Calprotectin expression in vitro by oral epithelial cells confers resistance to infection by Porphyromonas gingivalis

Abstract

Calprotectin, an S100 calcium-binding protein with broad-spectrum antimicrobial activity in vitro, is expressed in neutrophils, monocytes, and gingival keratinocytes. In periodontitis, calprotectin appears upregulated and is detected at higher levels in gingival crevicular fluid and tissue specimens. How calprotectin contributes to the pathogenesis of periodontal diseases is unknown. To isolate the effects of calprotectin, a calprotectin-negative oral epithelial cell line was transfected with calprotectin genes to enable expression. Porphyromonas gingivalis was permitted to bind and invade transfected cells expressing calprotectin and sham transfectants. Rates of invasion into both cell lines were compared using the antibiotic protection assay. Transfected cells expressing calprotectin showed 40 to 50% fewer internalized P. gingivalis than sham transfectants. Similarly, binding to calprotectin expressing cells was reduced approximately twofold at all time points (15, 30, 45, and 60 min) as estimated by immunofluorescence analysis. Independent of invasion, however, prolonged exposure to P. gingivalis induced epithelial cell rounding and detachment from the substratum. These morphological changes were delayed, however, in cells expressing calprotectin. Using P. gingivalis protease-deficient mutants, we found that Arg-gingipain and Lys-gingipain contributed to epithelial cell rounding and detachment. In conclusion, expression of calprotectin appears to protect epithelial cells in culture against binding and invasion by P. gingivalis. In addition, cells expressing calprotectin are more resistant to detachment mediated by Arg-gingipain and Lys-gingipain. In periodontal disease, calprotectin may augment both the barrier protection and innate immune functions of the gingival epithelium to promote resistance to P. gingivalis infection.

FIG. 1

P. gingivalis invasion into KB-EGFP and KB-MRP8/14. KB-EGFP or KB-MRP8/14 was incubated with P. gingivalis at an MOI of 100 for 1.5 h, followed by 1 h with antibiotics. The cell monolayers were then washed and lysed with distilled H₂O to release intracellular bacteria. Internalized bacteria were plated and counted. Values are means ± SD from a representative experiment. Each experiment was performed in triplicate wells. The experiments were repeated at least three times with similar results for each strain of P. gingivalis. The differences between KB-EGFP and KB-MRP8/14 from three experiments were statistically significant (∗, P < 0.05; †, P < 0.01).

FIG. 2

Binding of P. gingivalis ATCC 33277 to KB-EGFP and KB-MRP8/14. KB-EGFP and KB-MRP8/14 cell monolayers were incubated with P. gingivalis for 15, 30, 45, or 60 min; washed; fixed; and stained for adherent P. gingivalis by indirect immunofluorescence. At each time point, images from 10 random microscopic fields at a magnification of ×200 were captured and the adherent bacteria in each field were counted. The differences between KB-EGFP and KB-MRP8/14 were significant (∗, P < 0.001). Error bars, SD.

FIG. 3

Changes in epithelial cell morphology after exposure to P. gingivalis FDC381 for 0, 5, and 9 h. KB, KB-EGFP, or KB-MRP8/14 was incubated with P. gingivalis FDC381 at an MOI of 100. Changes in epithelial cell morphology over time were observed by phase-contrast microscopy. Magnification, ×850.

FIG. 4

Changes in epithelial cell morphology after 24 h of exposure to P. gingivalis FDC381, DPG3, or heat-killed FDC381. Epithelial cells that were not exposed to P. gingivalis served as controls. Magnification, ×800.

FIG. 5

Changes in epithelial cell morphology after exposure to P. gingivalis ATCC 33277 wild-type parental strain and protease-deficient mutants for 24 h. KDP129 is a Kgp-deficient mutant; KDP133 is an Rgp-deficient mutant; and KDP136 is a Kgp- and Rgp-deficient mutant. Magnification, ×1,000.