Local chemokine paralysis, a novel pathogenic mechanism for Porphyromonas gingivalis

Abstract

Periodontitis, which is widespread in the adult population, is a persistent bacterial infection associated with Porphyromonas gingivalis. Gingival epithelial cells are among the first cells encountered by both P. gingivalis and commensal oral bacteria. The chemokine interleukin 8 (IL-8), a potent chemoattractant and activator of polymorphonuclear leukocytes, was secreted by gingival epithelial cells in response to components of the normal oral flora. In contrast, P. gingivalis was found to strongly inhibit IL-8 accumulation from gingival epithelial cells. Inhibition was associated with a decrease in mRNA for IL-8. Antagonism of IL-8 accumulation did not occur in KB cells, an epithelial cell line that does not support high levels of intracellular invasion by P. gingivalis. Furthermore, a noninvasive mutant of P. gingivalis was unable to antagonize IL-8 accumulation. Invasion-dependent destruction of the gingival IL-8 chemokine gradient at sites of P. gingivalis colonization (local chemokine paralysis) will severely impair mucosal defense and represents a novel mechanism for bacterial colonization of host tissue.

FIG. 1

F. nucleatum induces IL-8 accumulation from GEC. Various amounts of F. nucleatum were coincubated with GEC as described in the text. After 18 h, the culture supernatant was removed and the level of IL-8 in the culture supernatant was determined. The amount of IL-8 in culture supernatants without the addition of bacteria was 40 (±9) ng/ml. Three separate experiments were performed, and the data are presented as the averages and interassay standard deviations (with 10⁸ F. nucleatum cells, more than 500 ng of IL-8 per ml was detected in each experiment).

FIG. 2

P. gingivalis inhibition of IL-8 accumulation. F. nucleatum (10⁸ bacteria) and various amounts of P. gingivalis 33277, MP4-504, 381, or DPG3 were mixed before addition to GEC. After coincubation with GEC for 18 h, the level of IL-8 was determined as described in the text. The amount of IL-8 inhibition was determined by comparing the amount of IL-8 detected after incubation with the combination of P. gingivalis and F. nucleatum to that obtained with F. nucleatum alone. Three separate experiments were performed for each strain. In each experiment, at most datum points, there was either complete or no inhibition of IL-8 accumulation, depending upon the concentration of bacteria examined. When partial inhibition occurred, an average of the three experiments is presented.

FIG. 3

P. gingivalis 33277 inhibition of additional IL-8 accumulation without degradation of preexisting IL-8. F. nucleatum (10⁸ bacteria) was added to GEC, and the amount of IL-8 in the culture supernatant was determined at the indicated times (◊). At 4 (□) and 8 h (▵) after F. nucleatum addition to GEC, P. gingivalis (10⁶ bacteria) was added to the wells (indicated by arrows). The cells were allowed to incubate for 18 h, and the level of IL-8 in the culture supernatant was determined. The data are presented as the means and standard deviations from at least three separate experiments.

FIG. 4

Relative levels of IL-8 mRNAs obtained from GEC treated with P. gingivalis or F. nucleatum. P. gingivalis 33277 (P. ging) (10⁶ cells), F. nucleatum (F. nuc) (10⁸ cells), or a combination of P. gingivalis (10⁶ cells) and F. nucleatum (10⁸ cells) (P. ging/F. nuc) was used. After 18 h of exposure to the bacteria, mRNA was extracted from GEC and the relative amounts of IL-8 and actin mRNAs were determined by RT-PCR with appropriate probes. Scanning densitometry and quantitation of the gel using NIH Image 1.6 revealed a 75% decrease in the amount of IL-8 mRNA compared to that of actin mRNA in P. gingivalis-infected GEC.

FIG. 5

Lack of P. gingivalis inhibition of IL-8 accumulation in oral epithelial KB cells. Oral epithelial KB cells were plated as described for GEC in the text, and IL-8 accumulation was examined as described in the text with 10⁸ F. nucleatum and 10⁶ P. gingivalis 33277 bacteria per well. The amount of IL-8 found in the supernatant after 18 h of incubation was determined as described in the text. Three separate experiments were performed. IL-8 accumulation induced by F. nucleatum varied in each experiment (experiment 1 [expt 1], 125 ng/ml; expt 2, 40 ng/ml; and expt 3, 25 ng/ml). No IL-8 was observed when P. gingivalis was added. The amount of IL-8 found when the combination of F. nucleatum and P. gingivalis was used was not significantly less (expt 1, 90 ng/ml; expt 2, 40 ng/ml; and expt 3, 30 ng/ml). The data for the combination are presented as the mean amount of IL-8 accumulation by F. nucleatum and P. gingivalis (90.66% ± 13%) compared to F. nucleatum alone (100%).