The presence of inflammatory infiltrates with B cells, specifically plasma cells, is the hallmark of periodontitis lesions. The composition of these infiltrates in various stages of homeostasis and disease development is not well documented. Human tissue biopsies from sites with gingival health (n = 29), gingivitis (n = 8), and periodontitis (n = 21) as well as gingival tissue after treated periodontitis (n = 6) were obtained and analyzed for their composition of B cell subsets. Ag specificity, Ig secretion, and expression of receptor activator of NF-κB ligand and granzyme B were performed. Although most of the B cell subsets in healthy gingiva and gingivitis tissues were CD19+CD27+CD38 memory B cells, the major B cell component in periodontitis was CD19+CD27+CD38+CD138+HLA-DRlow plasma cells, not plasmablasts. Plasma cell aggregates were observed at the base of the periodontal pocket and scattered throughout the gingiva, especially apically toward the advancing front of the lesion. High expression of CXCL12, a proliferation-inducing ligand, B cell–activating factor, IL-10, IL-6, and IL-21 molecules involved in local B cell responses was detected in both gingivitis and periodontitis tissues. Periodontitis tissue plasma cells mainly secreted IgG specific to periodontal pathogens and also expressed receptor activator of NF-κB ligand, a bone resorption cytokine. Memory B cells resided in the connective tissue subjacent to the junctional epithelium in healthy gingiva. This suggested a role of memory B cells in maintaining periodontal homeostasis.

The Global Burden of Disease Study (2010) ranked severe periodontitis as the sixth-most prevalent disease in the world, affecting 11.2% worldwide (1).

Chronic periodontitis is an advanced form of periodontal diseases and represents a chronic inflammation of tooth-supporting structures. It is characterized by gingival inflammation (redness, swelling, and bleeding) and loss of connective tissue attachment to the tooth that may result in resorption of the alveolar bone. Thus, an advanced stage of periodontitis may eventually lead to tooth loss. The precursor stage of periodontitis is limited to the marginal gingival region and is called gingivitis. It is characterized by gingival inflammation without the loss of attachment and alveolar bone.

In the 1960s to 70s, the etiologic role of dental plaque in periodontal diseases was firmly established. In the same period, an intense effort was made to understand periodontal immunopathogenesis, which involves the interplay between the dental plaque biofilm and the host response.

Classical human experimental gingivitis (2, 3) and experimental periodontitis (4) studies in animal models outlined changes in microbial plaque and tissue immunopathology during the transition from periodontal health to gingivitis and, later on, to periodontitis. Increases in the Gram-positive bacterial species in supragingival plaque biofilms such as Streptococcus and Actinomyces species were observed concomitant with the development of a T cell–dominated gingivitis infiltrate. The presence of Gram-negative bacterial species in subgingival plaque biofilms such as Porphyromonas gingivalis, Aggregatibacter actinomycetemcomitans, Tanerella forsythia, and Treponema denticola was later associated with the development of plasma cell–dominated periodontitis lesions (5, 6).

The T cell response to the presence of periodontal pathogens is considered to play a key role in the regulation of periodontal pathogenesis. It has been speculated that a Th2 response was associated with periodontitis, whereas Th1 responses are associated with gingivitis (7). However, such a hypothesis remains controversial. More recently, other Th subsets, including regulatory Th cells and Th17 cells, have been demonstrated in periodontal tissues, of which an inverse relationship between these two cell types could be established (8). Whereas Th17 cells promote periodontal inflammation and tissue destruction (9), T regulatory cells are implicated in guard against periodontitis progression (10).

To date, emphasis has been placed on the T cell response in controlling local immunity and causing chronic periodontal tissue destruction. However, the role of the B cell response in periodontal homeostasis or disease development has not been thoroughly studied. Since 1965, it has been known that Ig-producing plasma cells predominated the periodontitis lesions (11). B cells and plasma cells outnumbered T cells, when gingivitis progressed to periodontitis (5). However, detailed profiles of B cell subsets, including naive B cells, memory B cells, and Ab-secreting cells (ASCs), had not yet been investigated systematically in periodontal tissues of various healthy or diseased conditions.

Human B cell subsets may now be identified phenotypically based on the expression of different surface markers, such as CD19, CD20, CD27, CD38, and CD138 (12, 13). A pan B cell marker, CD19, is expressed in all B cell subsets, whereas CD20 is downregulated, when memory B cells differentiate into ASCs (14). CD27, a member of the TNFR family, which induces differentiation and promotes survival (15), is expressed on the majority of memory B cells (16). CD38 catalyzes the formation of cyclic ADP-ribose and NADP and regulates Ca+ signaling in lymphoid cells (17). Both CD27 and CD38 are highly expressed on ASCs. CD138, a heparin sulfate proteoglycan, also known as syndecan-1, is generally used as a plasma cell marker (18).

The purpose of this study was to identify different B cell subsets in clinically healthy and diseased periodontal tissues. Moreover, the morphological compartmental localization of the cells and hence their possible role in disease development were to be explored.

Medium for gingival cell culture.

The gingival cells were cultured in complete medium consisting of RPMI 1640 supplemented with nonessential amino acids, 2 mM l-glutamine, 1 mM sodium pyruvate, 100 μg/ml penicillin, 100 μg/ml streptomycin (Life Technologies Laboratories, Grand Island, NY), and 10 μg/ml 2-ME, supplemented with 10% FCS (Hyclone).

Abs.

Abs were used against the following proteins for flow cytometry: CD3 (SK7), CD19 (4G7), CD27 (M-T271), CD38 (HIT2), CD138 (MI15), HLA-DR (L243), and granzyme B (GB11) from BD Biosciences; receptor activator of NF-κB ligand (RANKL; MIH24) from BioLegend. Abs were used against the following proteins for immunohistochemistry analysis: CD20 (L26) and CD27 (137B4) from Abcam; CD138 (MI15) and peripheral node addressin (PNAd; MECA-79) from BioLegend; follicular dendritic cells (CNA.2) from eBioscience; ICAM-1 (23G12) from Thermo Scientific; and CD62L (B-8) from Santa Cruz Biotechnology.

The ethical approval was obtained from the Ethics Committee of Faculty of Dentistry, Chulalongkorn University (HREC-DCU 2013-016). Written informed consent of all participating subjects was obtained prior to inclusion in the study.

Periodontal tissues and heparinized peripheral blood samples were obtained from patients with severe chronic periodontitis and gingivitis and subjects with clinically healthy gingiva. These specimens were collected from patients at Periodontal Clinic and Oral Maxillofacial Surgery Clinic, Faculty of Dentistry, Chulalongkorn University (Bangkok, Thailand).

Each patient had at least 16 remaining teeth with no history of periodontal treatment for the past 6 mo. Subjects with clinically healthy gingiva showed no sign of gingival inflammation (no bleeding on probing, probing depth <4 mm) and no clinical attachment loss or bone loss. Healthy gingival tissue specimens were collected during crown-lengthening procedure for prosthetic or orthodontic reasons. Gingivitis tissue specimens were collected from inflamed sites (bleeding on probing, but no clinical attachment or bone loss) of extracted tooth due to tooth malposition, crowding, or pericoronitis. Periodontitis tissue specimens were collected from sites of extracted teeth irrational to treat (bleeding on probing, probing pocket depth >6 mm, and bone loss >60% of the root). After periodontal treatment of scaling and root planing in four subjects with severe chronic periodontitis, treated tissue specimens (negligible sign of marginal gingival inflammation, but advanced clinical attachment loss and bone loss >60% of the root) were collected from teeth extracted as being judged irrational to treat. All subjects were in good general health, and none of them had taken antimicrobial or anti-inflammatory drugs within the previous 3 mo.

The excised tissues were immediately placed in sterile tubes that contain RPMI 1640 medium. Three milliliters of peripheral blood was collected. The samples were transferred to the laboratory within a few hours for the study.

The method for obtaining single-cell suspensions from gingival tissues was modified from the method that was described by Mahanonda et al. (19). Briefly, the tissues were washed in RPMI 1640 medium and cut into small fragments (1–2 mm3). The fragments were incubated in medium that contained 2 mg/ml collagenase type I (Life Technologies). After 90-min incubation (37°C), residual tissue fragments were disaggregated by gentle flushing, until single-cell suspensions were obtained. These cells were filtered through filter (mesh size 70 μm).

To characterize the profiles of B cell subsets (naive B cells, memory B cells, ASCs) in isolated gingival cell suspension form, periodontal tissues and heparinized whole blood were stained with mouse anti-human CD19 (FITC), CD27 (PE), CD3 (PerCP), and CD38 (allophycocyanin) mAbs at 4°C for 30 min, whereas whole blood was stained at room temperature for 30 min. Naive B cells, memory B cells, and ASCs were characterized as CD19+CD27CD38, CD19+CD27+CD38, and CD19+CD27+CD38+, respectively. The stained gingival cells were washed with stain buffer (BD Biosciences). The stained cells were treated with RBC lysing solution for 10 min at room temperature in the dark, washed, and then fixed with 1% paraformaldehyde. Analysis of flow cytometry samples was performed by four-color flow cytometry (FACSCalibur; BD Biosciences).

Differential expression between plasmablasts and plasma cells was used to investigate ASC subsets. Unlike plasmablasts and memory B cells, plasma cells express very low level of MHC class II (HLA-DR) (2022) and also express CD138 (a well-known marker for plasma cell) (18, 2325).

To identify surface IgG and IgA on memory B cells, gingival cell suspension form periodontal healthy tissues were stained with mouse anti-human IgG (PE) or IgA1/IgA2 (PerCP) mAbs. IgG memory B cells and IgA memory B cells were characterized as CD19+CD27+CD38IgG+ and CD19+CD27+CD38IgA1/IgA2+, respectively.

The excised periodontal tissues were immediately washed in normal saline solution. For paraffin-embedded sections, they were fixed in 10% buffered formalin for a maximum of 24 h and subsequently embedded in paraffin. Microtome serial 4-μm–thick sections were cut and mounted on glass slides. Sections were deparaffinized. To inhibit endogenous peroxidase, they were incubated with 0.3% hydrogen peroxide solution for 20 min, and heated in 1 mM EDTA (pH 8.0) at 95°C for 20 min for Ag retrieval.

For identifying local plasma cells, single immunohistochemical staining was performed via polymer/HRP and diaminobenzidine (DAB)+ chromagen system (DAKO EnVisionTM G/2 Doublestain System) on the sections. They were stained with primary mouse anti-human CD138 or isotype control. Counterstaining was done with hematoxylin. They were investigated under light microscope. To identify gingiva memory B cells, double immunohistochemical staining with mAbs to CD20 and CD27 was performed via polymer/HRP and DAB+ chromagen system on the sections. Expression of PNAd and ICAM-1 on gingival blood vessel endothelial cells, tissue staining with mAbs to PNAd, and ICAM-1 was performed via polymer/HRP and DAB+ chromagen system on the sections.

To characterize Ag-specific Ab production, the frequency of Ag-specific ASCs from periodontal tissues was measured by ELISPOT assay. Briefly, Multiscreen 96-well filtration plates (Millipore) was coated with a variety of soluble bacterial Ags. These included key periodontal pathogens, as follows: 1) P. gingivalis ATCC33277; 2) A. actinomycetemcomitans Y4; 3) commensal plaque bacteria: Streptococcus gordonii DL-1 (from F. Yoshimura, Department of Microbiology, School of Dentistry, Aichi Gakuin University, Nagoya, Japan); and 4) self-tissue: type I collagen (Sigma-Aldrich) overnight at 4°C in a humidified chamber. Keyhole limpet hemocyanin was used as a negative control.

The plate was washed twice with Dulbecco PBS (DPBS; Life Technologies) and blocked with DPBS containing 10% FBS for 1 h at 37°C. After being washed twice with DPBS, gingival mononuclear cells were added and incubated overnight at 37°C. After that, the plate was washed three times with PBS and another three times with PBS with 0.05% Tween 20 (PBST). Goat anti-human IgG or IgA biotin conjugated (KPL) was added into the plate. After 2 h of incubation at 37°C, the plate was washed four times with PBST, and streptavidin-alkaline phosphatase was added to the plate at a 1:1000 dilution and incubated for 1 h at 37°C. Then the plate was washed three times with PBST and another three times with PBS; after that a substrate (5-bromo-4-chloro-3-indolyl phosphate/NBT; Sigma-Aldrich) was added to allow spots to develop for 5–15 min, and then spots were counted by using an ELISPOT reader (Cellular Technologies).

To investigate the ability to secrete IgG and IgA of the memory B cells, the gingival cell suspension from periodontal healthy tissue was activated with TLR7/8, R848 (Resiquimod; Innovagen), and human rIL-2 (R&D Systems) for 6 d in 37°C, 5% CO2. After 6 d of incubation, IgG- and IgA-secreting cells were detected by ELISPOT assay, as described above for plasma cells.

To study the role of local tissue mediators in B cell response, total RNA from periodontal tissue samples was isolated by using an RNeasy mini kit from Qiagen. One microgram of DNase I–treated total RNA was reverse transcribed using ImProm-II Reverse Transcription System for RT-PCR (Promega). The cDNA was then divided and used for PCR amplification of chemokines and survival factors for B cell responses. Real-time RT-PCR assays were performed on LightCycler System 480 (Roche Molecular Diagnostics) using SYBR Green PCR Master Mix (Roche Molecular Diagnostics). CXCL12, a proliferation-inducing ligand (APRIL), B cell–activating factor (BAFF), IL-10, IL-6, IL-21, and GAPDH were amplified using specific primers purchased from Operon. The PCR conditions have been described previously (2629), and the primer sequences are shown as follows: CXCL12 (5′-ATGCCCATGCCGATTCTT-3′/5′-GCCGGGCTACAATCTGAAGG-3′), APRIL (5′-AGAGTCTCCCGGAGCAGAGTT-3′/5′-CTGGTTGCCACATCACCTCTGT-3′), BAFF (5′-TGAAACACCAACCTATACAAAAAG-3′/5′-TCAATTCATCCCCAAAGACAT-3′), IL-10 (5′-GGCGCTGTCATCGATTTCTT-3′/5′-TGGAGCTTATTAAAGGCATTCTTCA-3′), IL-6 (5′-CCTGAACCTTCCAAAGATGG-3′/5′-ACCAGGCAAGTCTCCTCATT-3′), IL-21 (5′-TGTGAATGACTTGGTCCCTGAA-3′/5′-ACCAGGAAAAAGCTGACCACTCA-3′), and GAPDH (5′-GAAGGCTGGGGCTCATTT-3′/5′-CAGGAGGCATTGCTGATGAT-3′). Amplification conditions, sequences, and concentrations of the primers were similar to those of RT-PCR. After 45 reaction cycles, the melting curve analysis was performed at 95°C for 5 s, 65°C for 1 min, and heating to 97°C using a ramp rate of 0.11°C/s with continuous monitoring of fluorescence. The melting peak generated represented the specific amplified product. All samples had only a single peak, indicating a pure product and no primer/dimer formation. Amplicons of a single band with the expected sizes were also confirmed in all reactions by agarose gel electrophoresis. The amplification efficiencies were high (close to 100%) when multiple standard curves were performed using serial mRNA dilutions.

For periodontal tissue specimens, the relative mRNA expression was normalized to corresponding GAPDH for each sample, using the formula = 2−ΔCT, where ΔCT = CT−geneX − CT−GAPDH. The relative quantification of mRNA expression in periodontitis tissues was presented as the mean fold increase ± SEM, using the mean value obtained from the healthy tissue as a reference (relative quantification = 1).

To investigate the expression of RANKL and granzyme B in plasma cells, gingival cells were first stained for surface-expressing molecules with anti-human CD19, CD27, and CD38 mAbs at 4°C for 30 min. The stained gingival cells were washed with stain buffer and then fixed and permeabilized with BD Cytofix/Cytoperm kit (BD Biosciences) on ice for 20 min and washed with BD Perm/Wash buffer (BD Biosciences). The cells were then suspended in BD Perm/Wash buffer, and anti-human RANKL or anti-human granzyme B or isotype control mAbs were added. After another 30 min of incubation on ice, cells were washed and then fixed with 1% paraformaldehyde. Analysis of flow cytometry samples was performed by four-color flow cytometry, FACSCalibur.

Results were expressed as mean ± SEM. Comparisons between multiple clinical groups were analyzed using the Kruskal–Wallis test by rank, and post hoc comparisons were made using the Mann–Whitney U test with Bonferroni’s correction. Comparisons between multiple B cell subsets within each clinical group were analyzed using the Friedman test by rank, and post hoc comparisons were made using the Wilcoxon signed-rank test with Bonferroni’s correction. Comparisons between two variables in one group were analyzed using Wilcoxon signed-rank test. In cases in which data were normalized to healthy control, a one-sample Wilcoxon signed-rank test was used with an expected value of 1. Comparisons of one variable between two groups were analyzed using the Mann–Whitney U test. Two-tailed p values <0.05 were considered statistically significant (SPSS version 22.0).

A mixture of mAbs consisting of anti-CD19, anti-CD27, and anti-CD38 was employed to identify different subsets of B cells in gingival tissues from clinically healthy gingiva, gingivitis, and periodontitis. Naive B cells were characterized as CD19+CD27CD38, memory B cells were characterized as CD19+CD27+CD38, and ASCs as CD19+CD27+CD38+, respectively. Flow cytometry analysis of periodontal tissues showed very few naive B cells (<8%) in all stages of healthy or diseased tissues.

Memory B cells represented the majority in the CD19+ B cell population (86.59% ± 1.29) in the clinically healthy gingiva and in gingivitis tissues (85.90% ± 2.67). In periodontitis tissues, however, the density of memory B cells was 37.67% ± 3.39. The difference in the density of memory B cells in healthy and gingivitis tissues compared with periodontitis tissue was statistically significant (p < 0.01, Kruskal–Wallis test by rank, and post hoc comparisons by Mann–Whitney U test with Bonferroni’s correction).

In periodontitis tissue, ASCs were the major cell type in the CD19+ B cell population (58.44% ± 3.79). The mean percentage of ASCs was significantly higher than that of memory B cells (p < 0.01, Friedman test by rank, and post hoc comparisons by Wilcoxon signed-rank test with Bonferroni’s correction) (Fig. 1A). In contrast, in the healthy and the gingivitis tissues, the mean percentage of memory B cells was significantly higher than the mean percentage of ASCs (6.64% ± 1.19; 6.34% ± 1.98, respectively).

Unlike in gingival tissues, there were no differences in peripheral blood B cell subsets between the healthy or diseased stages. In contrast to the gingival tissues, peripheral blood-naive B cells represented a major population (>64 and 65% in healthy and periodontitis patients, respectively) in CD19+ B cell population, followed by memory B cells (34 and 33% in healthy and periodontitis patients, respectively), and very small proportions of ASCs (2.1 and 2.0% in healthy and periodontitis patients, respectively) (Fig. 1B).

In addition, gingival tissues from periodontitis patients (n = 6) who received initial periodontal treatment of scaling and root planing (removal of biofilms) and who were scheduled for some tooth extractions were analyzed. As depicted in Fig. 1C, the majority of B cell subsets at treated sites represented high proportions of memory B cells similar to those of clinically healthy gingiva and gingivitis tissues.

Because high proportions of CD19+CD27+CD38 memory B cells among the CD19+ B cell population were identified in clinically healthy gingiva (Fig. 1A), the anatomical compartmental location of this specific population in histological sections was investigated. Because CD19 staining in formaldehyde-fixed, paraffin-embedded tissues was not successful in our experiments, mAbs against CD20 and CD27 were used to identify memory B cells. A cluster of CD20+CD27+ memory B cells in clinically healthy gingiva was detected in the connective tissue subjacent to the apical region of the junctional epithelium (Fig. 2A). A representative flow cytometry histogram in Fig. 2B demonstrates that gingival memory B cells from clinically healthy human gingiva also expressed surface IgG and IgA, thus confirming their characteristics of memory B cells. Moreover, it was explored whether these gingival tissue-memory B cells could be differentiated in vitro into ASCs. Upon polyclonal B cell activation with TLR7/8 (R848) and IL-2, these cells transformed into IgG and IgA ASCs, as assessed by ELISPOT assay (Fig. 2C). A significantly higher frequency of IgG spot-forming cells (SFCs) was observed when compared with IgA-SFCs (n = 3, p < 0.05, Wilcoxon signed-rank test). It should be pointed out that endothelial cells of the blood vessels in the connective tissue subjacent to the junctional epithelium of healthy gingiva expressed peripheral node addressin (PNAd+) and ICAM-1+ (Fig. 3). Inducible PNAd and ICAM-1 are known to bind with lymphocyte endothelial cell adhesion molecule-1 and LFA-1, respectively, on B cells (30, 31).

CD19+CD27+CD38+ ASCs consist of two subsets: plasmablasts and plasma cells (20, 32). Unlike plasmablasts, plasma cells express very low level of HLA-DR (20, 33, 34) as compared with memory B cells. The levels of HLA-DR expression were then used to differentiate the two subpopulations of ASCs in periodontal tissues. It was found that ASCs in periodontitis tissues expressed ∼7-fold lower levels of HLA-DR (mean fluorescence intensity [MFI] 37.62 ± 7.54, n = 10) than memory B cells (mean MFI 271.19 ± 33.06, n = 10, p < 0.05, Wilcoxon signed-rank test) (Fig. 4A). These CD19+CD27+CD38+HLA-DRlow cells were also positive for CD138 (syndecan-1), a well-known plasma cell marker (35) (Fig. 4A). Memory B cells were in very low levels for the CD138 expression. In contrast, circulating ASCs in peripheral blood showed a high expression level of HLA-DR (mean MFI was 229.47 ± 31.46, n = 5), a phenotypic marker of plasmablasts (data not shown).

Subsequently, the anatomical compartmental location of plasma cells in periodontal tissues was examined by immunostaining using anti-CD138 Ab. Paraffin-embedded sections prepared from gingival tissue specimens were obtained from clinically healthy gingiva, gingivitis, and periodontitis (Fig. 4B–D). Anti-CD138 Ab is known to react not only with plasma cells, but also with gingival epithelium (36). An abundance of CD138+ plasma cells was observed in periodontitis tissues as compared with gingivitis and clinically healthy gingiva. Plasma cells were arranged in clusters that were detected at the base of the periodontal pocket area and scattered throughout the gingival connective tissue, especially apically toward the advancing front of the lesion (Fig. 4D). Small numbers of plasma cells were identified in gingivitis and, to a much lesser extent, in clinically healthy gingiva (Fig. 4B, 4C). A few smaller clusters of plasma cells were detected in the connective tissue subjacent to the junctional epithelium of gingivitis tissue, whereas they were loosely scattered through the gingival connective tissue in healthy tissues (Fig. 4B).

The expressions of CXCL12, BAFF, APRIL, IL-10, IL-6, and IL-21 molecules that are involved in B cell recruitment, survival, and differentiation (37) were assessed in periodontal tissues. The relative quantification expressed as fold change for each studied molecule is shown in Fig. 5. Although mRNA expression for CXCL12, APRIL, BAFF, IL-10, IL-6, and IL-21 was detected in all tissue specimens of clinically healthy gingiva, gingivitis, and periodontitis, a significant overexpression of IL-21 was detected in periodontitis tissues compared with clinically healthy gingiva (34-fold, p < 0.01, one-sample Wilcoxon signed-rank test). Similarly, CXCL12 (4-fold), APRIL (3-fold), BAFF (4-fold), and IL-10 (3.8-fold) were significantly overexpressed in periodontitis tissues when compared with healthy gingiva (p < 0.01, one-sample Wilcoxon signed-rank test). In addition, a significant overexpression of CXCL12 (9-fold) and IL-6 (21-fold) was detected in gingivitis tissues compared with healthy gingiva (p < 0.01, one-sample Wilcoxon signed-rank test). Even though gingivitis tissues highly expressed IL-21 expression (23-fold), no significant difference was found, and this may be due to a large variation in each sample. Moreover, there were no significant differences between periodontitis and gingivitis in all expressions (p > 0.05, Mann–Whitney U test).

Ag specificity of plasma cells in periodontitis tissues was evaluated using ELISPOT assays. Fig. 6A shows SFCs specific to P. gingivalis, A. actinomycetemcomitans, S. gordonii, and collagen type 1 in representative ELISPOT analyses. Plasma cells producing IgG and IgA to P. gingivalis soluble Ags in all five tissue specimens were consistently detected (Fig. 6B). The frequency of P. gingivalis-specific plasma cells producing IgG isotype (mean SFCs = 4532 ± 1908/106 input mononuclear immune cells; p < 0.05, Wilcoxon signed-rank test) was significantly higher compared with IgA isotype (mean SFCs = 842 ± 753/106 input mononuclear immune cells). Plasma cells producing IgG isotype specific to A. actinomycetemcomitans soluble Ags were detected in three of five specimens with mean frequencies of 78 ± 63/106 input mononuclear immune cells. A. actinomycetemcomitans-specific plasma cells producing IgA isotype were not detected (under assay sensitivity). The presence of plasma cells specific to commensal bacteria, S. gordonii, and collagen type 1 was immeasurable. Keyhole limpet hemocyanin protein-coated well was used as a negative control. Negligible numbers of SFCs in the keyhole limpet hemocyanin controls were consistently detected, suggesting a high specificity of the assay system.

To further extend the analysis of the possible role of periodontitis plasma cells in the pathogenesis process, the expression of RANKL, a key molecule in bone-resorbing activity, was assessed (38). RANKL could be expressed as a preform intracellular and cell surface protein (39). As shown in Fig. 6C, both cell surface and intracellular RANKL were detected on periodontitis plasma cells. The cell surface expression of RANKL was modest and consistently lower than that of the intracellular RANKL (n = 3). Moreover, the intracellular expression of granzyme B, a molecule that can degrade extracellular matrix (40), was measured with no detectable expression.

The present study has identified various subsets of B cells in human periodontal tissues obtained from clinically healthy, gingivitis, and periodontitis sites. Moreover, gingival biopsies were analyzed from patients that had suffered from periodontitis and were subsequently treated for the disease. This resulted in a resolution of the disease characteristics. Hence, the presence of various subsets of B cells could be clearly studied and their localization within the marginal gingival tissues enumerated in various conditions ranging from clinical health to periodontitis and reversed to treated periodontitis.

The most intriguing findings in the current study were the identification of CD19+CD27+ memory B cells in human clinically healthy gingiva. Unlike tissue-resident memory T cells, very little is known about memory B cells residing in human nonlymphoid tissues. The evidence is limited to a few reports of tissue memory B cells in healthy nonlymphoid tissues in animals, such as in the lung of mice after influenza infection (41). Other studies described the presence of B cells within adventitia of normal aortas in mice (42). Up to date, only one report dealt with the presence of memory B cells in normal human skin (43).

The observed memory B cells in human clinically healthy gingiva preferentially resided in the connective tissue subjacent to the junctional epithelium. In this vicinity, PNAd+ and ICAM-1+ were detected on blood vessels, suggesting a local low-grade inflammatory response due to constant challenge of the bacterial biofilm in the sulcular area. One could speculate that endothelial PNAd and ICAM-1 may be responsible for a sustained recruitment of memory B cells in clinically healthy gingiva. This memory B cell trafficking may take place along with the well-documented polymorphonuclear cell emigration, a well-recognized phenomenon in a clinically healthy sulcus (44, 45). Such gingival memory B cells were obviously functional, as they were able to secrete IgG and IgA after in vitro polyclonal stimulation in the current study. However, questions remain still unanswered about their role in the homeostasis of clinically healthy gingiva. For instance: 1) how did these memory B cells emigrate from the blood circulation and reside subjacent to the junctional epithelium; 2) what was the Ag specificity of these memory B cells; 3) did they, at a later stage, differentiate into Ab-secreting plasma cells that were found in large numbers in periodontitis; and 4) is there a role of these memory B cells in first line of defense to maintain gingival homeostasis?

Massive infiltrated B cells and plasma cells have been recognized in inflamed tissues of advanced periodontitis (4648). Although such features have been deep rooted in the vision of periodontitis pathogenesis, current knowledge of B cell responses in the diseased tissues has been limited. In the current study, previous reports of the dominance of B cells in periodontitis lesions (49, 50) have been confirmed. Owing to the fact that ASCs in periodontitis tissues expressed ∼7-fold lower levels of HLA-DR than memory B cells, it must be assumed that the observed majority of the ASCs in diseased tissues were actually plasma cells (CD19+CD27+CD38+CD138+HLA-DRlow), rather than plasmablasts (CD19+CD27+CD38+CD138HLA-DRhigh). Aggregates of plasma cells were found at the base of the periodontal pockets and scattered in periodontal connective tissue of marginal gingiva, especially apically toward the advancing front of the lesion. Unlike in periodontitis lesions, significant proportions of memory B cells (CD19+CD27+CD38) were detected in both clinically healthy gingiva and gingivitis tissues. Yet, very low numbers of naive B cells (CD19+CD27CD38) were enumerated in all clinical tissue specimens without any statistically significant differences. It remains unclear whether the observed naive B cells come from blood contamination or represent cells that truly reside in the tissue. The B cell subsets in gingival tissues obviously differ from those in peripheral blood of corresponding subjects. In the peripheral blood, two thirds of the B cells belonged to the subset of naive B cells. It should be pointed out that the number of follicular Th cells (CD4+PD-1+CXCR3+CXCR5+), which are critical for germinal center formation, was negligible in periodontitis tissue (data not shown). The data are in line with previous study, which argues against the formation of germinal center in periodontitis tissue by showing the lack of reticular organization by follicular dendritic cells (50).

Periodontal therapy for patients with periodontitis involving scaling and root planing (removal of the plaque biofilm) resulted in a reversal shift of the tissue B cell profile resembling closely that of the B cell response in clinically healthy and gingivitis tissues. Again, the majority of the B cells were memory B cells, and not plasma cells. These findings documented that the bacterial plaque biofilm was responsible for the induction of local B cell response in periodontitis tissues.

High expression of known molecules involved in B cell response was detected in diseased tissues. Among all expressions, IL-21 was the highest in gingivitis and periodontitis lesions. IL-21 has a variety of actions that influence on B cell response and its destiny (51). The major source of IL-21 is follicular Th cells (52) and Th17 cells (53). Very low numbers of CD4+PD1+CXCR5+ follicular Th cells were identified in the periodontitis tissues in the current study (<1%, n = 4, data not shown). This suggests that IL-21 may be derived from other cell types such as Th17 cells. Recent studies demonstrated the presence of Th17 cytokine in periodontitis tissues (9, 54). However, the ability of this T cell subset in producing IL-21 in periodontitis requires further investigation. Despite the high expression of IL-21 in gingivitis, plasma cell numbers were low compared with the periodontitis lesion. It is well established that IL-21 induces greater plasma cell differentiation in the presence of CD40 signaling (55). It may be speculated that large infiltration of activated T cells expressing CD40L in periodontitis, but not in gingivitis, may increase CD40 signaling on B cells, leading to enhancement of plasma cell differentiation.

Periodontitis tissue plasma cells in all specimens secreted Abs specific to P. gingivalis and, to a lesser extent, to A. actinomycetemcomitans. No commensal S. gordonii-specific Ab or tissue collagen type 1–specific Ab was detected. The data of the current study confirm those of earlier studies for the presence of different Ig isotypes specific for key pathogens in periodontitis tissue (56, 57). The presence of a large infiltrate of plasma cells and with constitutive high levels of Abs specific to pathogens does not seem to eliminate pathogens and hence stop the progression of disease. One explanation may be the nature of the plaque biofilm that made the biofilm more resistant to Ab-mediated protection. Staphylococcus epidermidis within the biofilm was found to be more resistant to opsonic killing mediated by Ab compared with a planktonic form of S. epidermidis (58).

It should be pointed out that the possible deposition of large Ag–Ab complexes may activate the complement classical pathway and neutrophils, resulting in periodontal tissue destruction (59). Deposits of Igs, complements, and the formation of immune complexes in inflamed human gingiva are involved in the acute phase of periodontal destruction in rats (60).

Recently, it has been shown that most CD20+ B cells in periodontitis lesions express RANKL (61). However, it is unclear whether those cells are plasma cells or memory B cells. In the current study, it has been shown that CD19+CD27+CD38+ plasma cells, the major B cell component in periodontitis tissues, expressed RANKL. Moreover, gut plasma cells in patients with inflammatory bowel disease were demonstrated to be positive for granzyme B (62). However, no granzyme B–positive plasma cells were detected in periodontitis tissues.

In conclusion, the current study detected large proportions of memory B cells residing subjacent to the junctional epithelium in clinically healthy gingiva and gingivitis. Unlike tissue-resident memory T cells, the understanding of human B cell immunology in tissues has been very limited. Moreover, the presence of numerous plasma cells in periodontitis tissue and, to a much lesser extent, in gingivitis and clinically healthy gingiva was confirmed in the current study. These periodontitis plasma cells secreted IgG and IgA to P. gingivalis and A. actinomycetemcomitans and also expressed bone-resorbing cytokine, RANKL. Gingival tissues from both healthy and diseased sites obtained during dental procedures represent invaluable samples to study local human B cell immunology. Furthermore, the role of these cells in homeostasis and disease development of periodontal tissue may serve as a model for studying other chronic inflammatory diseases.

We thank Dr. Kitti Torrungruang and Dr. Attawood Lertpimonchai for suggestions in statistical analysis.

This work was supported by the Thailand Research Fund and Chulalongkorn University (Grant BRG5880003); the Ratchadapiseksomphot Endowment Fund, Chulalongkorn University Grant RES560530242-AS; and the Chulalongkorn Academic Advancement into Its 2nd Century Project.

Abbreviations used in this article:

APRIL

a proliferation-inducing ligand

ASC

Ab-secreting cell

BAFF

B cell–activating factor

DAB

diaminobenzidine

DPBS

Dulbecco PBS

MFI

mean fluorescence intensity

PBST

PBS with 0.05% Tween 20

PNAd

peripheral node addressin

RANKL

receptor activator of NF-κB ligand

SFC

spot-forming cell.

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The authors have no financial conflicts of interest.