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RESEARCH REPORT |
Department of Periodontics, 110 Rockland Hall, School of Dental Medicine, State University of New York at Stony Brook, Stony Brook, NY 11794-8703;
* corresponding author, ccutler{at}notes.cc.sunysb.edu
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS & METHODSRESULTSDISCUSSIONREFERENCES |
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KEY WORDS: dendritic cells • Langerhans cells • dermal DCs • periodontitis • T-cells • immunohistochemistry
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS & METHODSRESULTSDISCUSSIONREFERENCES |
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Skin and most mucosal surfaces contain at least two subsets of DCs. Langerhans cells are the best-characterized immature DCs, located above the basal layer of epithelial cells in the skin, oral, nasal, esophageal, pulmonary, vaginal, and rectal mucosa (Girolomoni et al., 2002). Dermal DCs, of which less is known, are present in the dermis of the skin and in the lamina propria of the rectum, uterus, and cervix (Geijtenbeek et al., 2000; Jameson et al., 2002). The immune function of these DCs depends upon their stage in maturation. In their immature state, DCs are considered to be part of the "innate phase" of the immune response. In this phase, DCs encounter and capture infectious agents like viruses, bacteria, and bacterial products, resulting in the release of inflammatory cytokines such as TNF-
and IL-1ß. These cytokines, through autocrine effects, activate and mobilize DCs and initiate the process leading to DC maturation. They can also be activated and mobilized by the cytokines of other cells in the local environment (paracrine effects). The mobilization of DCs from peripheral tissues to lymph nodes is a coordinated event regulated by several chemokines and chemokine receptors. In the "adaptive phase" in the lymph nodes, mature DCs present captured and processed antigens and prime naïve helper/cytotoxic T-cells to undergo clonal expansion (Banchereau and Steinman, 1998; Girolomoni et al., 2002).
The unique ability of DCs to elicit strong T-cell immunity has been exploited in DC-based vaccines for immunotherapy of cancer (Nestle et al., 2001; Steinman and Dhodapkar, 2001). Although DCs have been investigated mostly for their immunogenic capacities, recent evidence indicates that they might contribute to peripheral T-cell tolerance (Hawiger et al., 2001; Steinman and Nussenzweig, 2002) and dissemination of various infections like HIV-1, dengue virus, and Venezuelan equine encephalitis virus within the host (Geijtenbeek et al., 2000; MacDonald and Johnston, 2000; Wu et al., 2000; Cutler et al., 2001). There is very little information, however, about the role of DCs in the pathogenesis of chronic periodontitis (CP).
The number of Langerhans cells in the gingiva epithelium is a topic of much speculation, with increased numbers (Saglie et al., 1987), decreased numbers (Seguier et al., 2000), and no quantitative change (Gemmell et al., 2002) being reported during inflammation. Discrepancies in different studies may have been due to differences in the stage of disease. This is evident in an experimental gingivitis study (Moughal et al., 1992) in which an increase in number of LCs was observed until 7 days, followed by a plateau and then a decrease by 21 days. Interestingly, we observed that the periodontal pathogen Porphyromonas gingivalis comes into contact with, and possibly infects, Langerhans cells in the gingival epithelium in situ, suggesting that the component of plaque biofilm might be responsible for Langerhans cell mobilization in situ (Cutler et al., 1999). Recent studies, including ours, suggest that mature DCs increase in number in diseased lamina propria (Jotwani et al., 2001; Cirrincione et al., 2002), but the cells contributing to this mature DC pool have not been identified. Also, there is no information on whether oral mucosa (i.e., gingiva), like other mucosal surfaces, harbors different DC subsets known to play important roles in inflammation and infection.
In the present study, to establish the DC subpopulations and their association with T-cells in chronic periodontitis, we used a double-immunofluorescence staining technique, revealed by image-enhanced fluorescence and confocal microscopy. We found that gingiva, in addition to Langerhans cells, also contains a dermal DC population in the lamina propria, and the number of dermal DCs increases during inflammation. Further, in diseased tissues, Langerhans cells, dermal DCs, and B-cells contribute to the CD83+ mature DC pool. Moreover, mature DCs are associated with large clusters of CD4+ T-cells, suggestive of in situ antigen presentation.
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MATERIALS & METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS & METHODSRESULTSDISCUSSIONREFERENCES |
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Double-immunofluorescence staining was performed on pre-fixed frozen sections. The primary mouse monoclonal antibodies and their dilutions used in the study are listed in the Table
. For double-immunofluorescence staining, slides were rehydrated, blocked, and incubated for 1 hr at room temperature with primary mouse antibodies to Langerin, CD1a, CD19, CD4, CD83, and mannose receptor (MR). Slides were washed and incubated for 30 min at room temperature with Texas Red/FITC conjugated goat antibodies to mouse immunoglobulin (Molecular Probes Inc., Eugene, OR, USA). In a subsequent secondary step, FITC/Texas Red-conjugated mouse monoclonal antibodies to CD1a-, CD4-, CD83-, and DC-specific ICAM-3-grabbing non-integrin (DC-SIGN) were used. We confirmed the specificity of the primary and secondary antibodies by substituting each with the respective isotype controls. Images (Figs. 1, 2) were acquired with a Nikon Eclipse E600 microscope equipped with light and epifluorescence illumination and a high-resolution CCD camera (RT Slider, Diagnostic Instruments, Inc., Sterling Heights, MI, USA) and a PC running Image-Pro software (Media Cybernetics, Inc., Silver Spring, MD, USA). Images were sharpened with the use of 2D-deconvolution software. Some fluorescence images (0.25-µ optical sections) were also acquired by confocal laser scanning microscope (CLSM) system (Fig. 3), with the use of an epifluorescence microscope (Nikon E800, Japan) integrated to a confocal laser scanning microscope system (BioRad Radiance 2000, Hercules, CA, USA).
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS & METHODSRESULTSDISCUSSIONREFERENCES |
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). Further, we observed that these dermal DCs are distributed throughout the lamina propria, and the vast majority express mannose receptor (MR) (Fig. 1B), as described in skin (Turville et al., 2002). We further quantitated dermal DCs in the lamina propria of healthy and chronic periodontitis gingiva by a single-immunoenzyme technique (Jotwani et al., 2001). Analysis of our data indicates that chronic periodontitis (CP) is associated with a statistically significant increase in the number of dermal DCs in the lamina propria, relative to gingival health (mean number DC-SIGN+ cells per 20x field ± SE mean in CP vs. health = 124 ± 29 vs. 35.4 ± 5.2, respectively, p = 0.019, two-sample Student’s t test).
Langerhans Cells, Dermal DCs, and B-cells Contribute to the Mature CD83+ DCs in Chronic Periodontitis
To investigate the contributions of Langerhans cells, dermal DCs, and B-cells to the mature DC subpopulation in lamina propria, we co-localized CD83 with cell-subset-specific markers. It was observed that some Langerhans cells at the junction of epidermis and lamina propria (Fig. 2A) and some dermal DCs (Fig. 2B) and CD19+ B cells (Fig. 2C) in the lamina propria co-expressed CD83. This suggests that these cell subsets all contribute to the CD83+ mature DC population. With regard to B-cells and mature DCs, the vast majority are single-positive for CD19 and CD83, respectively.
Mature DCs are Associated with Large Clusters of CD4+ T-cells in Chronic Periodontitis
We have previously shown, quantitatively, that the lamina propria is infiltrated with increasing numbers of CD83+ mature DCs and CD4+ lymphocytes in chronic periodontitis. In the present study, the relationship between these two cell types was analyzed by double-immunofluorescence staining. Shown in Figs. 3A–3C are CD83+ cells associated with large clusters of CD4+ T-cells as determined by confocal microscopy. Some CD83+ cells also co-express CD4 (Figs. 3A, 3C), but were surrounded by CD4+ (single-positive) T-cells. In health, similar infiltrates are sparse at best (not shown).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS & METHODSRESULTSDISCUSSIONREFERENCES |
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, 1B). Double-immunofluorescence labeling demonstrated that most DC-SIGN+ cells expressed high levels of mannose receptors (MR) (Fig. 1B). We observed that there was a significant increase in the number of DC-SIGN+ cells in chronic periodontitis. The literature surveyed indicates that this is probably the first demonstration of the presence of an additional subset of immature DCs, besides Langerhans cells, in the inflamed gingiva. Based on the staining pattern and their location, this DC subpopulation in the gingiva closely resembles a subpopulation of dermal DCs in the skin (Turville et al., 2002). Similar immature DC populations, also identified by DC-SIGN, have been reported in the lamina propria of other mucosal tissues like the rectum, uterus, and cervix and in lymphoid tissues such as lymph nodes, tonsils, and spleen (Geijtenbeek et al., 2000; Jameson et al., 2002). Studies with in vitro-cultured monocyte-derived DCs which closely resemble dermal DCs have shown that DC-SIGN is a major receptor for HIV envelope protein gp 120 which facilitate HIV transfer to CD4+ T-lymphocytes (Geijtenbeek et al., 2000). The presence of DCs expressing DC-SIGN at mucosal surfaces suggests that they may be among the first cells to become infected and potentiate viral entry to deeper tissues and to the lymph nodes (Spira et al., 1996). The endocytic potential of DC-SIGN+ DCs has also been reported with other pathogens, like Ebola virus and Leishmania amastigotes (Alvarez et al., 2002; Colmenares et al., 2002). However, further studies will be required to investigate the role and functions of this DC subpopulation in human gingiva.
We have previously shown that, in chronic periodontitis, immature LCs predominantly infiltrate the gingival epithelium, whereas CD83+ mature DCs specifically infiltrate the CD4+ lymphoid-rich lamina propria (Jotwani et al., 2001). Since then, the presence of mature DCs in the lamina propria has been confirmed by another group as well (Cirrincione et al., 2002). However, there is no information regarding the cells which might contribute to the pool of mature DCs, except a recent report in which CD19+ B-cells from the gingiva of periodontitis subjects were shown to express CD83 and the co-stimulatory molecule CD86 by flow cytometry (Mahanonda et al., 2002). In view of these and our earlier findings, we used a double-immunofluorescence technique to analyze expression of CD83 by different cells in the inflamed gingiva. We found that some Langerhans cells (CD1a+) (Fig. 2A) at the junction of epidermis and lamina propria and some dermal DCs (DC-SIGN+) (Fig. 2B) and B-cells (CD19+) (Fig. 2C) in the lamina propria also express CD83. The vast majority of B-cells, however, do not co-express CD83, and the vast majority of mature DCs do not co-express CD19.
We further observed that most Langerhans cells remain isolated anatomically from CD4+ T-cells in the healthy gingiva. In disease, CD83+ cells are associated with large clusters of CD4+ T-cells, suggestive of local antigen presentation (Figs. 3A–3C). That contact occurs between the mature DCs and lymphocytes in inflamed gingiva has previously been reported (Cirrincione et al., 2002). In that study, the investigators used transmission electron microscopy to show that mature DCs in the lamina propria were present adjacent to lymphocytes. However, phenotypic characterization of lymphocytes was not determined, because the cells were not immunostained with cell-subset-specific markers. Formation of mature DC and CD4+ T-cell clusters has also been demonstrated in chronically inflamed skin infected with Candida albicans (Katou et al., 2000). The mature DCs in the inflamed skin were shown to be contributed by Langerhans cells; moreover, most of the CD4+ T-cells belonged to memory/effector T-cells. Subsequently, it was shown that the formation of clusters between mature DCs and CD4 cells was due to the expression of macrophage-derived chemokine (MDC) by DCs and expression of CCR4 (chemokine receptor 4) by CD4+ memory T-cells (Katou et al., 2001). Formation of mature DC and CD4 clusters in the inflamed gingiva in the present study does not imply that there is no trafficking of mature DCs to the lymph nodes, but does suggest that local trafficking and in situ maturation might be occurring as well. The CD4+ T-cells involved in cluster formation in the present study are not yet well-characterized, but the importance of CD4+ T-cells is very well-established in periodontal diseases. There is now ample in vivo evidence to prove that antigen presentation and activated CD4+ T-cells are major players in alveolar bone loss (Taubman and Kawai, 2001), and it is also evidenced by study on H2-AB knockout mice (deficient in CD4+ T-cells). While wild-type mice orally infected with P. gingivalis were found to manifest significant alveolar bone loss, the CD4+ T-cell-deficient mice did not (Baker et al., 2001). Furthermore, it was shown that adoptive transfer of human T-cells from aggressive periodontitis subjects into SCID mice induced alveolar bone loss upon infectious challenge with the etiological agent of aggressive periodontitis (Teng et al., 2000).
In conclusion, we have demonstrated the presence of a dermal DC subpopulation in the lamina propria of gingiva from healthy and CP subjects. Both DC subpopulations (Langerhans cells and dermal DCs) as well as B-cells have been shown to contribute to the CD83+ population observed in the inflamed gingiva. It remains to be clarified how these subpopulations of DCs and B-cells are involved in local and systemic antigen presentation.
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ACKNOWLEDGMENTS |
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Received October 3, 2002; Last revision May 28, 2003; Accepted June 6, 2003
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS & METHODSRESULTSDISCUSSIONREFERENCES |
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