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J Dent Res 83(5): 384-387, 2004
© 2004 International and American Associations for Dental Research

RESEARCH REPORT
Clinical

Mast Cells in Human Periodontal Disease

E. Gemmell*, C.L. Carter, and G.J. Seymour

Oral Biology and Pathology, School of Dentistry, The University of Queensland, Brisbane 4072, Australia;

* corresponding author, e.gemmell{at}uq.edu.au


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS & METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Recently, mast cells have been shown to produce cytokines which can direct the development of T-cell subsets. The aim of the present study was to determine the relationship between mast cells and the Th1/Th2 response in human periodontal disease. Tryptase+ mast cell numbers were decreased in chronic periodontitis tissues compared with healthy/gingivitis lesions. Lower numbers of c-kit+ cells, which remained constant regardless of clinical status, indicate that there may be no increased migration of mast cells into periodontal disease lesions. While there were no differences in IgG2+ or IgG4+ cell numbers in healthy/gingivitis samples, there was an increase in IgG4+ cells compared with IgG2+ cells in periodontitis lesions, numbers increasing with disease severity. This suggests a predominance of Th2 cells in periodontitis, although mast cells may not be the source of Th2-inducing cytokines.

KEY WORDS: mast cells • periodontal disease • immunohistology


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS & METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Mast cells are a heterogeneous population which can be divided into two phenotypes on the basis of neutral serine proteases (Irani et al., 1986). Connective tissue mast cells (CTMC)s are found throughout the connective tissues of the skin and peritoneal cavity, whereas mucosal mast cells (MMC)s are present in the intestinal lamina propria and lung (Irani and Schwartz, 1989; Wasserman, 1994).

While the primary role for mast cells was thought to be in the innate defense against intestinal and cutaneous parasitic and bacterial infections, they are now believed also to play a role in the induction of acquired immune responses (Mécheri and David, 1997). Mast cells have been reported to reside close to T-cells (Mekori and Metcalf, 1999), can phagocytose and process bacterial antigens prior to presentation of antigens to T-cells (Malaviya et al., 1996), and are a source of Th1- and Th2-inducing cytokines (Plaut et al., 1989; Smith et al., 1994; Marietta et al., 1996).

The aim of the present study was to determine the relationship between mast cells and the Th1/Th2 response in human periodontal disease. Tryptase+ and c-kit+ mast cells were first demonstrated in biopsies from healthy/gingivitis and chronic periodontitis patients. Stem cell factor is a differentiation and proliferation factor for mast cells and is a ligand for the receptor that is encoded by the c-kit proto-oncogene (Galli et al., 1995). Second, mast cells produce IL-4, IL-10, and IL-13 (Plaut et al., 1989; Marietta et al., 1996), which direct Th2 responses, and IL-12 (Smith et al., 1994), which induces IFN-gamma production (Chan et al., 1991), contributing to the development of a Th1 response. Since interferon gamma and IL-4 induce T-cell-dependent isotype switching in B-cells to IgG2 and IgG4, respectively (Agresti and Vercelli, 2002; Tanaka et al., 2003), IgG2+ and IgG4+ B-cells/plasma cell numbers were also examined.


   MATERIALS & METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS & METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Patients
Gingival tissue was obtained from 53 subjects. Twenty-two biopsies taken from patients undergoing surgery for non-disease-related reasons, such as crown lengthening, displayed minimal periodontal disease (probing depths of < 4 mm). Sixteen of the 22 gingival biopsies were classified as healthy at the time of surgery. Clinically healthy gingiva usually displays histological evidence of inflammation similar to that seen in marginal gingivitis (Seymour et al., 1983), so that clinically healthy and marginal gingivitis samples were grouped together (healthy/gingivitis), as published previously (Gemmell and Seymour, 1995). Tissue from 31 patients showed moderate to advanced disease and were classified as a chronic periodontitis group with probing depths > 4 mm, the majority (20/31) being > 6 mm. All subjects in this group had previous oral hygiene instruction and scaling and root planing prior to surgery, but continued to have bleeding on probing from the base of the pocket. A written explanation of the purpose of the study and signed consent according to the Helsinki Declaration to use tissue which would otherwise have been discarded were obtained at the time of surgery. Institutional ethics review committee approval to carry out the study was also obtained.

Preparation of Tissue
Tissue samples were prepared, and cryostat sections were cut and fixed as described by Gemmell and Seymour (1995).

Immunoperoxidase Technique
Mast cells were labeled according to an avidin-biotin immunoperoxidase method described previously (Gemmell and Seymour, 1995). The antibodies used were mouse anti-human tryptase (1/20) and rabbit anti-human c-kit (1/20), followed by biotinylated rabbit anti-mouse and swine anti-rabbit immunoglobulins, respectively (1/50), and finally streptavidin peroxidase (1/50) (DAKO, Glostrup, Denmark). IgG2+ and IgG4+ B-cells/plasma cells were stained by a direct immunoperoxidase method described previously (Gemmell et al., 2003), with horseradish peroxidase conjugated mouse anti-human IgG2 and IgG4 antibodies (Zymed Laboratories Inc., South San Francisco, CA, USA).

Cell Analysis
Using hematoxylin-and-eosin-stained sections, we evaluated each healthy/gingivitis or chronic periodontitis tissue sample with respect to the size of the connective tissue inflammatory infiltrate as described by Seymour et al.(1983). As described previously (Reinhardt et al., 1988; Gemmell and Seymour, 1995), biopsies with small infiltrates confined to the upper 1/3 of the section in the region adjacent to the junctional and sulcular epithelium were placed in a group defined as "small". The "medium" group contained sections with infiltrates which occupied the upper 2/3 of the section, and infiltrates extended throughout the entire section in the "large" group.

Using a graticule, we counted the numbers of tryptase+ and c-kit+ mast cells and IgG2+ and IgG4+ cells in 10 representative high-power fields (X400) and determined the mean (± standard error of the mean) numbers/2.5 mm2 in the healthy/gingivitis and chronic periodontitis tissues, as well as those in the three groups within each of these two main categories.

Statistical Analysis
We used multivariate analysis of variance with the general linear model to test for differences between the numbers of tryptase+ and c-kit+ mast cells as well as numbers of IgG2+ and IgG4+ cells. Selected pairs of groups were then tested for significance by the Student t test. We used the Minitab statistical package (Minitab Inc., State College, PA, USA) to perform the analyses.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS & METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Tryptase+ and C-kit+ Mast Cells
Both tryptase+ and c-kit+ mast cells were scattered fairly evenly throughout the connective tissue, although occasionally there was a slight increase in numbers of positive cells immediately beneath the epithelium (Figs. 1AGo–Go1C).In some infiltrates, staining suggested that mast cells appeared to be in cell-cell contact with lymphocytes (Fig. 1CGo). No mast cells were detected in the epithelial tissues.



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  		Figure 1. Tryptase+ mast cells in the lesion of a biopsy from a chronic periodontitis patient with a "large" lesion (A,B). C-kit+ mast cells appearing to be in close contact with lymphocytes in a "small" lesion of a biopsy from a chronic periodontitis patient (C).

 
Numbers of tryptase+ mast cells decreased in chronic periodontitis tissue sections compared with healthy/gingivitis samples (p = 0.004). Tryptase+ cell numbers were higher than c-kit+ cells in both healthy/gingivitis and chronic periodontitis sections (p = 0.000) (Fig. 2AGo). C-kit+ mast cell numbers were reduced in comparison with tryptase+ mast cells in all "small", "medium", and "large groups (p < 0.007) (Figs. 2BGo, 2CGo).



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  		Figure 2. The mean percent (± standard error of the mean) tryptase+ and c-kit+ mast cells in (A) the lesions of healthy/gingivitis (n = 22) and periodontitis (n = 31) subjects; (B) healthy/gingivitis tissues placed into 3 groups on the basis of size of infiltrate ("small", n = 9; "medium", n = 7; "large", n = 6); and (C) periodontitis lesions placed into 3 groups on the basis of size of infiltrate ("small", n = 8; "medium", n = 12; "large", n = 11).

 
The ratio of numbers of tryptase+ cells to c-kit+ cells was determined for each individual tissue section, and the percentages of tissues with ratios lower than 2/1 were determined for each group. The percentages of tissue sections with ratios less than 2/1 increased with increasing inflammation in the healthy/gingivitis group. In the chronic periodontitis group, the percentages increased from the "small" to the "medium" group and then decreased slightly in the "large" group (TableGo).


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  		Table. Percentage of Gingival Tissues with a Tryptase/C-kit Ratio of Less than 2/1
 
IgG2+ and IgG4+ B-cells/Plasma Cells
Where larger numbers of immunoglobulin-positive B-cells and plasma cells were present in the lesions, they tended to occur in clusters within the infiltrates. While there was no difference in IgG2+ and IgG4+ cell numbers in healthy/gingivitis tissues, there was an increase in IgG4+ cell numbers compared with IgG2+ cells in periodontitis lesions (p = 0.000) (Fig. 3AGo). There were no differences in IgG2+ or IgG4+ cells among the 3 histological groups of healthy/gingivitis samples (Fig. 3BGo). However, increased numbers of IgG4+ cells were demonstrated in "large" periodontitis lesions compared with "small"- group (p = 0.007) and "medium"-group lesions (p = 0.004) (Fig. 3CGo).



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  		Figure 3. The mean percent (± standard error of the mean) IgG2+ and IgG4+ B-cells/plasma cells in (A) the lesions of healthy/gingivitis (n = 21) and periodontitis (n = 29) subjects; (B) healthy/gingivitis tissues placed into 3 groups on the basis of size of infiltrate ("small", n = 9; "medium", n = 6; "large", n = 6); and (C) periodontitis lesions placed into 3 groups on the basis of size of infiltrate ("small", n = 7; "medium", n = 12; "large", n = 10).

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS & METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The results of the present study have shown that mast cells were scattered throughout the connective tissue. In some specimens, mast cells were observed in close contact with lymphocytes, suggesting possible antigen presentation or other cell-cell communication. Mast cells begin development in the bone marrow, enter the peripheral circulation, and undergo maturation in the tissues. CTMCs are long-lived cells which appear to be dependent mainly on stem cell factor for their development and survival (Gurish and Boyce, 2002) and, unusually, generally retain expression of c-kit receptor, for which stem cell factor is a ligand (Shanas et al., 1998). MMCs, in contrast, maturate in the tissues after exposure to Th2 cytokines, resulting in MMC hyperplasia in response to stimuli such as intestinal helminth infection and allergy, numbers reducing on resolution of the activating agent (Gurish and Boyce, 2002). IL-3 induces an MMC-like phenotype from murine bone marrow cells expressing the IL-4 gene as well as IL-10 and IL-13, while stem cell factor induces a CTMC-like phenotype expressing IL-12 (Smith et al., 1994; Marietta et al., 1996), suggesting that IL-3 and MMC may influence Th2 cell development and stem-cell-factor-derived CTMCs the development of Th1 cells (Smith et al., 1994).

Mast cells reside close to T-cells (Mekori and Metcalfe, 1999), phagocytose and process antigens, and initiate acquired immune responses by presenting antigens to T-cells (Fox et al., 1994; Frandji et al., 1996; Malaviya et al., 1996). Since T-lymphocytes have been shown to predominate in the stable periodontal lesion with an increase in the numbers of B-cells and plasma cells in the progressive lesion, there has been the suggestion that T-cells with a Th1 cytokine profile may be the major mediator in periodontitis, while Th2 cells play a role in periodontitis (reviewed in Gemmell et al., 2002). Interferon gamma and IL-4 induce T-cell-dependent isotype switching in B-cells to IgG2 and IgG4, respectively (Agresti and Vercelli, 2002; Tanaka et al., 2003). In the present study, there was an increase in the numbers of IgG4+ B-cells and plasma cells in comparison with IgG2+ cells in periodontitis lesions, but not healthy/gingivitis tissues, and numbers increased with increasing inflammation, suggesting higher levels of IL-4 with disease progression. Since tryptase+ mast cell numbers decreased in periodontitis tissues, it is possible that these cells do not constitute an IL-4-producing subset. The constant numbers of c-kit+ mast cells, regardless of clinical status or degree of inflammation, also indicate that there may be no increased migration of mast cells into the lesion.

In conclusion, tryptase+ mast cell numbers decreased with increasing inflammation in periodontal disease lesions. The numbers of c-kit+ mast cells, while lower than those of tryptase+ cells, remained constant. There was an increase in the numbers of IgG4+ cells compared with IgG2+ cells in periodontitis lesions, and IgG4+ cell numbers increased with increasing inflammation, indicating a predominance of Th2 responses in progressive periodontitis. The association of decreased tryptase+ mast cells with increased Th2 responses suggests that mast cells in periodontitis may not be the source of Th2 cytokines. However, since there were no differences in IgG2+ cell numbers, the c-kit+ population of mast cells may influence Th1 responses and therefore the IgG2+ cell population in periodontal disease.


   ACKNOWLEDGMENTS
 
This work was supported by the National Health and Medical Research Council of Australia.

Received August 18, 2003; Last revision February 24, 2004; Accepted February 25, 2004


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS & METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Agresti A, Vercelli D (2002). c-Rel is a selective activator of a novel IL-4/CD40 responsive element in the human Ig gamma4 germline promoter. Mol Immunol 38:849–859.[ISI][Medline]

Chan SH, Perussia B, Gupta JW, Kobayashi M, Pospisil M, Young HA, et al. (1991). Induction of interferon gamma production by natural killer cell stimulatory factor: characterization of the responder cells and synergy with other inducers. J Exp Med 173:869–879.[Abstract/Free Full Text]

Fox CC, Jewell SD, Whitacre CC (1994). Rat peritoneal mast cells present antigen to a PPD-specific T-cell line. Cell Immunol 158:253–264.[ISI][Medline]

Frandji P, Tkaczyk C, Oskeritzian C, David B, Desaymard C, Mecheri S (1996). Exogenous and endogenous antigens are differentially presented by mast cells to CD4+ T lymphocytes. Eur J Immunol 26:2517–2528.[ISI][Medline]

Galli SJ, Tsai M, Wershil BK, Tam SY, Costa JJ (1995). Regulation of mouse and human mast cell development, survival and function by stem cell factor, the ligand for the c-kit receptor. Int Arch Allergy Immunol 107:51–53.[ISI][Medline]

Gemmell E, Seymour GJ (1995). Gamma delta T lymphocytes in human periodontal disease tissue. J Periodontol 66:780–785.[ISI][Medline]

Gemmell E, Yamazaki K, Seymour GJ (2002). Destructive periodontitis lesions are determined by the nature of the lymphocytic response. Crit Rev Oral Biol Med 13:17–34.[Abstract/Free Full Text]

Gemmell E, Winning TA, Carter CL, Ford PJ, Bird PS, Ashman RB, et al. (2003). Differences in mouse strain influence leukocyte and immunoglobulin phenotype response to Porphyromonas gingivalis. Oral Microbiol Immunol 18:364–370.[ISI][Medline]

Gurish MF, Boyce JA (2002). Mast cell growth, differentiation, and death. Clin Rev Allergy Immunol 22:107–118.[ISI][Medline]

Irani AM, Schwartz LB (1989). Mast cell heterogeneity. Clin Exp Allergy 19:143–155.[ISI][Medline]

Irani AA, Schechter NM, Craig SS, DeBlois G, Schwartz LB (1986). Two types of human mast cells that have distinct neutral protease compositions. Proc Natl Acad Sci USA 83:4464–4468.[Abstract/Free Full Text]

Malaviya R, Twesten NJ, Ross EA, Abraham SN, Pfeifer JD (1996). Mast cells process bacterial Ags through a phagocytic route for class I MHC presentation to T cells. J Immunol 156:1490–1496.[Abstract]

Marietta EV, Chen Y, Weis JH (1996). Modulation of expression of the anti-inflammatory cytokines interleukin-13 and interleukin-10 by interleukin-3. Eur J Immunol 26:49–56.[ISI][Medline]

Mécheri S, David B (1997). Unravelling the mast cell dilemma: culprit or victim of its generosity? Immunol Today 18:212–215.[ISI][Medline]

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Plaut M, Pierce JH, Watson CJ, Hanley-Hyde J, Nordan RP, Paul WE (1989). Mast cell lines produce lymphokines in response to cross-linkage of Fc epsilon RI or to calcium ionophores. Nature 339:64–67.[Medline]

Reinhardt RA, Bolton RW, McDonald TL, DuBois LM, Kaldahl WB (1988). In situ lymphocyte subpopulations from active versus stable periodontal sites. J Periodontol 59:656–670.[ISI][Medline]

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Shanas U, Bhasin R, Sutherland AK, Silverman AJ, Silver R (1998). Brain mast cells lack the c-kit receptor: immunocytochemical evidence. J Neuroimmunol 90:207–211.[ISI][Medline]

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