Mast cells--a role in periodontal diseases?

OBJECTIVES: Limited attention has been given to the role mast cells may play in periodontal diseases. BACKGROUND: Mast cells are indeed found abundantly below and within several types of mucosal epithelia. On the basis of their proteinase content, mast cells are divided into connective tissue (CT) and mucosal phenotypes. The CT phenotype contains both tryptase and chymase (MC(TC)), while the mucosal phenotype contains only tryptase (MC(T)). The in vivo significance of different mast cell phenotypes has not yet been fully established. Mast cells are able to phagocytose, process and present antigens as effectively as macrophages. RESULTS: Recently mast cells were found in high numbers in chronically inflamed gingival tissue taken from patients with chronic marginal periodontitis (CMP). The number of mast cells was found to be even higher in HIV(+) patients with CMP. Furthermore, mast cells also express strongly matrix metalloproteinases (MMPs), which are key enzymes in degradation of gingival extracellular matrix. Mast cells may release preformed cytokines directing local innate and adaptive immune responses. The present review will focus on possible roles for mast cells in periodontal diseases. CONCLUSIONS: We certainly feel that this is a key cell in inflamed periodontal tissue and its role in periodontitis needs to be revisited.

Matrix metalloproteinases and their inhibitors in gingival mast cells in persons with and without human immunodeficiency virus infection.

BACKGROUND: Mast cells are a prominent cell type in the gingival infiltrate in periodontitis. In this study we examined the expression by gingival mast cells of matrix metalloproteinases, MMP-1, MMP-2, MMP-8 and the tissue inhibitors of metalloproteinases, TIMP-1 and TIMP-2. METHODS: Gingival specimens from 12 human immunodeficiency virus-negative (HIV-) and 15 HIV-positive (HIV+) patients with chronic marginal periodontitis (CMP), and from 10 HIV- and four HIV+ controls with clinically healthy gingiva (HG) were examined after double immunofluorescence staining for mast cell tryptase, combined with antibodies for MMP-1, MMP-2, MMP-8 or their inhibitors TIMP-1 and TIMP-2. RESULTS: In the HIV+CMP, HIV+HG and HIV-CMP groups, all mast cells expressed MMP-1 and MMP-8, whereas a smaller proportion (40-60%) in the HIV-HG controls displayed such staining. The former groups also displayed a significantly higher proportion (39-64%) of mast cells expressing MMP-2 as compared with the HIV-HG group (21-31%). All groups displayed similar proportions of TIMP-1 expressing mast cells (86-100%), whereas significantly increased proportions of TIMP-2+ mast cells were seen in the HIV+CMP, HIV+HG and HIV-CMP groups (18-25%) as compared with the HIV-HG group (8-13%). Mast cells were the cell type that most prominently expressed MMP-1 and MMP-8. MMP-2 expression was also strong in mast cells, but was also similarly expressed in other cell types. CONCLUSION: The chronically inflamed periodontal lesions in the present study appeared with little evidence of mast cell degranulation. The results show, however, that mast cells in inflamed gingiva have the potential to degrade extracellular matrix if appropriately triggered.

Fc(alpha) receptor I (CD89) on neutrophils in periodontal lesions.

AIMS: In this study, we have examined the occurrence of FcalphaRI-bearing cells in gingival tissue, gingival fluid and blood, in search for possible roles of IgA and FcalphaRI in periodontal lesions. METHODS: Gingival biopsies from inflamed and healthy sites were obtained from patients with chronic marginal periodontitis. Sections of inflamed gingiva were examined by immunofluorescence techniques and compared to sections from healthy sites. Smears were made from blood and gingival crevicular fluid and similarly studied. RESULTS: Dense infiltrates of neutrophils with strong expression of FcalphaRI (and FcgammaRIII) were found in connective tissue and epithelium of the apical part of periodontal pockets from diseased sites. In contrast, only few such cells were found in healthy gingiva from the same patients. Neutrophils in gingival fluid, tissue and blood expressed FcalphaRI with similar intensity, whereas the expression of FcgammaRIII was significantly decreased in gingival crevicular fluid. Considerable numbers of bacteria from gingival plaque were found to be covered by IgA. CONCLUSION: It is suggested that FcalphaRI on neutrophils may play an important rôle in elimination of IgA-opsonized bacteria, both in periodontal tissue and the adjacent pockets.

Increased levels of soluble Fc gamma receptor III in gingival fluid from periodontal lesions.

Enzyme-linked immunosorbent assay was used for determination of the concentration of soluble Fc gamma receptor III (Fc gamma RIII) in 40 samples of gingival fluid obtained from periodontal pockets in 30 patients with periodontitis. The assay was based on a monoclonal immobilized antibody binding Fc gamma RIII and a polyclonal Fc gamma RIII rabbit antibody for its quantification. The results indicate a substantially increased concentration of soluble Fc gamma RIII in gingival fluid as compared to the serum level. This increased concentration of soluble Fc gamma RIII may interfere with phagocytosis and immune homeostasis in the periodontal lesions.

Topical distribution of Fc gammaRI, Fc gammaRII and Fc gammaRIII in inflamed human gingiva.

The topical distribution of Fc gamma receptor types I, II and III (Fc gammaRI-III) was analyzed by means of immunohistochemistry in human gingival tissue obtained from 12 patients with chronic periodontitis. CD68+ macrophages expressing all three classes of Fc gammaR were found throughout the whole gingival connective tissue (CT), whereas dense infiltrates of polymorphonuclear granulocytes (identified by staining for neutrophil elastase) with strong staining for Fc gammaRIII and Fc gammaRII were found subjacent to the apical part of the pocket epithelium (PE) and in the PE itself. CD19+ B lymphocytes with variable staining intensity for Fc gammaRII were observed in clusters subjacent to the PE and extending into the central part of the CT. Only a few scattered CD3+ T lymphocytes stained for Fc gammaRIII. Some spindle-shaped cells (CD68-, therefore non-macrophages) and apparently non-cellular fibrous tissue elements stained for Fc gammaRI and Fc gammaRII. In the epithelium, Fc gammaRII+ dendritic cells were frequently observed in the entire oral gingival epithelium and in the coronal part of the PE. Occasionally, some keratinocytes which stained for Fc gammaRII and Fc gammaRIII were found. The observations indicate that Fc gammaR of the various classes are amply expressed on numerous cell types in inflamed gingival tissue. The specific distribution pattern detected suggests that Fc gammaRs may play a role in the mediation of chronic inflammation in the periodontal lesion.

LPS from Actinobacillus actinomycetemcomitans and the expression of beta2 integrins and L-selectin in an ex vivo human whole blood system.

Actinobacillus actinomycetemcomitans is assumed to be an important etiological agent in localized juvenile periodontitis (LJP) and to have the ability to invade periodontal tissues. This bacterium has also been noted for its potential to cause serious extraoral infections. In this study, the effect of lipopolysaccharides (LPS) extracted from A. actinomycetemcomitans on the expression of the leukocyte adhesion molecules CD11a/CD18, CD11b/CD18, CD11c/CD18 and L-selectin (CD 62L) were measured in an ex vivo whole blood system by use of fluorescent antibodies followed by flow cytometry. LPS from Escherichia coli, which is known to elicit a strong inflammatory response was used as a reference. The expression of the beta2 integrins CD11a/CD18, CD11b/CD18, and CD11c/CD18 were significantly upregulated in granulocytes and monocytes. This expression was dose-dependent. The baseline levels of L-selectin was high on all three types of leukocytes, but on granulocytes and monocytes it decreased dramatically after stimulation with LPS. The LPS from A. actinomycetemcomitans was equally potent as LPS from E. coli in its ability to affect the expression of the leukocyte integrins and L-selectin.

Lipopolysaccharide from Actinobacillus actinomycetemcomitans stimulates production of interleukin-1beta, tumor necrosis factor-alpha, interleukin-6 and interleukin-1 receptor antagonist in human whole blood.

Actinobacillus actinomycetemcomitans (A. actinomycetemcomitans) is supposed to be an important etiological agent in localized juvenile periodontitis (LJP). We have studied the effect of lipopolysaccharide (LPS) extracted from these periodontopathogenic bacteria on synthesis of the proinflammatory cytokines, interleukin-1beta(IL-1beta), tumor necrosis factor-alpha (TNF-alpha), interleukin-6 (IL-6) and the anti-inflammatory cytokine IL-1 receptor antagonist (IL-1ra) in human whole blood. LPS from A. actinomycetemcomitans in concentrations > or =1 ng/ml induced a significant production of all these proinflammatory cytokines, whereas LPS from Escherichia coli (E. coli), strain 026:B6 had to be added in concentrations > or =1 microg/ml to obtain a similar effect. Similarly, LPS from A. actinomycetemcomitans > or =0.1 ng/ml resulted in production of IL-1ra, while LPS from E. coli 026:B6 had to be added at > or =10 ng/ml to obtain similar effects. It has been suggested that the ratio between production of proinflammatory and anti-inflammatory cytokines may influence the outcome of periodontal diseases. Other in vitro and in vivo studies have, however, indicated that very large excesses (100-1000 times) of IL-1ra compared to IL-1beta are required to shift the IL-1ra:IL-1beta ratio in favor of an inhibition of IL-1 bioactivity. In our ex vivo system, we found that stimulation with extremely low doses of A. actinomycetemcomitans LPS (0.1-1 ng/ml) resulted in IL-1ra production solely, without concomitant production of IL-1beta, the excess of IL-1ra over IL-1beta peaking at 1 ng/ml, which accordingly should suggest that LPS from A. actinomycetemcomitans primarily has proinflammatory effects.

The gingival plasma cell infiltrate in renal transplant patients on an immunosuppressive regimen.

Treatment with immunosuppressive agents inhibits gingival inflammation and progression of periodontitis in humans. We examined the numbers and the isotype distribution of immunoglobulin-producing plasma cells by immunohistochemistry in gingival specimens taken from renal transplant transplant recipients receiving immunosuppressive agents (IS), and from otherwise comparable systemically healthy patients. The immunosuppressed patient group had significantly (P< 0.05) fewer IgG-, IgA-, IgG1-, IgG2-, and IgG4-producing plasma cells in the connective tissue adjacent to the pocket epithelium. The reduced numbers of such patents with quiescent periodontal disease support the contention that high counts of plasma cells are indicative of more severe disease.

Soluble Fc gamma receptors in periodontal lesions.

Soluble Fc gamma-binding components were detected in gingival fluid from periodontal lesions by incubation with biotinylated human Fc gamma fragments. Fc gamma III receptor was identified by incubation of gingival fluid with monoclonal antibody. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western transfer showed that most of the Fc gamma-binding components had minimal mobility in a 4-15% gradient gel under nonreducing conditions. Under reducing conditions, the main band of Fc gamma-binding components in gingival fluid migrated corresponding to protein A of 49 kDa. The pattern of Fc gamma-binding components was similar in serum and gingival fluid except for the observation in gingival fluid of Fc gamma-binding components migrating like standard proteins of 19 to 20 kDa, a size that corresponds to the polypeptide part of Fc gamma II receptor and Fc gamma III receptor.

LPS from Actinobacillus actinomycetemcomitans and production of nitric oxide in murine macrophages J774.

Nitric oxide (NO) plays a complex role in the modulation of the inflammatory response, having either a pro-inflammatory or a protective role. Actinobacillus actinomycetemcomitans is considered an important etiological agent in localized juvenile periodontitis. We have studied the effect of lipopolysaccharide (LPS) extracted from this periodontopathogenic bacterium on NO synthesis in an in vitro murine macrophage system. LPS from A. actinomycetemcomitans induced a significant production of NO even at concentrations as low as 1 ng/ml, whereas LPS from E. coli had to be added in concentrations of 100 ng/ml to obtain similar effects. Production of NO was blocked by NG-nitro-L-arginine methylester, and pre-treatment of LPS from A. actinomycetemcomitans with polymyxin B abolished the production of NO, while prostaglandin E2 enhanced the synthesis of NO.

Fc gamma-binding bacteria in periodontal lesions.

The proportion of bacteria exhibiting surface Fc gamma-binding proteins was determined in periodontal pockets of 20 patients diagnosed with periodontal disease and in subgingival areas of 20 patients without periodontal lesions. Bacterial smears were examined by fluorescence microscopy based on DNA staining (Hoechst 33256) and staining of Fc gamma-binding proteins by human biotin-labelled Fc gamma and Texas red-conjugated streptavidin. Fc gamma-binding proteins were observed in all smears from the patients diagnosed with periodontitis, and in a majority of the smears high proportions of the bacteria were positive for Fc gamma-binding proteins. In contrast, most smears from patients without periodontal lesions included low or undetectable proportions of bacteria with Fc gamma-binding proteins.

In vitro cleavage of serum complement protein C3: a comparison between patients with adult periodontitis and periodontally healthy persons.

This study tested the hypothesis that in vitro cleavage of C3 could be triggered with similar case in serum samples from patients with adult periodontitis (n = 26) as in samples from periodontally healthy subjects (n = 13). A lipoteichoic acid, a lipopolysaccharide and an aggregated IgG served as activators of complement. On the average, the periodontitis group generated significantly (p < 0.01) more C3d activation fragments than did the healthy group, as judged from rocket immunoelectrophoresis measurements. Cleavage of C4 and factor B were then assayed through immunoblotting, without prior purification of the sera. C4c fragments were seen in all activated samples, the healthy group causing significantly (p < 0.05) more C4 conversion than did the periodontitis group. Cleavage of factor B, taken as a measure of soluble amplification convertase formation, was about equal between the groups. We inferred therefore that the 2 groups produced comparable amounts of C3b. The results suggested, however, that periodontitis sera favour breakdown of the opsonin C3b, most likely by activating the regulatory proteins factor H and I. Lipoteichoic acid, causing moderate depletion of C4 and factor B, produced significantly (p < 0.01) more C3d fragments than the other two activators examined. It may be that complement activation is down-regulated in periodontitis sera, perhaps at the expense of adequate local opsonic function.

In vitro activation of the classical pathway of complement by a streptococcal lipoteichoic acid.

The purpose of this study was to find whether a glycerolphosphate-containing lipoteichoic acid prepared from Streptococcus sobrinus OMZ 176 cells would activate the classical pathway of complement while in solution. Reference activators were lipopolysaccharide from Escherichia coli 0111:B4 and heat-aggregated immunoglobulin G. Serum samples were taken from healthy students. Analysis through crossed immunoelectrophoresis showed that lipoteichoic acid caused an almost complete dissociation of the C1qrs macromolecule. All activators decreased the area of and slowed the electrophoretic mobility of the C4 protein peaks, with lipoteichoic acid causing the most pronounced alterations. Electroimmunoassays showed that lipoteichoic acid separately, yielded detectable amounts of free C1r2s2 subunits; it also generated significantly more trimer complexes between C1r, C1s and C1 inhibitor (C1INH) than did the other two activators. Lipoteichoic acid was, however, a comparatively weak inducer of tetramer C1INH-C1r-C1s-C1INH complexes. Analysis through Western blotting showed that all activators accelerated consumption of C1r, induced complex formations between C1INH and C1s and produced cleavage products of C2. Altogether, the immunochemical analysis gave clear evidence of classical pathway activation by lipoteichoic acid, but its activation profile differed from those seen with lipopolysaccharide and aggregated immunoglobulin G.

Specificity and levels of oral and systemic antibodies to Actinobacillus actinomycetemcomitans.

Salivary and gingival crevicular fluid antibodies and systemic antibodies were analysed for levels and specificity against Actinobacillus actinomycetemcomitans components. The major reactivity of salivary and serum IgA1 and IgA2 antibodies to the periodontal pathogen A. actinomycetemcomitans was against bands between 14 and 83 kD for IgA1 and bands between 14 and 68 kD for IgA2 in Western blot. In addition to specific binding, there was also a hitherto unrecognized Fc-mediated binding of IgG antibodies to an A. actinomycetemcomitans component around 50 kD. Serum IgG antibodies to A. actinomycetemcomitans leukotoxin displayed the highest median value and only 1 individual showed salivary IgM antibodies in ELISA. Elevated levels of gingival crevicular fluid IgA2 antibodies indicated a local production of IgA from periodontal tissues. Using synthetic peptides, several distinct epitopes on the leukotoxin were recognized by both salivary and serum IgA antibodies.

Cell cycle-specific growth inhibitory effect on human gingival fibroblasts of a toxin isolated from the culture medium of Actinobacillus actinomycetemcomitans.

A toxin isolated from the growth medium of Actinobacillus actinomycetemcomitans by ammonium sulfate precipitation was shown to inhibit irreversibly the multiplication of human gingival fibroblasts. DNA histograms from flow cytometric measurements showed that the cells accumulated preferentially in the G2 phase of the cell cycle. Such cells exhibited sheetlike protrusions, and an increased frequency of micronuclei was evident in cells treated with low concentrations of the toxin. Toxin-treated cells were viable for several weeks, as shown by staining with trypan blue and fluorescein diacetate, and the general cell metabolism as measured by oxygen consumption was unimpaired.

Entrance of Actinobacillus actinomycetemcomitans into HEp-2 cells in vitro.

A strain of actinobacillus actinomycetemcomitans, freshly isolated from a juvenile periodontitis patient, and the FDC Y4 laboratory strain of Aa were tested for their capacity to adhere to and enter the epithelial cell line HEp-2 cells in vitro. Immunofluorescence microscopy as well as scanning electron microscopy (SEM) and transmission electron microscopy (TEM) showed that both strains adhered to the outer surface of the HEp-2 cells. In the TEM studies, the specimens were also treated with Aa specific antibodies and gold labeled protein A. These examinations showed that only the freshly isolated strain of Aa was found within the HEp-2 cells. The intracellular Aa were found to be viable, and in one case one of them was seen to undergo division. It is concluded that freshly isolated Aa has the ability to enter epithelial HEp-2 cells in vitro, and it is tentatively suggested that this may play a role in the pathogenesis of periodontal disease.

Fc-binding components: a virulence factor in Actinobacillus actinomycetemcomitans?

Actinobacillus actinomycetemcomitans (ATCC 33384) can produce and release components that bind to the Fc part of IgG. Fc-binding components were observed in whole bacteria, capsular material and medium from broth cultures. The components were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotted with biotinylated Fc-fragments and myeloma proteins. In a phagocytosis assay with human granulocytes and sheep erythrocytes, preincubation of opsonized erythrocytes with protein A reduced phagocytosis by 90%. In contrast, preincubation of the opsonizing antibody with medium components from a culture of A. actinomycetemcomitans enhanced the opsonizing effect of the antibody. The enhanced binding of erythrocytes may be caused by formation of aggregates between opsonizing antibody and bacterial Fc-binding components. Aggregated IgG can bind to low-affinity Fc gamma II and gamma III receptors that cannot bind monomeric IgG. Release of Fc-binding components from bacteria may contribute to the periodontal lesion through interference with the phagocytic activity of granulocytes and with the complement system. Fc-binding components may also interfere with downregulation of the B-cell response.