B and T Lymphocytes Are the Primary Sources of RANKL in the Bone Resorptive Lesion of Periodontal Disease
2006; Elsevier BV; Volume: 169; Issue: 3 Linguagem: Inglês
10.2353/ajpath.2006.060180
ISSN1525-2191
AutoresToshihisa Kawai, Takashi Matsuyama, Yoshitaka Hosokawa, Seicho Makihira, Makoto Seki, Nadeem Y. Karimbux, Reginaldo Bruno Gonçalves, Paloma Valverde, Serge Dibart, Yiping Li, Letícia Algarves Miranda, Cory W.O. Ernst, Yuichi Izumi, Martin A. Taubman,
Tópico(s)Oral microbiology and periodontitis research
ResumoReceptor activator of nuclear factor-κB (RANKL)-mediated osteoclastogenesis plays a pivotal role in inflammatory bone resorption. The aim of this study was to identify the cellular source of RANKL in the bone resorptive lesions of periodontal disease. The concentrations of soluble RANKL, but not its decoy receptor osteoprotegerin, measured in diseased tissue homogenates were significantly higher in diseased gingival tissues than in healthy tissues. Double-color confocal microscopic analyses demonstrated less than 20% of both B cells and T cells expressing RANKL in healthy gingival tissues. By contrast, in the abundant mononuclear cells composed of 45% T cells, 50% B cells, and 5% monocytes in diseased gingival tissues, more than 50 and 90% of T cells and B cells, respectively, expressed RANKL. RANKL production by nonlymphoid cells was not distinctly identified. Lymphocytes isolated from gingival tissues of patients induced differentiation of mature osteoclast cells in a RANKL-dependent manner in vitro. However, similarly isolated peripheral blood B and T cells did not induce osteoclast differentiation, unless they were activated in vitro to express RANKL; emphasizing the osteoclastogenic potential of activated RANKL-expressing lymphocytes in periodontal disease tissue. These results suggest that activated T and B cells can be the cellular source of RANKL for bone resorption in periodontal diseased gingival tissue. Receptor activator of nuclear factor-κB (RANKL)-mediated osteoclastogenesis plays a pivotal role in inflammatory bone resorption. The aim of this study was to identify the cellular source of RANKL in the bone resorptive lesions of periodontal disease. The concentrations of soluble RANKL, but not its decoy receptor osteoprotegerin, measured in diseased tissue homogenates were significantly higher in diseased gingival tissues than in healthy tissues. Double-color confocal microscopic analyses demonstrated less than 20% of both B cells and T cells expressing RANKL in healthy gingival tissues. By contrast, in the abundant mononuclear cells composed of 45% T cells, 50% B cells, and 5% monocytes in diseased gingival tissues, more than 50 and 90% of T cells and B cells, respectively, expressed RANKL. RANKL production by nonlymphoid cells was not distinctly identified. Lymphocytes isolated from gingival tissues of patients induced differentiation of mature osteoclast cells in a RANKL-dependent manner in vitro. However, similarly isolated peripheral blood B and T cells did not induce osteoclast differentiation, unless they were activated in vitro to express RANKL; emphasizing the osteoclastogenic potential of activated RANKL-expressing lymphocytes in periodontal disease tissue. These results suggest that activated T and B cells can be the cellular source of RANKL for bone resorption in periodontal diseased gingival tissue. Periodontal disease (or periodontitis) is an inflammatory lesion that is accompanied by soft tissue destruction and bone resorption in the tooth-supporting structures. A positive correlation between the occurrence of disease and elevated serum antibody response to the oral bacteria colonizing the gingival crevice1McArthur WP Clark WB Specific antibodies and their potential role in periodontal diseases.J Periodontol. 1993; 64: 807-818Crossref PubMed Google Scholar, 2Taubman MA Ebersole JL Smith DJ Genco RJ Mergenhangen SE Association between systemic and local antibody and periodontal diseases. Host Parasite Interactions in Periodontal Diseases. American Society of Microbiology, Washington DC1982: 283-298Google Scholar, 3Ebersole JL Taubman MA Smith DJ Frey DE Haffajee AD Socransky SS Human serum antibody responses to oral microorganisms. IV. Correlation with homologous infection.Oral Microbiol Immunol. 1987; 2: 53-59Crossref PubMed Scopus (88) Google Scholar suggests the involvement of an immune response to the multiple bacteria in the onset and development of periodontal disease. In general, immune responses to bacteria are considered to be a host protective mechanism to pathogenic bacteria. However, despite the elevated IgG antibody response to certain disease-associated bacteria colonizing the periodontal crevice, inflammation and/or bone resorption proceed in the periodontitis lesions. The question is posed as to whether immune response to periodontal bacteria is protective, or otherwise pathogenic, in the context of periodontal disease. The fundamental cytokine system that underlies bone resorption processes is dependent on the osteoclast differentiation, activation, and survival factor, receptor activator of nuclear factor-κB (RANKL), and its soluble decoy receptor osteoprotegerin (OPG).4Wu X Pan G McKenna MA Zayzafoon M Xiong WC McDonald JM RANKL regulates Fas expression and Fas-mediated apoptosis in osteoclasts.J Bone Miner Res. 2005; 20: 107-116Crossref PubMed Google Scholar, 5Hofbauer LC Khosla S Dunstan CR Lacey DL Boyle WJ Riggs BL The roles of osteoprotegerin and osteoprotegerin ligand in the paracrine regulation of bone resorption.J Bone Miner Res. 2000; 15: 2-12Crossref PubMed Scopus (1011) Google Scholar, 6Kong YY Boyle WJ Penninger JM Osteoprotegerin ligand: a common link between osteoclastogenesis, lymph node formation and lymphocyte development.Immunol Cell Biol. 1999; 77: 188-193Crossref PubMed Scopus (115) Google Scholar Involvement of immune cells in the course of bone resorption has been demonstrated by the expression of RANKL on activated T cells.7Kong YY Feige U Sarosi I Bolon B Tafuri A Morony S Capparelli C Li J Elliott R McCabe S Wong T Campagnuolo G Moran E Bogoch ER Van G Nguyen LT Ohashi PS Lacey DL Fish E Boyle WJ Penninger JM Activated T cells regulate bone loss and joint destruction in adjuvant arthritis through osteoprotegerin ligand.Nature. 1999; 402: 304-309Crossref PubMed Scopus (1570) Google Scholar RANKL expressed by T cells, as well as by osteoblasts and bone marrow stromal cells, triggers signaling in osteoclast precursor cells that elicits the differentiation into their mature form.8Boyle WJ Simonet WS Lacey DL Osteoclast differentiation and activation.Nature. 2003; 423: 337-342Crossref PubMed Scopus (4727) Google Scholar OPG is expressed ubiquitously by many types of cells and tissues, and it counterregulates the excessive bone loss by antagonizing the RANKL-binding to its receptor RANK.9Simonet WS Lacey DL Dunstan CR Kelley M Chang MS Luthy R Nguyen HQ Wooden S Bennett L Boone T Shimamoto G DeRose M Elliott R Colombero A Tan HL Trail G Sullivan J Davy E Bucay N Renshaw-Gegg L Hughes TM Hill D Pattison W Campbell P Boyle WJ Osteoprotegerin: a novel secreted protein involved in the regulation of bone density.Cell. 1997; 89: 309-319Abstract Full Text Full Text PDF PubMed Scopus (4261) Google Scholar The paradigm of osteoclast differentiation regulation is based on the RANKL/OPG ratio expressed in the microenvironment surrounding osteoclast precursor cells.10Lacey DL Timms E Tan HL Kelley MJ Dunstan CR Burgess T Elliott R Colombero A Elliott G Scully S Hsu H Sullivan J Hawkins N Davy E Capparelli C Eli A Qian YX Kaufman S Sarosi I Shalhoub V Senaldi G Guo J Delaney J Boyle WJ Osteoprotegerin ligand is a cytokine that regulates osteoclast differentiation and activation.Cell. 1998; 93: 165-176Abstract Full Text Full Text PDF PubMed Scopus (4546) Google Scholar An active periodontal lesion is characterized by the prominent infiltration of B cells11Mackler BF Frostad KB Robertson PB Levy BM Immunoglobulin bearing lymphocytes and plasma cells in human periodontal disease.J Periodontal Res. 1977; 12: 37-45Crossref PubMed Scopus (170) Google Scholar, 12Seymour GJ Greenspan JS The phenotypic characterization of lymphocyte subpopulations in established human periodontal disease.J Periodontal Res. 1979; 14: 39-46Crossref PubMed Scopus (139) Google Scholar and T cells.13Taubman MA Stoufi ED Ebersole JL Smith DJ Phenotypic studies of cells from periodontal disease tissues.J Periodontal Res. 1984; 19: 587-590Crossref PubMed Scopus (83) Google Scholar, 14Okada H Kida T Yamagami H Identification and distribution of immunocompetent cells in inflamed gingiva of human chronic periodontitis.Infect Immun. 1983; 41: 365-374PubMed Google Scholar Although the T cells infiltrating the inflamed gingival tissues express activation markers such as CD45RO15Yamazaki K Nakajima T Aoyagi T Hara K Immunohistological analysis of memory T lymphocytes and activated B lymphocytes in tissues with periodontal disease.J Periodontal Res. 1993; 28: 324-334Crossref PubMed Scopus (85) Google Scholar or CD29,16Seymour GJ Taubman MA Eastcott JW Gemmell E Smith DJ CD29 expression on CD4+ gingival lymphocytes supports migration of activated memory T lymphocytes to diseased periodontal tissue.Oral Microbiol Immunol. 1997; 12: 129-134Crossref PubMed Scopus (24) Google Scholar functional roles of these activated T cells are not completely clear. We demonstrated that adoptive transfer of RANKL+, antigen-specific T cells can induce bone loss in rat periodontal tissue that received local injection of the T-cell antigen.17Valverde P Kawai T Taubman MA Selective blockade of voltage-gated potassium channels reduces inflammatory bone resorption in experimental periodontal disease.J Bone Miner Res. 2004; 19: 155-164Crossref PubMed Google Scholar, 18Valverde P Kawai T Taubman MA Potassium channel-blockers as therapeutic agents to interfere with bone resorption of periodontal disease.J Dent Res. 2005; 84: 488-499Crossref PubMed Scopus (26) Google Scholar Teng and colleagues19Teng YA Nguyen H Gao X Kong Y Gorczynski RM Singh B Ellen RP Penninger JM Functional human T-cell immunity and osteoprotegerin ligand control alveolar bone destruction in periodontal infection.J Clin Invest. 2000; 106: R59-R67Crossref PubMed Scopus (410) Google Scholar reported that adoptive transfer of an Actinobacillus actimomycetemcomitans-specific human T cell line isolated from patients with aggressive (juvenile) periodontal disease could induce significant periodontal bone loss in NOD/SCID mice orally inoculated with A. actinomycetemcomitans every 3 days.19Teng YA Nguyen H Gao X Kong Y Gorczynski RM Singh B Ellen RP Penninger JM Functional human T-cell immunity and osteoprotegerin ligand control alveolar bone destruction in periodontal infection.J Clin Invest. 2000; 106: R59-R67Crossref PubMed Scopus (410) Google Scholar Although the latter report showed that systemic administration of OPG-Fc could reduce periodontal bone loss, it is not clear whether the transferred human T cells or bystander cells that might be secondarily stimulated by the transferred human T cells are the source of RANKL. It has been reported that RANKL produced by B cells is responsible for the devastating bone resorption in multiple myeloma.20Croucher PI Shipman CM Lippitt J Perry M Asosingh K Hijzen A Brabbs AC van Beek EJ Holen I Skerry TM Dunstan CR Russell GR Van Camp B Vanderkerken K Osteoprotegerin inhibits the development of osteolytic bone disease in multiple myeloma.Blood. 2001; 98: 3534-3540Crossref PubMed Scopus (318) Google Scholar Activation of B cells in vitro can induce expression of RANKL, but these cells are deficient in production of OPG.21Choi Y Woo KM Ko SH Lee YJ Park SJ Kim HM Kwon BS Osteoclastogenesis is enhanced by activated B cells but suppressed by activated CD8(+) T cells.Eur J Immunol. 2001; 31: 2179-2188Crossref PubMed Scopus (142) Google Scholar Recently, we reported that antigen-specific activated B cells can induce periodontal bone resorption in a rat model.22Han X Kawai T Eastcott JW Taubman MA Bacterial-responsive B lymphocytes induce periodontal bone resorption.J Immunol. 2006; 176: 625-631PubMed Google Scholar However, it is unclear if B cells accumulating in periodontal diseased tissue express RANKL. To determine the cellular source of RANKL in bone resorptive periodontitis, enzyme-linked immunosorbent assay (ELISA) and double-color confocal microscopic analyses were used. Results of ELISA demonstrated that soluble RANKL (sRANKL) production was significantly elevated in gingival tissues with periodontal disease compared to healthy gingival tissues. Confocal microscopic analyses showed that both T cells and B cells, but not monocytes or fibroblasts, are the cellular source of RANKL in the bone resorptive lesion of periodontal disease. Importantly, RANKL expressed by periodontal T cells and B cells appeared to be the osteoclastogenic functional component, as determined by in vitro RANKL-dependent osteoclast differentiation assays. Patients diagnosed with chronic periodontitis [n = 32 (including three smokers), 12 males and 21 females; average age, 46.9 years; range of ages, 33 to 67 years] were otherwise systemically healthy patients. These patients had periodontal bone resorption diagnosed by X-ray examination, bleeding on probing, and clinical gingival crevice probe depths of greater than 3 mm at the diseased site. Informed consents from all patients were obtained before sample collection. The diseased gingival tissue lesions and healthy tissues were sampled during surgical treatment. Healthy gingival tissues were collected from patients with gingival crevice depth of equal to or less than 3 mm and with no X-ray indication of bone loss, at surgery for tooth restorative purposes including crown lengthening [n = 12 (including one smoker), five males and seven females; average age, 43.4 years; range of ages, 25 to 72 years]. Total RNA was extracted from gingival tissues and RT-PCR was performed as previously described.23Takeichi O Haber J Kawai T Smith DJ Moro I Taubman MA Cytokine profile of T lymphocytes from gingival tissues with pathological pocketing.J Dent Res. 2000; 79: 1548-1555Crossref PubMed Scopus (128) Google Scholar, 24Kawai T Seki M Hiromatsu K Eastcott JW Watts GFM Sugai M Smith DJ Porcelli SA Taubman MA Selective diapedesis of Th1 cells induced by endothelial cell RANTES.J Immunol. 1999; 163: 3269-3278PubMed Google Scholar Primer pairs for human RANKL and OPG were as follows: RANKL forward primer, 5′-TCAGAAGATGGCACTCACTG-3′ and RANKL reverse primer, 5′-AACATCTCCCACTGGCTGTA-3′ (PCR product size 879 bp),25Hofbauer LC Gori F Riggs BL Lacey DL Dunstan CR Spelsberg TC Khosla S Stimulation of osteoprotegerin ligand and inhibition of osteoprotegerin production by glucocorticoids in human osteoblastic lineage cells: potential paracrine mechanisms of glucocorticoid-induced osteoporosis.Endocrinology. 1999; 140: 4382-4389Crossref PubMed Scopus (544) Google Scholar OPG forward primer, 5′-GCCCTGACCACTACTACACA-3′ and OPG reverse primer, 5′-TCTGCTCCCACTTTCTTTCC-3′ (PCR product size 736 bp). Total RNA isolated from gingivae (1 μg) was synthesized to cDNA. The resulting cDNA was subject to PCR by amplifying 30 cycles for RANKL or for OPG along with β-actin23Takeichi O Haber J Kawai T Smith DJ Moro I Taubman MA Cytokine profile of T lymphocytes from gingival tissues with pathological pocketing.J Dent Res. 2000; 79: 1548-1555Crossref PubMed Scopus (128) Google Scholar as an internal control (94°C for 30 second, 60°C for 1 minute, 72°C for 1 minute, and final elongation at 72°C for 10 minutes). PCR products were separated in 1.7% agarose gels and stained with ethidium bromide. Gingival tissues were homogenized with a Dounce glass homogenizer in phosphate-buffered saline supplemented with 0.05% Tween 20, phenylmethyl sulfonyl fluoride (1 mmol/L; Sigma, St. Louis, MO), and protease inhibitor cocktail (Sigma), as published with slight modification.26Smith DJ Gadalla LM Ebersole JL Taubman MA Gingival crevicular fluid antibody to oral microorganisms. III. Association of gingival homogenate and gingival crevicular fluid antibody levels.J Periodontal Res. 1985; 20: 357-367Crossref PubMed Scopus (34) Google Scholar For ELISA, the concentration of sRANKL, interleukin (IL)-1β, and OPG in the tissue homogenates or culture supernatants were measured with ELISA kits for human soluble RANKL (sRANKL) (Peprotech, Rocky Hill, NJ) and DuoSet ELISA for human IL-1β, OPG, IL-10, IL-12 p70, and GM-CSF (R&D Systems, Minneapolis, MN). The method for detection of IgG response to oral bacteria has been previously published.27Kawai T Ito H Sakado N Okada H A novel approach for detecting an immunodominant antigen of Porphyromonas gingivalis in diagnosis of adult periodontitis.Clin Diagn Lab Immun. 1998; 5: 11-17PubMed Google Scholar, 28Ebersole JL Frey DE Taubman MA Smith DJ An ELISA for measuring serum antibodies to Actinobacillus actinomycetemcomitans.J Periodontal Res. 1980; 15: 621-632Crossref PubMed Scopus (109) Google Scholar To detect anti-bacterial IgG levels in the gingival homogenates, the following formalin-fixed bacteria were used as antigen in ELISA: A. actinomycetemcomitans strain Y4 (American Type Culture Collection no. 43718), Fusobacterium nucleatum str. 25586, Eikenella corrodens str. 23834, Porphyromonas gingivalis str. W83, and Prevotella intermedia str. 25611. PBMCs were collected from healthy patients under informed consent agreement (n = 4; 28- to 40-year-old males; average age, 34.5 years). The mononuclear cell (MC) fraction was separated from blood by gradient centrifugation using Histopaque-1077 (Sigma) and incubated in RPMI 1640 supplemented with 10% fetal bovine serum, 2-mercaptoethanol, l-glutamine, and penicillin and streptomycin. T cells were enriched from PBMCs by glass-wool and nylon-wool column purification.24Kawai T Seki M Hiromatsu K Eastcott JW Watts GFM Sugai M Smith DJ Porcelli SA Taubman MA Selective diapedesis of Th1 cells induced by endothelial cell RANTES.J Immunol. 1999; 163: 3269-3278PubMed Google Scholar B cells were enriched from PBMCs by negative selection using magnetic beads coated with a mixture of anti-CD3 monoclonal antibody (mAb) (OKT3: American Type Culture Collection, Rockville, MD; UCTH1: R&D Systems), CD4 (OKT4), CD8 (OKT8), and CD28 (CD28.2; BD Pharmingen, San Diego, CA). The enriched T cells or B cells were stimulated with immobilized antibodies on a 96-well culture plate with mouse mAb to CD3 (OKT3) and CD28 (CD28.2) or mAb to CD40 (5C3, BD Pharmingen) and goat polyclonal anti-human IgM (Serotec, Oxford, UK), respectively. Basic fluorescent staining technique was performed as previously reported.24Kawai T Seki M Hiromatsu K Eastcott JW Watts GFM Sugai M Smith DJ Porcelli SA Taubman MA Selective diapedesis of Th1 cells induced by endothelial cell RANTES.J Immunol. 1999; 163: 3269-3278PubMed Google Scholar Sectioned tissues mounted on glass slides were fixed with 2% paraformaldehyde. T cells, B cells, and monocytes were stained with the mouse monoclonal antibodies to CD3 (UCTH1), CD20 (H1; BD Pharmingen), and CD14 (3C10; American Type Culture Collection), respectively, followed by fluorescein isothiocyanate-conjugated anti-mouse IgG (Jackson Immunoresearch Laboratories, West Grove, PA) as a secondary reagent. RANKL expression was monitored by biotinylated-OPG-Fc followed by Texas Red-Avidin (Invitrogen, Carlsbad, CA). To block FcRn and FcγRII, which can bind to monomeric γ chain of human IgG-Fc, human IgG was prereacted with some of the gingival tissue sections. OPG-Fc was generously provided by Dr. Colin Dunston (Amgen, Thousand Oaks, CA) under mutual material transfer agreement and was conjugated with biotin using EZ-Link sulfo-NHS-biotin (Pierce, Rockford, IL). A human IgG-Fc fusion protein (L6) conjugated with biotin was used as a negative control. After washing, the cover glass was set on the sample with Fluoromount-G mounting medium (Southern Biotechnology, Birmingham, AL). The staining pattern was analyzed by 0.3 μm sequential optical sectioning at ×400 or ×1000 magnification with a Leica TCS/SP-2 laser-scanning confocal microscope (Leica, Wetzlar, Germany). MCs were isolated from patient gingival tissue biopsies as previously described.23Takeichi O Haber J Kawai T Smith DJ Moro I Taubman MA Cytokine profile of T lymphocytes from gingival tissues with pathological pocketing.J Dent Res. 2000; 79: 1548-1555Crossref PubMed Scopus (128) Google Scholar Patient gingival MCs, fresh PBMCs, stimulated PB T cells, or stimulated PB B cells in vitro were fixed with formalin. Human PB CD14+ monocytes were isolated from PBMCs using a magnetic bead-based monocyte-negative isolation kit (Dynal Biotech, Oslo, Norway). In some experiments, patient gingival MCs were incubated in 96-well plates for 3 hours, and nonadherent cells were separated from the adherent cells on the culture plate. The adherent gingival MCs were ∼80% CD14+ monocytes, whereas nonadherent gingival MCs contained less than 2% CD14+ cells by immunostaining. The resulting adherent cells in the wells and nonadherent gingival MCs were fixed with formalin and were used in the following co-culture system. The fixed human lymphocytes were co-cultured in 96-well plates with the mouse osteoclast precursor cell line MOCP-529Chen W Li Y-P Generation of mouse osteoclastogenic cell lines immortalized with SV40 large T antigen.J Bone Miner Res. 1998; 13: 1112-1123Crossref PubMed Scopus (38) Google Scholar or PB CD14+ monocytes in the presence of M-CSF (10 ng/ml; Peprotech). Recombinant human RANKL (50 ng/ml; R&D Systems) was added to the MOCP-5 culture, as a positive control. To assess the involvement of RANKL in the osteoclast differentiation, OPG-Fc (10 μg/ml) was added to antagonize RANKL in some cultures. The culture medium was changed every 3 days by replacing half of the volume. After 6 to 8 days of culture for MOCP-5, or 14 to 16 days of culture for PB CD14+ monocytes, when multinuclear osteoclast-like cells are observed by phase-contrast microscopy, cells were fixed with 5% formalin-saline. Differentiated osteoclasts were identified as tartrate-resistant acid phosphatase (TRAP)-positive cells with three or more nuclei as described previously.30Kawai T Eisen-Lev R Seki M Eastcott JW Wilson ME Taubman MA Requirement of B7 costimulation for Th1-mediated inflammatory bone resorption in experimental periodontal disease.J Immunol. 2000; 164: 2102-2109PubMed Google Scholar The TRAP+ cells with more than three nuclei were counted as osteoclasts using phase contrast microscopy and expressed as cell number/well of 96-well plates. The bone resorption activity of osteoclasts was evaluated by a pit formation assay using a calcium phosphate-coated tissue culture vessel system (Biocoat Osteologic System; BD Biosciences, San Jose, CA) or dentin disks (Alpco Diagnostics, Windham, NH) according to the manufacturers' instructions. The concentration of sRANKL protein in gingival tissue was significantly elevated (P < 0.02, t-test) in diseased tissues as compared to healthy gingival tissues (Figure 1A). In addition, there was a positive correlation between the level of sRANKL in tissues and the depth of the gingival crevice where the tissues were sampled (Figure 1B). Differences in OPG concentration between diseased and healthy tissues were not statistically significant (Figure 1C). The proinflammatory cytokine IL-1β was monitored in the same groups of gingival tissues (Figure 1D), as an indicator of the degree of inflammation in each tissue. All gingival tissues from diseased lesions expressed significantly higher IL-1β amounts than the healthy gingival tissues (Student's t-test, P < 0.05). We also evaluated the concentrations of the osteoclastogenesis inhibitory cytokines, IL-10, IL-12 p70, and GM-CSF and the osteoclast precursor chemoattractant factor, MIP-1α, compared to healthy tissues. However, although the diseased gingival tissues had higher mean concentrations of each of the four factors, none of the differences were statistically significant. Compared to healthy patients' antibody levels, the tissue homogenates of patient gingival tissues also demonstrated significantly higher IgG antibody levels to three of five periodontal disease-associated bacteria examined in this study (F. nucleatum, P. gingivalis, and P. intermedia; data not shown). RT-PCR was performed to detect mRNA expression of RANKL and OPG in the gingival tissue RNA samples (Figure 2). RANKL mRNA expression was detected from 67% of the diseased gingival tissues (four of six) examined, whereas no expression of RANKL mRNA was observed from the healthy gingival tissues tested (zero of four). OPG mRNA message was observed in tissue from periodontally diseased patients (two of six) and one healthy subject (one of four). To identify the cell types that express RANKL in inflamed gingival tissue, double-color confocal microscopy (Figure 3) was used after staining for RANKL (red) and lymphocyte-specific CD makers (green). Images of RANKL and CD marker-positive cells were merged in the computer system, and double-positive cells were displayed as yellow staining. Gingival tissues from periodontitis lesions demonstrated marked expression of RANKL (Figure 3, A–C). The cellular infiltrates in the diseased gingival tissue were predominantly T (Figure 3A) and B lymphocytes (Figure 3B), with few CD14+ monocytes (Figure 3C). RANKL was expressed in these cellular infiltrates, especially by T cells and by B cells, and to a much lesser extent by monocytes. Although most B cells were positive for RANKL staining, not all T cells expressed RANKL. Very few lymphocytes expressing RANKL were present in healthy gingival tissues (Figure 3D). RANKL expression by other nonlymphoid cell types, such as fibroblasts, was not distinctly observed. The numbers of CD3+ and CD20+ lymphocytes infiltrating the gingival tissues were significantly higher than those in the healthy gingival tissues, whereas the number of CD14+ cells did not show a remarkable increase (Figure 4). Importantly, the percentage of CD14+ cells to the number of total lymphocytes remained low irrespective of the gingival condition of disease/healthy (19% in healthy versus 5% in disease gingival tissue; Figure 4). These data strongly indicate that T cells and B cells are a much more significant source of RANKL than CD14+ cells in the diseased gingival tissues. The percentage of RANKL+ cells in the population of T cells or B cells was also determined (Figure 5) based on counts of the images obtained in the confocal microscopic analyses (Figure 3). The percentage of RANKL-expressing T cells (Figure 5A) or B cells (Figure 5B) demonstrated a positive correlation with the depth of the gingival crevice. Importantly, in the diseased gingival tissues, RANKL+ cells were greatly elevated compared to the healthy tissues, and a significantly higher percentage of B cells expressed RANKL (94 ± 5% SD) than T cells (51 ± 18% SD) (Figure 5C). To investigate whether naïve or activated lymphocytes express RANKL, peripheral blood T cells or B cells from healthy patients were stimulated with immobilized anti-CD3 and anti-CD28 mAbs or with immobilized anti-CD40 and/or anti-IgM, respectively (Figure 6). The stimulation via CD3/CD28 or CD40/BCR (B-cell receptor; anti-IgM) induced proliferation and sRANKL expression by the purified T cells or B cells [Figure 6, A and C (T cells) and B and D (B cells)]. Although production of OPG by the activated T cells or B cells was also monitored (by ELISA), the level of OPG production was lower than the detection limit (4 pg/ml). An oral bacterial stimulus (fixed A. actinomycetemcomitans) also activated peripheral blood lymphocytes to produce both sRANKL and cellular RANKL from T cells and B cells (examined by confocal microscopy, not shown). The activated T cells and B cells isolated from peripheral blood were able to induce osteoclast (TRAP+, multinucleated) cell differentiation in a RANKL-dependent manner (Figure 6E), whereas activated CD14+ monocytes did not induce such osteoclastogenesis (Figure 6E). Of note, most published literature also supports the finding that CD14+ cells express of RANK but not RANKL.31Boyle WJ Oppenheim JJ Feldmann M Durum SK Hirano T Vilcek J Nicola NA RANK ligand. Cytokine Reference. Academic Press, San Diego2001: 489-497Google Scholar, 32Boyle WJ Oppenheim JJ Feldmann M Durum SK Hirano T Vilcek J Nicola NA RANK. Cytokine Reference. Academic Press, London2001: 1691-1709Google Scholar These data suggested that T cells and B cells that express RANKL in gingival tissues are probably in the activated form and that these activated T and B cells are the major source of RANKL. To evaluate the functional aspects of RANKL expressed by MCs isolated from the diseased gingival tissue, an in vitro differentiation assay was outperformed using the osteoclast precursor cell line MOCP-5 (Figure 7A) or peripheral blood CD14+ monocytes (Figure 7B). Both MOCP-5 cells and CD14+ monocytes differentiated into TRAP+ multinucleated cells in response to recombinant human sRANKL in the presence of M-CSF. The isolated diseased gingival MCs as well as recombinant RANKL induced TRAP+ multinucleated cells (Figure 7A). However, only nonadherent diseased gingival MCs, but not adherent gingival MC cells, induced RANKL-dependent osteoclastogenesis (Figure 7B), indicating that any RANKL expression that might have been detected on CD14+ cells (Figure 3C) is not functionally active. The morphological appearance of the differentiated MOCP-5 cell line is shown [Figure 8, A (sRANKL) and C (patient gingival MC)]. Induction of TRAP+ multinucleated cells was abolished by the addition of OPG-Fc into the culture [Figure 7, Figure 8 (sRANKL + OPG-Fc) and D (patient gingival MC + OPG-Fc)], demonstrating that RANKL expressed by MCs from diseased tissues can induce RANKL-dependent osteoclast differentiation. The pit formation assay was performed to assess the functional capabilities of differentiated multinucleated cells (Figure 8, E–J). Resorption pits shown as positively stained by toluidine blue (indicated by arrows) were observed in the disks from wells in which MOCP-5 cells were co-cultured with sRANKL or MCs from diseased gingival tissue (Figure 8, F and G, respectively). The formation of resorption pits induced by MOCP-5 co-cultured w
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