Cytokine responses of oral epithelial cells exposed to Porphyromonas gingivalis

Objectives: The periodontopathogen Porphyromonas gingivalis (Pg) adheres to, invades and replicates within human oral epithelial cells. The pro-inflammatory cytokine interleukin-8 (IL-8) is a potent chemoattractant inducing the influx of neutrophils into periodontal lesions. We quantified the IL-8 secreted by human oral squamous HSC-3 cells after exposure to live and heat-inactivated Pg, and to lipopolysaccharide (LPS) from Pg and E. coli. Methods: The Pg strain 2561 (ATCC 33277) was sub-cultivated on blood agar plates and suspended in Medium 199 (4x108 Pg/ml). HSC-3 cells were challenged with live or heat-killed Pg at 107 or 108 bacteria/well, and incubated at 37°C for 6, 24 and 48 h. The Multi-Analyte Profiler ELISArray kit was used to profile pro-inflammatory cytokines and chemokines. IL-8 was determined by ELISA. LPS from E. coli and Pg were tested at 5 µg/ml. Results: Analysis by the Profiler ELISA indicated the stimulation of IL-6 and IL-8. Secretion of IL-8 was affected by the number and heat killing of bacteria, and the time of incubation. Untreated HSC-3 cells produced 5.2±0.9 ng IL-8/ml within 24 h, while cells incubated with 107 and 108 live Pg secreted 9.6±2.3 and 12.9±2.8 ng IL-8/ml, respectively. Cells stimulated with heat-killed Pg produced higher levels of IL-8: 12.0±2.1 and 18.1±1.9 ng IL-8/ml. Higher IL-8 stimulation by heat-killed Pg was also evident at 48 h. Treatment with LPS from Pg and E. coli at 5 µg/ml resulted in the production of 11.8±6.1 and 8.6±3.2 ng IL-8/ml, respectively in 24 h. Conclusion: Pg induced significant IL-8 secretion by HSC-3 cells. Degradation of IL-8 by cysteine proteinases (gingipains) produced by live Pg may be responsible for the higher up-regulation of IL-8 observed with heat-killed bacteria. Supported by Research Pilot Project Award 03-Activity 069 from the University of the Pacific, School of Dentistry (K. Konopka)