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Mast Cells in Human Periodontal DiseaseOral Biology and Pathology, School of Dentistry, The University of Queensland, Brisbane 4072, Australia; Correspondence: * corresponding author, e.gemmell{at}uq.edu.au
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
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.
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
Immunoperoxidase Technique
Cell Analysis 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
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. 1A  –1C).In some infiltrates, staining suggested that mast cells appeared to be in cell-cell contact with lymphocytes (Fig. 1C). No mast cells were detected in the epithelial tissues.
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. 2A ). C-kit+ mast cell numbers were reduced in comparison with tryptase+ mast cells in all "small", "medium", and "large groups (p < 0.007) (Figs. 2B, 2C).
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 (Table ).
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. 3A ). There were no differences in IgG2+ or IgG4+ cells among the 3 histological groups of healthy/gingivitis samples (Fig. 3B). 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. 3C).
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.
This work was supported by the National Health and Medical Research Council of Australia. Received for publication August 18, 2003. Revision received February 24, 2004. Accepted for publication February 25, 2004.
Journal of Dental Research, Vol. 83, No. 5,
384-387 (2004)
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