High expression levels of receptor activator of nuclear factor-kappa B ligand associated with human chronic periodontitis are mainly secreted by CD4+ T lymphocytes.
BACKGROUND
Chronic periodontitis is an infectious disease characterized by alveolar bone destruction and teeth loss. Receptor activator of nuclear factor-kappa B ligand (RANKL) is an osteoclastogenic cytokine, a central regulatory factor in the osteoclast's lifespan, and a participant in physiological and pathological bone resorption. Gingival T cells synthesize RANKL, contributing to molecular local imbalance that entails the alveolar bone resorption seen in periodontitis. Our study was aimed at associating the levels of RANKL with the CD4(+) T-cell activity present in gingival tissues of chronic periodontitis patients.


METHODS
Gingival biopsies were obtained from 33 chronic periodontitis patients and 20 healthy controls. Specimens were either formalin fixed and paraffin embedded for real-time reverse transcription-polymerase chain reaction (RT-PCR) and histologic analysis or tissue digestion processed for cell culture and flow-cytometry analysis. RANKL mRNA and protein levels were determined by quantitative RT-PCR and enzyme-linked immunosorbent assay (ELISA) in gingival-cell culture supernatants. Gingival leukocytes were quantified by flow cytometry. RANKL and CD4 immunoreactivity were analyzed by flow cytometry and confocal microscopy.


RESULTS
RANKL mRNA levels were higher in patients with periodontitis than in healthy subjects, and spontaneous and lipopolysaccharide (LPS)- and phytohemagglutinin (PHA)-stimulated RANKL synthesis were higher also in patients than controls. CD4(+) T lymphocytes were the predominant infiltrate cell subset present in gingival tissues of periodontitis patients. Furthermore, an association between RANKL and CD4(+) T cells was determined by double-staining flow cytometry and confocal microscopy.


CONCLUSIONS
Taken together, these data demonstrate that gingival CD4(+) T cells are the main cells responsible for higher levels of RANKL observed in human chronic periodontitis patients.
Vernal
Rolando
R
Periodontal Biology Laboratory, Conservative Department, Dentistry Faculty, University of Chile, Santiago, Chile. rvernal@uchile.cl
Dutzan
Nicolás
N
Hernández
Marcela
M
Chandía
Sabrita
S
Puente
Javier
J
León
Rubén
R
García
Leyre
L
Del Valle
Ignacio
I
Silva
Augusto
A
Gamonal
Jorge
J
D000328:Adult; D000368:Aged; D001706:Biopsy; D015496:CD4-Positive T-Lymphocytes / Q000276:immunology* / Q000378:metabolism; D002478:Cells, Cultured; D002908:Chronic Disease; D005260:Female; D005434:Flow Cytometry; D005881:Gingiva / Q000276:immunology / Q000378:metabolism; D006801:Humans; D008070:Lipopolysaccharides / Q000494:pharmacology; D008297:Male; D018613:Microscopy, Confocal; D008875:Middle Aged; D008934:Mitogens / Q000494:pharmacology; D010518:Periodontitis / Q000276:immunology* / Q000473:pathology; D010835:Phytohemagglutinins / Q000494:pharmacology; D053245:RANK Ligand / Q000032:analysis* / Q000235:genetics / Q000378:metabolism; D012333:RNA, Messenger / Q000032:analysis; D020133:Reverse Transcriptase Polymerase Chain Reaction
D008070:Lipopolysaccharides; D008934:Mitogens; D010835:Phytohemagglutinins; D053245:RANK Ligand; D012333:RNA, Messenger