte subsets during pregnancy were compared, not only to those of the same individuals post-partum, but also to the normal values derived from over 1,000 healthy individuals, determined over a period of 2.5 years. The data found during pregnancy appeared to be within the range of these normal values. At
DISCUSSION A decreased ratio of helper (CD4) to suppressor (CD8) cells, as a result of decreased proportions and numbers of helper cells, has been observed during the second and third trimesters of pregnancy.6 It has been suggested that this decreased CD4/CD8 ratio indicates the presence of immunodeficiency during pregnancy, as measured in vitro by
an
decreased lymphocyte proliferation.16 Clinically, gestational changes in immunity result in an increased overall susceptibility to infection.1 There are numerous reports dealing with pregnancy gingivitis, which is characterized by increased redness, edema, and a marked tendency to bleeding as compared to the gingival state post-partum.10,17'18 In addition to gestational changes in plasma levels of steroid hormones and a concomitant shift in the subgingival bacterial flora,3 there is evidence for a diminished in vitro response of PBL when cultured in the presence of preparations of oral microorganisms during pregnancy.2'19 Responses to preparations of P. intermedia, which is associated with pregnancy gingivitis, were reported to be elevated at the post-partum evaluation as compared to the responses to this antigen during the second and third trimesters of pregnancy. More recently, Stashenko and coworkers7 found that low CD4/CD8 ratios were predictive for low cell responses to oral microorganisms in patients with Periodontitis. In addition, patients with a low CD4/CD8 ratio had significantly more redness and bleeding on probing in the presence of approximately equal amounts of dental plaque, as compared to Periodontitis patients with a high CD4/CD8 ratio. It was hypothesized that the helper cell response plays an important protective role in limiting bacteria involved in gingival inflammation. Moreover, it was suggested that pregnancy gingivitis may reflect an analogous situation. In the present study, in which only a limited number of gravidae were investigated, we did not find evidence for a decrease of the mean CD4/CD8 ratio during pregnancy, although the numbers of CD4 positive cells seemed lower during gestation. However, it must be emphasized that
Lymphocyte Stimulation
In the test groups indicated above, lymphocyte reactivity towards mitogens and antigens was determined. The results obtained with lymphocytes from 8 subjects are summarized in Table 2. In this table, the mean (± SD) cpm 3H-thymidine incorporation to each stimulant on days 0 and 14 of both periods of experimental gingivitis is given. Because there are substantial inter-donor variations in the absolute magnitude of the cpm, no statistical analysis could be applied to these data. However, by expressing individual results as ratios (cpm during pregnancy, relative to cpm postpartum), inter-individual variations are circumvented. These data are also shown in Table 2. Statistical analysis, performed by means of the student t test and the Wilcoxon test, revealed no significant differences in the lymphocyte reactivity during pregnancy and
post-partum. Table 2. Mean Post-Partum
Lymphocyte Responses
at
Day 0 and Day 14
to Several
During Pregnancy SD
mean ±
Stimulant PHA ALS Con A CD 3 intermedia sonicate Unstimulated cultures
day 0 45.8 40.9 3.4 6.8 5.7 62.4
±
day 49.1 42.5 4.0 7.6 5.2
59.1
38.6
SD
14
26.0 15.2 3.7
±
SD
mean ±
day 0
4.1 4.1
40.6 33.9 2.8 8.3 4.2
20.1
37.0
±
22.2 16.8 2.3 3.7 3.4
33.8 29.1 1.8 8.3 3.4
26.1
46.0
±
Preparation
of P. intermedia
During Pregnancy and
Mean Ratio
Mean Ratio
day 0 pregnancy day 0 post-partum
day 14 pregnancy day 14 post-partum
15.3 12.4 0.9 3.9 2.9
1.23 1.47 1.88 0.90 3.65
39.8
3.15
mean ±
day
a
SD 14
Post-Partum
mean ±
20.7 14.8 1.9 3.4 4.2
Mitogens And
±
SD
0.39 0.81 1.55 0.41 5.97
1.63 1.83 3.20 0.98 2.35
4.10
0.99
SD
0.87 1.18 3.17 0.51 2.88 ±
0.32
In the stimulated cultures the responses are expressed as cpm IO3. In the unstimulated cultures the responses are expressed as cpm (no standard deviations are given). Because of the high inter-individual variation in the subjects investigated (n 8), the response ratios (response on day 0 during pregnancy/response on day 0 post-partum) were calculated for each individual: the mean values ± SD of these ratios are presented. Statistical analysis on these data revealed no significant difference in the magnitude of the responses during pregnancy and post-partum (P > 0.1). =
Volume 62 Number 11
RABER-DURLACHER, ZEULEMAKER, MEINESZ, ABRAHAM-INPLJN
Stashenko did not find that periodontal patients as a group exhibited lower CD4/CD8 ratios than normal individuals. Low responders; e.g., patients with a low CD4/CD8 ratio could only be identified when the experimental material was divided into subgroups. In our study, we could not find any evidence that the 2 individuals which displayed relatively low CD4/CD8 ratios during pregnancy, as compared to these ratios assessed post-partum, developed more gingivitis during pregnancy than the other pregnant subjects. In addition, we did not find any significant alterations in the percentages of CDllb-positive cells (corresponding to NK cells, monocytes, macrophages, and granulocytes). However, the absolute numbers of cells appeared to be decreased during pregnancy. When the PBL responses on day 0 and day 14 of experimental gingivitis were compared to these responses 6 months post-partum, the in vitro PBL response to several mitogens and a preparation of P. intermedia appeared not to be decreased during pregnancy. However, 14 days of plaque accumulation might be too short to develop a full range of PBL responses for all individuals.20 Furthermore, we were not able to assess any relation between the number of CD4 positive cells and the magnitude of the PBL response. The presence of a higher tendency towards bleeding during pregnancy indicates that the subjects investigated developed more inflammation during the experimental period. However, the PPBI at day 0 during pregnancy, when all efforts were made to obtain a state of optimal gingival health appeared to be increased as compared to the PPBI, associated with similar low plaque scores at day 0 post-partum. Thus, it is possible that pregnancy gingivitis is not only an exaggerated inflammatory response as a result of a disturbed host-parasite equilibrium; it cannot be excluded that the gingiva undergoes physiological changes resulting in increased redness, edema, and higher bleeding tendency, clinically resembling inflammation. Taken together, it may be concluded that in the present study no evidence was found for an overall lower CD4/ CD8 ratio during pregnancy. The numbers of cells, however, were decreased during pregnancy as compared to postpartum. Furthermore, the in vitro PBL responses to cell mitogens and a preparation of P. intermedia seemed not to be decreased during 14 days of experimental gingivitis during pregnancy as compared to the responses post-partum. It should be noted that this study is performed on a limited number of subjects. Possibly, a more extensive group of pregnant subjects and controls would reveal statistically significant differences. It is clear, however, that no gross differences occurred between our study groups.
Acknowledgments
The authors thank the staff of the Department of Biochemistry (Dr. Annemarie Lütjens, director), Andreas Hospital, Amsterdam for determining hématologie parameters. We are also grateful to Dr. Jan Dijkstra, Department of Ob-
stetrics and
667
Gynecology, Andreas Hospital, Amsterdam for
granting permission to examine his patients. We would also
like to thank Dr. John Raber, Dr. Erwin van der Zee, and Wietske Levinson for their editorial help. This study was supported by the Dutch Praeventiefonds grant 28-1176. REFERENCES 1. Brabin BJ.
2.
Kornman
of infection in pregnancy. Rev
KS, Loesche
Infect Dis
WJ. Modulation of immuno-
periodontal disease-associated microorganisms during pregnancy. Infect Immun 1980; 28:713. Kornman KS, Loesche WJ. The subgingival microbial flora during reactivity
3.
Epidemiology
1985; 7:579. Lopatin DE, to
pregnancy. J Periodont Res 1980; 15:111. 4. Slots J. Importance of Black-Pigmented Bacteroides in human periodontal disease. In: Host-Parasite Interactions in Periodontal Diseases. Genco RJ and Mergenhagen SE, eds. Washington, DC; American Society for Microbiology, 1982; 17-45. 5. Loesche WJ, Syed SA. Bacteriology of human experimental gingivitis: effect of plaque and gingivitis score. Infect Immun 1978; 21:830839. 6. Sridama V, Pacini F, Yang SL, Moawad A, Reilly M, DeGroot LJ. Decreased levels of helper cells. A possible cause of immunodeficiency in pregnancy. New Eng J Med 1982; 307:352. 7. Stashenko P, Resmini LM, Haffajee AD, Socransky SS. Helper and suppressor cells in periodontal disease. /Periodont Res 1985; 20:515. 8. Raber-Durlacher JE, Zeijlemaker WP, Meinesz AAP, Abraham-Inpijn L. Stimulation of lymphocytes in vitro by Bacteroides intermedius and Porphyromonas (Bacteroides) gingivalis sonicates. J Periodontol 1990; 61:217. 9. Löe , Theilade E, Jensen SB. Experimental gingivitis in man. / Periodontol 1965; 36:177. 10. Löe H, Silness J. Periodontal disease in pregnancy. Part I: Prevalence and severity. Acta Odontol Scand 1963; 21:533. 11. Van der Veiden U. Probing force and the relationship of the probe tip to the periodontal tissues. / Clin Periodontol 1979; 6:106. 12. DuBois MJGJ, Schellekens PThA, DeWit JJFM, Eijsvoogel VP. In vitro reactivity of human lymphocytes after cryopreservation using a programmed cooling device. Scand J Immunol 1976; 5(suppl):17. 13. Van Winkelhoff AJ, Carlee AW, de Graaff J. Bacteroides endodontalis and other black-pigmented Bacteroides species in odontogenic abscesses. Infect Immun 1985; 49:494. 14. Baker JJ, Tondreau SP. The stimulation of human peripheral blood lymphocytes by oral bacteria: Macrophage and T-cell dependence. J Dent Res 1985; 64:909. 15. Redinbaugh MJ, Turley RB. Adaptation of the bicinchoninic acid protein assay for use with microtiter plates and sucrose gradient fractions. Anal Biochem 1986; 153:267. 16. Weinberg ED. Pregnancy-associated depression of cell-mediated immunity. Rev Infect Dis 1984; 6:814. 17. Cohen DW, Friedman L, Shapiro J, Kyle GC. A longitudinal investigation of the periodontal changes during pregnancy. J Periodontol 1969; 40:563. 18. Silness J, Löe H. Periodontal disease in pregnancy. Part II: Correlation between oral hygiene and periodontal condition. Acta Odontol Scand 1964; 22:121. 19. O'Neil TCA. Maternal T-lymphocyte response and gingivitis in pregnancy. J Periodontol 1979; 50:178. 20. Patters MR, Sedransk N, Genco RJ. The lymphoproliferative response during human experimental gingivitis. J Periodont Res 1979; 14:269. Send reprint requests to: Dr. J.E. Raber-Durlacher, Department of General Pathology and Internal Medicine, Academic Center for Dentistry Amsterdam, (ACTA) Louwesweg 1, 1066 EA Amsterdam, The Netherlands. Accepted for publication June 4, 1991.
CD4 to CD8 Ratio and In Vitro
Lymphoproliferative Responses During Experimental Gingivitis in Pregnancy and Post-Partum
J.E.
Raber-Durlacher,"
W.P.
Zeijlemaker,* A.A. P. Meinesz,' and L. Abraham-Inpijn
The absolute numbers and percentages of peripheral , B, and NK cells were assessed in 7 women, both during the second trimester of pregnancy and 6 months Postpartum. Furthermore, the in vitro responses of peripheral blood lymphocytes (PBL) to several mitogens and a preparation of Prevotella intermedia were compared in a period of experimentally-induced gingivitis during pregnancy and post-partum. Clinically, the periodontal pocket bleeding index (PPBI) was found to be higher during pregnancy than post-partum. The absolute numbers of CD3, CD4, and CD19 positive cells appeared to be decreased during pregnancy as compared to post-partum. However, the results did not indicate any evidence for a reduced in vitro PBL response to several mitogens and a preparation of P. intermedia during pregnancy. J Periodontol 1991; 62:663-667.
Key Words: Lymphocytes; pregnancy; gingivitis; Prevetolla intermedia.
Pregnancy gingivitis may be partly due to changes in maternal immunity. The existence of such changes that may
result in an overall increased susceptibility to infection1 can be inferred from diminished in vitro proliferative responses of peripheral blood lymphocytes (PBL) to preparations of Prevotella intermedia.2 This black-pigmented anaerobic microorganism is associated with pregnancy gingivitis,3 acute necrotizing gingivitis,4 and experimental gingivitis.5 In addition, a decrease in the ratio of peripheral helper cells to suppressor-cytotoxic cells (CD4/CD8 ratio) has been observed during pregnancy.6 It has been suggested that this phenomenon may contribute to increased gingival inflammation during pregnancy, as helper cells are thought to play a primarily protective role in the pathogenesis of periodontal diseases.7 Recently, we found evidence for the contention that the in vitro stimulation of lymphocytes by preparations of P. intermedia is antigen-specific, although a secondary cell-dependent polyclonal cell activation may occur.8 The present study forms a part of an investigation in which the development of experimentally induced gingival inflammation9 was studied during pregnancy, and 6 months post-partum. The aims of this study were to determine whether: 1) An alteration in the levels of peripheral and cells, and a decrease in the numbers of helper
'Department of General Pathology and Internal Medicine, Academic Cenfor Dentistry Amsterdam, (ACTA) Amsterdam, The Netherlands. Department of Clinical Immunobiology and Hybridoma Laboratory, Central Laboratory of the Netherlands Red Cross Blood Transfusion Service, ter
Amsterdam, The Netherlands.
resulting in lower CD4/CD8 ratios could be observed during the second trimester of pregnancy as compared to the levels of these cell populations post-partum; and 2) if any evidence for immunosuppression during pregnancy could be found when the in vitro proliferation of PBL to mitogens and preparations of P. intermedia was assessed during the various phases of the study. cells
MATERIALS AND METHODS Human Subjects and Study Design Nine gravidae (mean age 25.3 years, range 20 to 34) were selected to participate in this study before their 12th week of pregnancy (initial evaluation). The outline of the investigation is presented in Figure 1. All participants were in good physical health and no medication was taken that could be expected to influence the immune system. No features of periodontal diseases were present. Furthermore, there were no inadequate restorations interfering with the removal of dental plaque. After informed consent was secured, all patients were subjected to a carefully controlled oral hygiene program in order to reduce pre-existent inflammation as much as possible. The amount of dental plaque and gingival health were evaluated at least every 3 weeks, until experimental gingivitis was induced. At day 0, in their 25th week of pregnancy, the subjects were instructed to abstain from all oral hygiene procedures for a period of 14 days. Clinical examinations were performed at the initial evaluation and at day 0, 4, 8, and 14. These examinations in-
664
J Periodontol November 1991
LYMPHOCYTES AND PREGNANCY
POSTPARTUM
PREGNANT EXPERIMENTAL
EXPERIMENTAL
GINGIVITIS
GINGIVITIS
OPTIMAL
CESSATION OF
OPTIMAL
ORAL HYGIENE
ORAL HYGIENE
ORAL HYGIENE
CESSATION OF ORAL HYGIENE
-M-
Figure
1. Outline
of the investigation.
eluded the assessment of the
Plaque Index, (PI)10 and the index periodontal pocket bleeding (PPBI)11 at the mesial and distal sites of the teeth. At day 0 and day 14, peripheral venous blood was obtained to isolate mononuclear cells. At the end of the oral hygiene abstention period, a thorough dental prophylaxis was performed, and oral hygiene measures were reinstituted. About 12 weeks post-partum the participants in this study were seen again and the procedures as described above were repeated. Unfortunately, 1 subject was unable to participate in the investigation after parturition.
of the bicinchoninic acid protein assay.15 The protein concentration was 0.85 mg/ml in a dilution of 1:64. In addition, PBL were cultured in the presence of the following cell mitogens: PHA (purified phytohaemagglutinin HA 16)11 in a final (optimal) concentration of 12 pg/ml, ALS1 (Horse anti-human lymphocyte serum), in a final dilution of 1:32, Con A,# (Concanavalin A no. C-2010 type IV) in a final concentration of 60 µg/ ll. Furthermore, cells CD3 (subclass IgG2a)1 were cultured in the presence of in a final dilution of 1:10.
Isolation of Mononuclear Cells Sixty ml of blood was collected by venipuncture of the median cubital vein, between 9:00 and 9:30 a.m. Peripheral blood mononuclear cells (PBL) were isolated from the heparinized blood by Ficoll-Isopaque density layer centrifugádon and preserved in liquid nitrogen.12
Culture Procedures All cultures were performed in round-bottom microtiter plates (200 µ per well).** Lymphocytes were cultured in Iscove's modified Dulbecco's medium (IMDM) supplemented with 20% heat-inactivated pooled human serum and standard antibiotics at 37°C in a humidified 95% air, 5% C02 atmosphere. Cultures stimulated by PHA, ALS, Con A, and 104 lymphocytes per well aCD3 were performed with 4 and harvested after 4 days. On the basis of earlier findings for measurement of antigen-specific lymphocyte stimulation, a higher cell number (20 105 per well), and a longer culture period (8 days) were used for cultures stimulated by P. intermedia? Proliferative responses were determined by measuring the incorporation of 3H-thymidine (0.4 µ . : specific activity 200 Ci/mol) added 24 hours before harvesting. The cells were harvested onto glass fiber filters and the radioactivity was measured with a liquid scintillation counter. All cultures were performed in triplicate. Variability among triplicate values was always less than 10%. In unstimulated cultures, 3H-thymidine incorporation was nearly always less than 100 cpm; therefore, no standard deviation is given. Lymphocyte cultures of 1 subject were performed in one experiment on the same day, thus avoiding day-to-day variations.
Determination of Lymphocyte Subsets Lymphocyte subsets were determined by FACS cytofluorimetry with monoclonal antibodies against the following surface markers: CD3, CD4, CD8, CDllb,* and CD19.§ Peripheral blood mononuclear cells were incubated with 30 µ of these monoclonal antibodies. Subsequently, the cells were washed twice with PBS containing 0.5% bovine serum albumin and 0.01% sodium azide, incubated on ice for a further 30 minutes with fluorescein-conjugated-goat antimouse IgG. After 2 washings, the number of positive cells was counted in a flow cytometer system. The absolute numbers of cell subsets were calculated from the total number of leukocytes, the percentage of lymphocytes, and the distribution of lymphocyte subpopulations.
Lymphocyte Stimulants A preparation of P. intermedia strain H6 110 (ATCC 25611) was kindly provided by Dr. T.J.M. van Steenbergen, Academic Center for Dentistry Amsterdam (ACTA). Bacteria were cultured in liquid BM medium,13 centrifuged, and collected in phosphate buffered saline. An extract was prepared by freeze-thawing, sonication, and centrifugádon of cell wall fragments as described by Baker and Tondreau.14 Protein concentration of bacterial sonicate was measured by *Becton-Dickinson, Mountain View, CA. 'Central Laboratory of The Netherlands Service, Amsterdam.
Red Cross Blood Transfusion
means
Statistical Procedures The student r-test (double tailed) for matched samples and the Wilcoxon signed rank test were used for the statistical evaluation of the different parameters. Values of < 0.05 were
accepted as statistically significant.
Wellcome Diagnostics, Amsterdam, The Netherlands. "CLB, Amsterdam, The Netherlands. 'Sigma Chemical Co., St. Louis, MO. "Greiner, Langenthal, Switzerland.
Volume 62 Number 11
RABER-DURLACHER, ZEULEMAKER, MEINESZ, ABRAHAM-INPUN
PPBI
665
As seen in Figure 2, all subjects had gingivitis the initial evaluation in the first trimester of pregnancy which decreased after a period of optimal oral hygiene. During the 14-day period of cessation of all oral hygiene measures, the PPBI increased in all subjects tested. On day 0 as well as on day 14 of experimental gingivitis the mean PPBI was higher in the group during pregnancy than in the post-partum group. The amount of dental plaque that accumulated during both experimental phases of the study was rather similar (data not shown).
gingivitis. at
day
day
0
day
day ß
4
during
14
post partum
pregnancy
Figure 2. The mean periodontal pocket bleeding index (PPBI) + SD for the interproximal sites of the investigated subjects (n 8) during preg=
nancy and post-partum.
RESULTS Induction of Experimental Gingivitis In 8 individuals, some parameters of immunocompetence were studied in relation to the development of experimental gingivitis during pregnancy and post-partum (see Fig. 1 for an outline of the study). The periodontal pocket bleeding index (PPBI) was taken as a measure for the severity of
Leukocyte Subsets Leukocyte subsets in peripheral blood, as assessed in the experiment outlined above, are shown in Table 1. However,
in one donor we were not able to determine the distribution of these subsets, due to a lack of cells. In the pregnant group the absolute numbers of CD3-positive cells seemed to be slightly decreased during pregnancy as compared to the numbers of these cells assessed post-partum (marginally significant 0.05). The same was true for the mean absolute numbers of CD4-positive cells (P 0.07). Howthe number of CD4 cells decreased was not ever, (absolute) in each pregnant individual. The mean numbers of CD8positive suppressor/cytotoxic cells were found to be not =
=
Phenotypic Characterization of Cells Isolated From Peripheral Blood in 7 Subjects During Pregnancy and Post-Partum in Percentages and Absolute Values Expressed in lO'/Liter
Table 1.
During Pregnancy
CD4
CD3 abs
CD8 abs.
CD19 abs
CD4/CD8
Patient
%
A
79.2 85.2 86.2 74.3 89.0 93.7 89.7
1.13 1.98 1.99 1.04 0.77 1.47 0.76
52.5 48.2 52.6 50.7 65.0 61.4 65.9
0.73 1.09 1.21 0.71 0.56 0.96 0.65
32.5 37.3 33.3 26.1 23.0 33.6 22.1
0.82 0.77 0.37 0.20 0.53 0.22
ratio 1.59 1.29 1.57 1.92 2.80 1.81 2.98
85.30 6.62
1.31* 0.52
56.60 7.28
0.84* 0.24
29.70 5.91
0.48 0.25
1.99 0.65
1.23 2.78 3.01 0.74 1.13 1.70 1.69
52.7 69.5 77.1 49.9 59.0 55.0 55.1
0.76 2.13 1.96 0.51 0.80 1.10 1.08
39.4 23.3 33.8 26.5 17.0 33.3 31.3
0.57 0.72 1.15 0.27 0.23 0.67 0.62
1.33 2.96 1.70 1.88 3.48 1.64 1.74
1.75 0.85
59.76 9.89
1.19 0.24
29.23 7.50
0.60 0.31
2.10 0.79
C D E F G mean
SD
±
no.
Post-Partum 84.7 A 90.6 C 88.5 D 72.1 E 83.0 F 85.0 G 86.0 mean
SD
±
84.30 5.94
%
no.
%
no.
0.46
abs
4.0 9.0
0.26 0.14 0.21 0.11 0.07 0.06 0.12
CDllb % 17.6 9.4 11.5 9.3 29.0 11.1 12.0
8.84 4.87
0.14t 0.07
14.27 7.06
31.3 5.2 7.7 24.0 13.0 5.6 8.1
0.45 0.16 0.26 0.20 0.18 0.11 0.19
12.5 19.5 9.3 12.1 28.0 20.4 14.3
13.56 10.18
0.22 0.11
16.59 6.43
%
no.
18.8 4.6 9.4 8.1 8.0
Normal Values
CD3
CD4
CD8
CD4/CD8
abs
abs
abs
ratio
CD19 abs
0.77-2.84
0.10-0.72
no.
no.
no.
0.87-2.38
0.50-1.56
0.31-1.11
'Marginally significant. fVery significant.
no.
CD3: total number of T-cells; CD4: helper cells; CD8: suppressor cells, cytototoxic cells; CDllb: natural killer cells, monocytes, polymorphonuclear cells; and CD19: cells. The normal values were derived from over 1,000 healthy individuals.
J Periodontol November 1991
LYMPHOCYTES AND PREGNANCY
666
significantly
different during pregnancy and post-partum. individual level, however, there was some variation present in the numbers of these cells during pregnancy and 6 months after parturition. As a result the CD4/CD8 ratio was decreased only in subjects and E during pregnancy as compared to the CD4/CD8 ratio found in these individuals post-partum. The mean CD4/CD8 ratio calculated for the pregnant and the post-partum groups did not differ significantly. With regard to the CD19 positive cells, the numbers were found to be decreased during pregnancy as compared to the numbers of these cells post-partum (P < 0.01). In addition, no significant differences were observed during pregnancy and post-partum with respect to CDllb positive cells. The values of lymphocyte subsets during pregnancy were compared, not only to those of the same individuals post-partum, but also to the normal values derived from over 1,000 healthy individuals, determined over a period of 2.5 years. The data found during pregnancy appeared to be within the range of these normal values. At
DISCUSSION A decreased ratio of helper (CD4) to suppressor (CD8) cells, as a result of decreased proportions and numbers of helper cells, has been observed during the second and third trimesters of pregnancy.6 It has been suggested that this decreased CD4/CD8 ratio indicates the presence of immunodeficiency during pregnancy, as measured in vitro by
an
decreased lymphocyte proliferation.16 Clinically, gestational changes in immunity result in an increased overall susceptibility to infection.1 There are numerous reports dealing with pregnancy gingivitis, which is characterized by increased redness, edema, and a marked tendency to bleeding as compared to the gingival state post-partum.10,17'18 In addition to gestational changes in plasma levels of steroid hormones and a concomitant shift in the subgingival bacterial flora,3 there is evidence for a diminished in vitro response of PBL when cultured in the presence of preparations of oral microorganisms during pregnancy.2'19 Responses to preparations of P. intermedia, which is associated with pregnancy gingivitis, were reported to be elevated at the post-partum evaluation as compared to the responses to this antigen during the second and third trimesters of pregnancy. More recently, Stashenko and coworkers7 found that low CD4/CD8 ratios were predictive for low cell responses to oral microorganisms in patients with Periodontitis. In addition, patients with a low CD4/CD8 ratio had significantly more redness and bleeding on probing in the presence of approximately equal amounts of dental plaque, as compared to Periodontitis patients with a high CD4/CD8 ratio. It was hypothesized that the helper cell response plays an important protective role in limiting bacteria involved in gingival inflammation. Moreover, it was suggested that pregnancy gingivitis may reflect an analogous situation. In the present study, in which only a limited number of gravidae were investigated, we did not find evidence for a decrease of the mean CD4/CD8 ratio during pregnancy, although the numbers of CD4 positive cells seemed lower during gestation. However, it must be emphasized that
Lymphocyte Stimulation
In the test groups indicated above, lymphocyte reactivity towards mitogens and antigens was determined. The results obtained with lymphocytes from 8 subjects are summarized in Table 2. In this table, the mean (± SD) cpm 3H-thymidine incorporation to each stimulant on days 0 and 14 of both periods of experimental gingivitis is given. Because there are substantial inter-donor variations in the absolute magnitude of the cpm, no statistical analysis could be applied to these data. However, by expressing individual results as ratios (cpm during pregnancy, relative to cpm postpartum), inter-individual variations are circumvented. These data are also shown in Table 2. Statistical analysis, performed by means of the student t test and the Wilcoxon test, revealed no significant differences in the lymphocyte reactivity during pregnancy and
post-partum. Table 2. Mean Post-Partum
Lymphocyte Responses
at
Day 0 and Day 14
to Several
During Pregnancy SD
mean ±
Stimulant PHA ALS Con A CD 3 intermedia sonicate Unstimulated cultures
day 0 45.8 40.9 3.4 6.8 5.7 62.4
±
day 49.1 42.5 4.0 7.6 5.2
59.1
38.6
SD
14
26.0 15.2 3.7
±
SD
mean ±
day 0
4.1 4.1
40.6 33.9 2.8 8.3 4.2
20.1
37.0
±
22.2 16.8 2.3 3.7 3.4
33.8 29.1 1.8 8.3 3.4
26.1
46.0
±
Preparation
of P. intermedia
During Pregnancy and
Mean Ratio
Mean Ratio
day 0 pregnancy day 0 post-partum
day 14 pregnancy day 14 post-partum
15.3 12.4 0.9 3.9 2.9
1.23 1.47 1.88 0.90 3.65
39.8
3.15
mean ±
day
a
SD 14
Post-Partum
mean ±
20.7 14.8 1.9 3.4 4.2
Mitogens And
±
SD
0.39 0.81 1.55 0.41 5.97
1.63 1.83 3.20 0.98 2.35
4.10
0.99
SD
0.87 1.18 3.17 0.51 2.88 ±
0.32
In the stimulated cultures the responses are expressed as cpm IO3. In the unstimulated cultures the responses are expressed as cpm (no standard deviations are given). Because of the high inter-individual variation in the subjects investigated (n 8), the response ratios (response on day 0 during pregnancy/response on day 0 post-partum) were calculated for each individual: the mean values ± SD of these ratios are presented. Statistical analysis on these data revealed no significant difference in the magnitude of the responses during pregnancy and post-partum (P > 0.1). =
Volume 62 Number 11
RABER-DURLACHER, ZEULEMAKER, MEINESZ, ABRAHAM-INPLJN
Stashenko did not find that periodontal patients as a group exhibited lower CD4/CD8 ratios than normal individuals. Low responders; e.g., patients with a low CD4/CD8 ratio could only be identified when the experimental material was divided into subgroups. In our study, we could not find any evidence that the 2 individuals which displayed relatively low CD4/CD8 ratios during pregnancy, as compared to these ratios assessed post-partum, developed more gingivitis during pregnancy than the other pregnant subjects. In addition, we did not find any significant alterations in the percentages of CDllb-positive cells (corresponding to NK cells, monocytes, macrophages, and granulocytes). However, the absolute numbers of cells appeared to be decreased during pregnancy. When the PBL responses on day 0 and day 14 of experimental gingivitis were compared to these responses 6 months post-partum, the in vitro PBL response to several mitogens and a preparation of P. intermedia appeared not to be decreased during pregnancy. However, 14 days of plaque accumulation might be too short to develop a full range of PBL responses for all individuals.20 Furthermore, we were not able to assess any relation between the number of CD4 positive cells and the magnitude of the PBL response. The presence of a higher tendency towards bleeding during pregnancy indicates that the subjects investigated developed more inflammation during the experimental period. However, the PPBI at day 0 during pregnancy, when all efforts were made to obtain a state of optimal gingival health appeared to be increased as compared to the PPBI, associated with similar low plaque scores at day 0 post-partum. Thus, it is possible that pregnancy gingivitis is not only an exaggerated inflammatory response as a result of a disturbed host-parasite equilibrium; it cannot be excluded that the gingiva undergoes physiological changes resulting in increased redness, edema, and higher bleeding tendency, clinically resembling inflammation. Taken together, it may be concluded that in the present study no evidence was found for an overall lower CD4/ CD8 ratio during pregnancy. The numbers of cells, however, were decreased during pregnancy as compared to postpartum. Furthermore, the in vitro PBL responses to cell mitogens and a preparation of P. intermedia seemed not to be decreased during 14 days of experimental gingivitis during pregnancy as compared to the responses post-partum. It should be noted that this study is performed on a limited number of subjects. Possibly, a more extensive group of pregnant subjects and controls would reveal statistically significant differences. It is clear, however, that no gross differences occurred between our study groups.
Acknowledgments
The authors thank the staff of the Department of Biochemistry (Dr. Annemarie Lütjens, director), Andreas Hospital, Amsterdam for determining hématologie parameters. We are also grateful to Dr. Jan Dijkstra, Department of Ob-
stetrics and
667
Gynecology, Andreas Hospital, Amsterdam for
granting permission to examine his patients. We would also
like to thank Dr. John Raber, Dr. Erwin van der Zee, and Wietske Levinson for their editorial help. This study was supported by the Dutch Praeventiefonds grant 28-1176. REFERENCES 1. Brabin BJ.
2.
Kornman
of infection in pregnancy. Rev
KS, Loesche
Infect Dis
WJ. Modulation of immuno-
periodontal disease-associated microorganisms during pregnancy. Infect Immun 1980; 28:713. Kornman KS, Loesche WJ. The subgingival microbial flora during reactivity
3.
Epidemiology
1985; 7:579. Lopatin DE, to
pregnancy. J Periodont Res 1980; 15:111. 4. Slots J. Importance of Black-Pigmented Bacteroides in human periodontal disease. In: Host-Parasite Interactions in Periodontal Diseases. Genco RJ and Mergenhagen SE, eds. Washington, DC; American Society for Microbiology, 1982; 17-45. 5. Loesche WJ, Syed SA. Bacteriology of human experimental gingivitis: effect of plaque and gingivitis score. Infect Immun 1978; 21:830839. 6. Sridama V, Pacini F, Yang SL, Moawad A, Reilly M, DeGroot LJ. Decreased levels of helper cells. A possible cause of immunodeficiency in pregnancy. New Eng J Med 1982; 307:352. 7. Stashenko P, Resmini LM, Haffajee AD, Socransky SS. Helper and suppressor cells in periodontal disease. /Periodont Res 1985; 20:515. 8. Raber-Durlacher JE, Zeijlemaker WP, Meinesz AAP, Abraham-Inpijn L. Stimulation of lymphocytes in vitro by Bacteroides intermedius and Porphyromonas (Bacteroides) gingivalis sonicates. J Periodontol 1990; 61:217. 9. Löe , Theilade E, Jensen SB. Experimental gingivitis in man. / Periodontol 1965; 36:177. 10. Löe H, Silness J. Periodontal disease in pregnancy. Part I: Prevalence and severity. Acta Odontol Scand 1963; 21:533. 11. Van der Veiden U. Probing force and the relationship of the probe tip to the periodontal tissues. / Clin Periodontol 1979; 6:106. 12. DuBois MJGJ, Schellekens PThA, DeWit JJFM, Eijsvoogel VP. In vitro reactivity of human lymphocytes after cryopreservation using a programmed cooling device. Scand J Immunol 1976; 5(suppl):17. 13. Van Winkelhoff AJ, Carlee AW, de Graaff J. Bacteroides endodontalis and other black-pigmented Bacteroides species in odontogenic abscesses. Infect Immun 1985; 49:494. 14. Baker JJ, Tondreau SP. The stimulation of human peripheral blood lymphocytes by oral bacteria: Macrophage and T-cell dependence. J Dent Res 1985; 64:909. 15. Redinbaugh MJ, Turley RB. Adaptation of the bicinchoninic acid protein assay for use with microtiter plates and sucrose gradient fractions. Anal Biochem 1986; 153:267. 16. Weinberg ED. Pregnancy-associated depression of cell-mediated immunity. Rev Infect Dis 1984; 6:814. 17. Cohen DW, Friedman L, Shapiro J, Kyle GC. A longitudinal investigation of the periodontal changes during pregnancy. J Periodontol 1969; 40:563. 18. Silness J, Löe H. Periodontal disease in pregnancy. Part II: Correlation between oral hygiene and periodontal condition. Acta Odontol Scand 1964; 22:121. 19. O'Neil TCA. Maternal T-lymphocyte response and gingivitis in pregnancy. J Periodontol 1979; 50:178. 20. Patters MR, Sedransk N, Genco RJ. The lymphoproliferative response during human experimental gingivitis. J Periodont Res 1979; 14:269. Send reprint requests to: Dr. J.E. Raber-Durlacher, Department of General Pathology and Internal Medicine, Academic Center for Dentistry Amsterdam, (ACTA) Louwesweg 1, 1066 EA Amsterdam, The Netherlands. Accepted for publication June 4, 1991.
