Intragingival injection of Porphyromonas gingivalis-derived lipopolysaccharide induces a transient increase in gingival tumour necrosis factor-α, but not interleukin-6, in anaesthetised rats

Abstract

This study used in vivo microdialysis to examine the effects of intragingival application of lipopolysaccharide (LPS) derived from Porphyromonas gingivalis (Pg-LPS) on gingival tumour necrosis factor (TNF)-α and interleukin (IL)-6 levels in rats. A microdialysis probe with an injection needle attached to the surface of the dialysis membrane was implanted into the gingiva of the upper incisor. For comparison, the effects of LPS derived from Escherichia coli (Ec-LPS) on IL-6 and TNF-α levels were also analysed. Pg-LPS (1 μg/1 μL) or Ec-LPS (1 or 6 μg/1 μL) was applied by microsyringe, with gingival dialysates collected every hour. Enzyme-linked immunosorbent assay (ELISA) revealed that gingival dialysates contained approximately 389 pg·mL⁻¹ of IL-6 basally; basal TNF-α levels were lower than the detection limit of the ELISA. Pg-LPS failed to alter IL-6 levels but markedly increased TNF-α levels, which remained elevated for 2 h after treatment. Neither IL-6 nor TNF-α were affected by Ec-LPS. Reverse transcriptase-polymerase chain reaction (RT-PCR) analysis revealed that the gingiva expresses Toll-like receptor (TLR) 2 and TLR4 mRNA. Immunohistochemical examination showed that TLR2 and TLR4 are expressed by gingival epithelial cells. The present study provides in vivo evidence that locally applied Pg-LPS, but not Ec-LPS, into the gingiva transiently increases gingival TNF-α without affecting IL-6. The present results suggest that TLR2 but not TLR4 expressed on gingival epithelial cells may mediate the Pg-LPS-induced increase in gingival TNF-α in rats.

Figure 1

Microdialysis system equipments. (a) Microdialysis probe with a needle for drug microinjection. (b) Schematic illustration of the in vivo microdialysis system using a push-pull pump.

Figure 2

Effects of intragingival injection of Pg-LPS or Ec-LPS on the gingival levels of IL-6. All values are presented as a percentage of the baseline level of IL-6, which was the mean of the IL-6 level in the two samples measured immediately prior to LPS treatment. The data are expressed as the mean change in the 1-hr observation period (ordinate) after intragingival injection of LPS (abscissa). Vertical bars indicate SEM. The basal gingival IL-6 level is control (100%). Neither injection of Pg-LPS nor Ec-LPS into the gingiva-altered gingival levels of IL-6 (n=6–7). Ec-LPS, lipopolysaccharide derived from Escherichia coli; IL, interleukin; LPS, lipopolysaccharide; Pg-LPS, lipopolysaccharide derived from Porphyromonas gingivalis; SEM, standard error of the mean.

Figure 3

Effects of intragingival injection of Pg-LPS or Ec-LPS on the gingival levels of TNF-α. (a) Intragingival injection of Pg-LPS (1 μg/1 μL) induced a transient increase in gingival levels of TNF-α (n=5). Intragingival injection of Ec-LPS did not alter gingival levels of TNF-α. Since the basal levels of TNF-α were below the detection limit of the ELISA, the data are expressed as the mean absolute amount of TNF-α in gingival dialysates (pg·mL⁻¹). (b) Average maximum increase in gingival levels of TNF-α induced by intragingival injection of Pg-LPS. The maximum increase in TNF-α in each rate was observed 1–3 h after the injection (n=5). Ec-LPS, lipopolysaccharide derived from Escherichia coli; Pg-LPS, lipopolysaccharide derived from Porphyromonas gingivalis; TNF, tumor necrosis factor.

Figure 4

RT-PCR analysis of expression of TLR2 and TLR4 mRNA in rat gingival tissue. Total RNA was extracted from rat gingival tissue. After cDNA was generated, PCR was performed with specific primers against TLR2, TLR4 and GAPDH. The PCR products were loaded onto a 1.5% agarose gel and photographed. The expected PCR products of TLR-2, TLR-4 and GAPDH are 177, 105 and 155 bp, respectively. GAPDH, glyceraldehyde-3-phosphate dehydrogenase; PCR, polymerase chain reaction; RT-PCR, reverse transcriptase-polymerase chain reaction; TLR, Toll-like receptor.

Figure 5

Immunohistochemical staining for TLR2 and TLR4 in the periodontal tissue of the upper incisors. A high magnification view of the boxed region in the left panel is shown in the right panel. TLR2 expression in epithelial cells (arrowheads in a); TLR2 expression in periodontal ligament fibroblasts (arrowheads in b); TLR4 expression in epithelial cells (arrowheads in c); and TLR4 expression in periodontal ligament fibroblasts (arrowheads in d). Bar = 20 μm. AB, alveolar bone; C, the cementum; D, dentine; PDL, periodontal ligament; TLR, Toll-like receptor.