Seon-Ah Kim; Sung-Sam Lim

doi:10.5395/JKACD.2002.27.1.001

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Original Article The levels of interleukin-2, interferon-γ, interleukin-4 and T lymphocyte subpopulations in rat pulpal inflammation induced experimentally by specific bacteria: Seon-Ah Kim, Sung-Sam Lim; 2002;27(1):-11.
DOI: https://doi.org/10.5395/JKACD.2002.27.1.001
Published online: January 31, 2002

Department of Conservative Dentistry, Graduate School, Seoul National University, Korea.

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Abstract
REFERENCES

Abstract

Immune responses associated with bacterial infection involve various inflammatory cells. Clinical symptoms and pathologic features are particularly influenced by the predominant cells. Among inflammatory cells, T cells have the heterogenity. T cells may develop into the mature cells expressing the cell surface markers with different functions and T helper cells are categorized into Th1 and Th2 cells based on their different patterns of cytokine production. The objective of this study was to investigate the change of expression of surface markers on T cells and the Th1/Th2 immune response in pulpal inflammation associated with specific bacteria.

We experimentally induced pulpal inflammation in rat incisors by drilling without coolant and innoculated with Streptococcus mutans (S.M. group), Porphyromonas endodontalis (P.E. group), or only sterile cotton (control group). After 1, 2, and 5 days, mandibular incisors were extracted and the pulp tissues were extirpated. The expressions of IL-2 recepters (CD25) and ICAM-1 (CD54) on CD4+ and CD8+ cells in the pulps were determined using a flow cytometer, and the concentration of IL-2, IFN-γ and IL-4 was measured by enzyme-linked immunosorbent assay.

The results were as follows;

1. In the S.M. group, CD4+ cells were more increased at 2nd day than 1st day and in the P.E. group, CD8+ cells were more increased at 2nd day than 1st day.

2. The percentages of CD4+, CD4+25+ and CD4+54+ cells were decreased in the pulp tissues at 5th day after irritation in all groups.

3. The ratios of CD4+/CD8+, CD4+/CD4+25+ and CD4+/CD4+54+ in the pulps at 2nd day after irritation by P. endodontalis were significantly lower than the other groups.

4. The higher concentrations of IFN-γ than IL-4 in the pulps at 2nd day after irritation by P. endodontalis showed that T helper 1 reaction were predominant in the early stage of the pulpal inflammation induced by P. endodontalis.

5. The higher concentrations of IL-4 than IFN-γ in the pulps at 1st day and 5th day after irritation by S. mutans were measured but the differences were not significant.
Keywords: Pulp; Streptococcus mutans; Porphyromonas endodontalis; T cells

Fig. 1

Lymphocytes were gated(R1) on FSC(cell size) versus SSC(cell granularity) plot.

Fig. 2

Two-color fluorescence analysis of CD25 (FL1-Height) and CD4 (FL3-Height) in left plot and two-color fluorescence analysis of CD54 (FL2-Height) and CD4 (FL3-Height) is shown in right plot. UL: upper left, UR: upper right, LL: lower left, LR: lower right, % Gated: percentage in the gated area, % Total: percentage in all particles.

Fig. 3

The percentages of CD4+ cells.

Fig. 4

The percentages of CD4+25+ cells.

Fig. 5

The percentages of CD8+ cells.

Fig. 6

The percentages of CD8+25+ cells.

Fig. 7

The ratios of CD4+/CD8+.

Fig. 8

The ratios of CD4+/CD4+25+.

Fig. 9

The concentration of IFN-γand IL-4.

Fig. 10

The pulp tissue in the P.E. group at 2nd day after irritation (H&E stain ×400).

Fig. 11

The pulp tissue in the P.E. group at 5th day after irritation (H&E stain ×400).

Table 1

The percentages and standard deviations of CD4+ cells in the gated area

*,#,∾,$ in the same line; statistically significant (p<0.05; by One-way ANOVA) between groups.

+,‡ in the same line; statistically significant (p<0.05; by One-way ANOVA) in the group.

Table 2

The percentages and standard deviations of CD8+ cells in the gated area

*,# in the same line; statistically significant (p<0.05; by One-way ANOVA) between groups.

+,‡ in the same line; statistically significant (p<0.05; by One-way ANOVA) in the group.

Table 3

The mean ratios of cell populations

*,#,∾,$ in the same line; statistically significant (p<0.05; by One-way ANOVA) between groups.

+,‡ in the same line; statistically significant (p<0.05; by One-way ANOVA) in the group.

Table 4

The average concentrations of cytokines

*,# in the same line; statistically significant (p<0.05; by One-way ANOVA) between groups.

+,‡ in the same line; statistically significant (p<0.05; by paired t-test) in the group.

REFERENCES

1. Marton IJ, Kiss C. Characterization of inflammatory cell infiltrate in dental periapical lesions. Int Endod J. 1993;26: 131-136.Article PubMed
2. Fouad AF. IL-1alpha and TNF-alpha expression in early periapical lesions of normal and immunodeficient mice. J Dent Res. 1997;76(9):1548-1554.Article PubMed PDF
3. Back SH, Lim SS. An immunohistochemical study on the immunoglobulin g subclasses of the experimentally induced rat pulp and periapical pathoses. J Korean Acad Conserv Dent. 1991;16: 41-59.
4. Kopp W, Schwarting R. Differentiation of T lymphocyte subpopulations, macrophages, and HDA-DR-restricted cells of apical granulatuib tissue. J Endod. 1989;15: 72-75.PubMed
5. Stashenko P, Yu SM, Wang CY. Kinetics of immune cell and bone resorptive responses to endodontic infections. J Endod. 1992;18: 422-426.Article PubMed
6. Kawashima N, Okiji T, Kosaka T. Kinetics of macrophages and lymphoid cells during the development of experimentally induced periapical lesions in rat molars; a quantitative immunohistochemcial study. J Endod. 1996;22: 311-316.PubMed
7. Matsuo T, Ebisu S, Shimabukuro Y. Quantitative analysis of immunocompetent cells in human periapical lesions; Correlations with clinical findings of the involved teeth. J Endod. 1992;8: 497-500.Article PubMed
8. Oh TS, Lim SS. Flow cytometric analysis of lymphocyte and cycling cell distribution in periapical lesions. J Korean Acad Conserv Dent. 1993;18: 317-340.
9. Sol MA, Tkaczuk J. Voigt JJ Characterization of lymphocyte subpopulations in periapical lesions by flow cytometry. Oral Microbiol Immunol. 1998;13: 253-258.PubMed
10. Abbs AK, Murphy KM, Sher A. Functional diversity of helper T lymphocytes. Nature. 1996;383: 787-793.Article PubMed PDF
11. Yamasaki M, Nakane A, Kumazawa M. Endotoxin and Gram-negative bacteria in the rat periapical lesions. J Endod. 1992;18(10):501-504.Article PubMed
12. Gemmell E, Winning TA, Bird PS. Cytokine profiles of lesional and splrenic T cells in Porphyromonas gingivalis infection in a murine model. J Periodontol. 1998;69: 1131-1138.PubMed
13. Byun HY, Lim SS, Park DS. Levels of tnf-α,-β, il-1β, tgf-β1 and their relationship with the presence of specific black pigmented bacteria in periapical and pulpal diseases. J Korean Acad Conserv Dent. 1999;24: 1-12.
14. Hren NI, Gubina M, Ihan A. Cytotoxic T lymphocytes versus streptococcal colonization in periapical granulomas. J Endod. 1999;25: 239-242.Article PubMed
15. Speer ML, Madonia JV, Heuer MA. Quantitative evaluation of the immunocompetence of the dental pulp. J Endod. 1977;3: 418-423.Article PubMed
16. Jontell M, Gunraj N, Bergenholtz G. Immunocompetent cells in normal dental pulp. J Dent Res. 1986;66: 1149-1153.Article PubMed PDF
17. Pulver WH, Taubman MA, Smith OJ. Immune components in normal and inflamed human dental pulp. Arch Oral Biol. 1977;22: 103-111.Article PubMed
18. Lee WC, Lim SS. Lymphocytes population in relation to clinical symptoms in irreversible pulpitis. J Korean Acad Conserv Dent. 1995;20: 235-249.
19. Hahn CL, Falkler WA, Siegel MA. A study of T and B cells in pulpal pathosis. J Endod. 1989;15: 20-26.Article PubMed
20. Mangkornkarn C, Steiner JC, Bohman R, Lindemann RA. Flow cytometric analysis of human dental pulp. J Endod. 1991;17: 49-53.PubMed
21. Kotylo HA, Sample RB, Redmond NL. Reference ranges for lymphocyte subsets. A comparison of standard vs rapid whole-blood lysis techniques. Arch Pathol Lab Med. 1991;115: 181-184.PubMed
22. Kim SA, Bae KS, Lim SS. Flow cytometric analysis of lymphocytes in normal and inflamed pulp. J Korean Acad Conserv Dent. 1997;22: 374-387.
23. Okiji T, Morita I, Sunada I, Murota S. Involvement of arachidonic acid metabolites in increases in vascular permeability in experimental dental pulpal inflammation in the rat. Arch Oral Biol. 1989;34: 523-528.Article PubMed
24. Bergenholtz G, Nagaoka S, Jontell M. Class II antigen expressing cells in experimentally induced pulpitis. Int Endod J. 1991;24: 8-14.Article PubMed
25. Okiji T, Morita I, Kobayashi C, Sunada I, Murota S. Arachidonic-acid metabolosm in normal and experimentally-inflamed rat dental pulp. Arch Oral Biol. 1987;32(10):723-727.PubMed
26. Hashimoto S, Maeda M, Yamakita J, Nakamura Y. Effects of zinc oxide-eugenol on leucocyte number and lipoxygenase products in artificially inflamed rat dental pulp. Arch Oral Biol. 1990;35: 87-93.Article PubMed
27. Suzuki N, Okiji T, Suda H. Enhanced expansion of activation - association molecules on macrophages of heterogeneous populations in expanding periapical lesions in rat molars. Arch Oral Biol. 1999;44: 67-79.PubMed
28. Rauschenberger CR, Bailey JC, Cootauco CJ. Detection of human IL-2 in normal and inflamed dental pulps. J Endod. 1997;23: 366-370.Article PubMed
29. Nguyen T, Wang R, Russeu JH. IL-12 enhances IL-2 function by inducing CD2 expression through a p38 mitogen-activated protein kinases pathway. Eur J Immunol. 2000;30: 1445-1452.Article PubMed
30. Okiji T, Morita I, Sunada I, Murota S. The role of Leukotriene B4 in neutrophil infiltration in experimentally-induced inflammation of rat tooth pulp. J Dent Res. 1991;70(1):34-37.Article PubMed PDF
31. Wedenberg C, Borstein R. Pulpal reactions in rat incisors to Caridex. Aust Dent J. 1990;35(6):505-508.Article PubMed
32. Kettering JD, Torbinejad M, Jones SL. Specificity of antibodies present in human periapical lesions. J Endod. 1991;17: 213-216.Article PubMed

Tables & Figures

Fig. 1

Lymphocytes were gated(R1) on FSC(cell size) versus SSC(cell granularity) plot.

Fig. 2

Fig. 3

The percentages of CD4+ cells.

Fig. 4

The percentages of CD4+25+ cells.

Fig. 5

The percentages of CD8+ cells.

Fig. 6

The percentages of CD8+25+ cells.

Fig. 7

The ratios of CD4+/CD8+.

Fig. 8

The ratios of CD4+/CD4+25+.

Fig. 9

The concentration of IFN-γand IL-4.

Fig. 10

The pulp tissue in the P.E. group at 2nd day after irritation (H&E stain ×400).

Fig. 11

The pulp tissue in the P.E. group at 5th day after irritation (H&E stain ×400).

Table 1

The percentages and standard deviations of CD4+ cells in the gated area

*,#,∾,$ in the same line; statistically significant (p<0.05; by One-way ANOVA) between groups.

+,‡ in the same line; statistically significant (p<0.05; by One-way ANOVA) in the group.

Table 2

The percentages and standard deviations of CD8+ cells in the gated area

*,# in the same line; statistically significant (p<0.05; by One-way ANOVA) between groups.

+,‡ in the same line; statistically significant (p<0.05; by One-way ANOVA) in the group.

Table 3

The mean ratios of cell populations

*,#,∾,$ in the same line; statistically significant (p<0.05; by One-way ANOVA) between groups.

+,‡ in the same line; statistically significant (p<0.05; by One-way ANOVA) in the group.

Table 4

The average concentrations of cytokines

*,# in the same line; statistically significant (p<0.05; by One-way ANOVA) between groups.

+,‡ in the same line; statistically significant (p<0.05; by paired t-test) in the group.

REFERENCES

1. Marton IJ, Kiss C. Characterization of inflammatory cell infiltrate in dental periapical lesions. Int Endod J. 1993;26: 131-136.Article PubMed
2. Fouad AF. IL-1alpha and TNF-alpha expression in early periapical lesions of normal and immunodeficient mice. J Dent Res. 1997;76(9):1548-1554.Article PubMed PDF
3. Back SH, Lim SS. An immunohistochemical study on the immunoglobulin g subclasses of the experimentally induced rat pulp and periapical pathoses. J Korean Acad Conserv Dent. 1991;16: 41-59.
4. Kopp W, Schwarting R. Differentiation of T lymphocyte subpopulations, macrophages, and HDA-DR-restricted cells of apical granulatuib tissue. J Endod. 1989;15: 72-75.PubMed
5. Stashenko P, Yu SM, Wang CY. Kinetics of immune cell and bone resorptive responses to endodontic infections. J Endod. 1992;18: 422-426.Article PubMed
6. Kawashima N, Okiji T, Kosaka T. Kinetics of macrophages and lymphoid cells during the development of experimentally induced periapical lesions in rat molars; a quantitative immunohistochemcial study. J Endod. 1996;22: 311-316.PubMed
7. Matsuo T, Ebisu S, Shimabukuro Y. Quantitative analysis of immunocompetent cells in human periapical lesions; Correlations with clinical findings of the involved teeth. J Endod. 1992;8: 497-500.Article PubMed
8. Oh TS, Lim SS. Flow cytometric analysis of lymphocyte and cycling cell distribution in periapical lesions. J Korean Acad Conserv Dent. 1993;18: 317-340.
9. Sol MA, Tkaczuk J. Voigt JJ Characterization of lymphocyte subpopulations in periapical lesions by flow cytometry. Oral Microbiol Immunol. 1998;13: 253-258.PubMed
10. Abbs AK, Murphy KM, Sher A. Functional diversity of helper T lymphocytes. Nature. 1996;383: 787-793.Article PubMed PDF
11. Yamasaki M, Nakane A, Kumazawa M. Endotoxin and Gram-negative bacteria in the rat periapical lesions. J Endod. 1992;18(10):501-504.Article PubMed
12. Gemmell E, Winning TA, Bird PS. Cytokine profiles of lesional and splrenic T cells in Porphyromonas gingivalis infection in a murine model. J Periodontol. 1998;69: 1131-1138.PubMed
13. Byun HY, Lim SS, Park DS. Levels of tnf-α,-β, il-1β, tgf-β1 and their relationship with the presence of specific black pigmented bacteria in periapical and pulpal diseases. J Korean Acad Conserv Dent. 1999;24: 1-12.
14. Hren NI, Gubina M, Ihan A. Cytotoxic T lymphocytes versus streptococcal colonization in periapical granulomas. J Endod. 1999;25: 239-242.Article PubMed
15. Speer ML, Madonia JV, Heuer MA. Quantitative evaluation of the immunocompetence of the dental pulp. J Endod. 1977;3: 418-423.Article PubMed
16. Jontell M, Gunraj N, Bergenholtz G. Immunocompetent cells in normal dental pulp. J Dent Res. 1986;66: 1149-1153.Article PubMed PDF
17. Pulver WH, Taubman MA, Smith OJ. Immune components in normal and inflamed human dental pulp. Arch Oral Biol. 1977;22: 103-111.Article PubMed
18. Lee WC, Lim SS. Lymphocytes population in relation to clinical symptoms in irreversible pulpitis. J Korean Acad Conserv Dent. 1995;20: 235-249.
19. Hahn CL, Falkler WA, Siegel MA. A study of T and B cells in pulpal pathosis. J Endod. 1989;15: 20-26.Article PubMed
20. Mangkornkarn C, Steiner JC, Bohman R, Lindemann RA. Flow cytometric analysis of human dental pulp. J Endod. 1991;17: 49-53.PubMed
21. Kotylo HA, Sample RB, Redmond NL. Reference ranges for lymphocyte subsets. A comparison of standard vs rapid whole-blood lysis techniques. Arch Pathol Lab Med. 1991;115: 181-184.PubMed
22. Kim SA, Bae KS, Lim SS. Flow cytometric analysis of lymphocytes in normal and inflamed pulp. J Korean Acad Conserv Dent. 1997;22: 374-387.
23. Okiji T, Morita I, Sunada I, Murota S. Involvement of arachidonic acid metabolites in increases in vascular permeability in experimental dental pulpal inflammation in the rat. Arch Oral Biol. 1989;34: 523-528.Article PubMed
24. Bergenholtz G, Nagaoka S, Jontell M. Class II antigen expressing cells in experimentally induced pulpitis. Int Endod J. 1991;24: 8-14.Article PubMed
25. Okiji T, Morita I, Kobayashi C, Sunada I, Murota S. Arachidonic-acid metabolosm in normal and experimentally-inflamed rat dental pulp. Arch Oral Biol. 1987;32(10):723-727.PubMed
26. Hashimoto S, Maeda M, Yamakita J, Nakamura Y. Effects of zinc oxide-eugenol on leucocyte number and lipoxygenase products in artificially inflamed rat dental pulp. Arch Oral Biol. 1990;35: 87-93.Article PubMed
27. Suzuki N, Okiji T, Suda H. Enhanced expansion of activation - association molecules on macrophages of heterogeneous populations in expanding periapical lesions in rat molars. Arch Oral Biol. 1999;44: 67-79.PubMed
28. Rauschenberger CR, Bailey JC, Cootauco CJ. Detection of human IL-2 in normal and inflamed dental pulps. J Endod. 1997;23: 366-370.Article PubMed
29. Nguyen T, Wang R, Russeu JH. IL-12 enhances IL-2 function by inducing CD2 expression through a p38 mitogen-activated protein kinases pathway. Eur J Immunol. 2000;30: 1445-1452.Article PubMed
30. Okiji T, Morita I, Sunada I, Murota S. The role of Leukotriene B4 in neutrophil infiltration in experimentally-induced inflammation of rat tooth pulp. J Dent Res. 1991;70(1):34-37.Article PubMed PDF
31. Wedenberg C, Borstein R. Pulpal reactions in rat incisors to Caridex. Aust Dent J. 1990;35(6):505-508.Article PubMed
32. Kettering JD, Torbinejad M, Jones SL. Specificity of antibodies present in human periapical lesions. J Endod. 1991;17: 213-216.Article PubMed

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The levels of interleukin-2, interferon-γ, interleukin-4 and T lymphocyte subpopulations in rat pulpal inflammation induced experimentally by specific bacteria

J Korean Acad Conserv Dent. 2002;27(1):1-11. Published online January 31, 2002

DOI: https://doi.org/10.5395/JKACD.2002.27.1.001

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Figure

The levels of interleukin-2, interferon-γ, interleukin-4 and T lymphocyte subpopulations in rat pulpal inflammation induced experimentally by specific bacteria

Fig. 1 Lymphocytes were gated(R1) on FSC(cell size) versus SSC(cell granularity) plot.

Fig. 2 Two-color fluorescence analysis of CD25 (FL1-Height) and CD4 (FL3-Height) in left plot and two-color fluorescence analysis of CD54 (FL2-Height) and CD4 (FL3-Height) is shown in right plot. UL: upper left, UR: upper right, LL: lower left, LR: lower right, % Gated: percentage in the gated area, % Total: percentage in all particles.

Fig. 3 The percentages of CD4+ cells.

Fig. 4 The percentages of CD4+25+ cells.

Fig. 5 The percentages of CD8+ cells.

Fig. 6 The percentages of CD8+25+ cells.

Fig. 7 The ratios of CD4+/CD8+.

Fig. 8 The ratios of CD4+/CD4+25+.

Fig. 9 The concentration of IFN-γand IL-4.

Fig. 10 The pulp tissue in the P.E. group at 2nd day after irritation (H&E stain ×400).

Fig. 11 The pulp tissue in the P.E. group at 5th day after irritation (H&E stain ×400).

Fig. 1

Fig. 2

Fig. 3

Fig. 4

Fig. 5

Fig. 6

Fig. 7

Fig. 8

Fig. 9

Fig. 10

Fig. 11

The levels of interleukin-2, interferon-γ, interleukin-4 and T lymphocyte subpopulations in rat pulpal inflammation induced experimentally by specific bacteria

Table 1

The percentages and standard deviations of CD4+ cells in the gated area

*,#,∾,$ in the same line; statistically significant (p<0.05; by One-way ANOVA) between groups.

+,‡ in the same line; statistically significant (p<0.05; by One-way ANOVA) in the group.

Table 2

The percentages and standard deviations of CD8+ cells in the gated area

*,# in the same line; statistically significant (p<0.05; by One-way ANOVA) between groups.

+,‡ in the same line; statistically significant (p<0.05; by One-way ANOVA) in the group.

Table 3

The mean ratios of cell populations

*,#,∾,$ in the same line; statistically significant (p<0.05; by One-way ANOVA) between groups.

+,‡ in the same line; statistically significant (p<0.05; by One-way ANOVA) in the group.

Table 4

The average concentrations of cytokines

*,# in the same line; statistically significant (p<0.05; by One-way ANOVA) between groups.

+,‡ in the same line; statistically significant (p<0.05; by paired t-test) in the group.

Table 1 The percentages and standard deviations of CD4+ cells in the gated area

*,#,∾,$ in the same line; statistically significant (p<0.05; by One-way ANOVA) between groups.

+,‡ in the same line; statistically significant (p<0.05; by One-way ANOVA) in the group.

Table 2 The percentages and standard deviations of CD8+ cells in the gated area

*,# in the same line; statistically significant (p<0.05; by One-way ANOVA) between groups.

+,‡ in the same line; statistically significant (p<0.05; by One-way ANOVA) in the group.

Table 3 The mean ratios of cell populations

*,#,∾,$ in the same line; statistically significant (p<0.05; by One-way ANOVA) between groups.

+,‡ in the same line; statistically significant (p<0.05; by One-way ANOVA) in the group.

Table 4 The average concentrations of cytokines

*,# in the same line; statistically significant (p<0.05; by One-way ANOVA) between groups.

+,‡ in the same line; statistically significant (p<0.05; by paired t-test) in the group.