Arginine-specific protease from Porphyromonas gingivalis activates protease-activated receptors on human oral epithelial cells and induces interleukin-6 secretion

Abstract

Periodontitis is a chronic inflammatory disease affecting oral tissues. Oral epithelial cells represent the primary barrier against bacteria causing the disease. We examined the responses of such cells to an arginine-specific cysteine proteinase (RgpB) produced by a causative agent of periodontal disease, Porphyromonas gingivalis. This protease caused an intracellular calcium transient in an oral epithelial cell line (KB), which was dependent on its enzymatic activity. Since protease-activated receptors (PARs) might mediate such signaling, reverse transcription-PCR was used to characterize the range of these receptors expressed in the KB cells. The cells were found to express PAR-1, PAR-2, and PAR-3, but not PAR-4. In immunohistochemical studies, human gingival epithelial cells were found to express PAR-1, PAR-2, and PAR-3 on their surface, but not PAR-4, indicating that the cell line was an effective model for the in vivo situation. PAR-1 and PAR-2 expression was confirmed in intracellular calcium mobilization assays by treatment of the cells with the relevant receptor agonist peptides. Desensitization experiments strongly indicated that signaling of the effects of RgpB was occurring through PAR-1 and PAR-2. Studies with cells individually transfected with each of these two receptors confirmed that they were both activated by RgpB. Finally, it was shown that, in the oral epithelial cell line, PAR activation by the bacterial protease-stimulated secretion of interleukin-6. This induction of a powerful proinflammatory cytokine suggests a mechanism whereby cysteine proteases from P. gingivalis might mediate inflammatory events associated with periodontal disease on first contact with a primary barrier of cells.

FIG. 1

Expression of mRNAs encoding PARs in KB cells. RNA isolated from KB cells or platelets was analyzed for PAR transcripts by RT-PCR as described in Materials and Methods. Since the PAR-1 primers did not span an intron, RNA from platelets was used as a positive control for expression of PAR-1; RNA which had not undergone RT (−RT) was used as a negative control in relation to RNA which had (+RT). (A) KB cells express PAR-2 and PAR-3 (the sizes of the expected products of RT-PCR are shown below the lanes, in comparison to the sizes of selected markers to the right). (B) KB cells and platelets express PAR-1 (the size of the expected product for PAR-1 is shown to the right of panel B).

FIG. 2

Expression of PAR-1, -2, -3, and -4 by human gingival epithelium. Serial sections of human gingival tissue were immunolocalized with rabbit polyclonal antibodies against the receptors indicated in the upper right of each picture: PAR-1 (A), PAR-2 (B), PAR-3 (C), PAR-4 (D), and nonimmune rabbit immunoglobulin G (E), using the avidin-horseradish peroxidase-biotin complex system and a hematoxylin counterstain. There was specific staining for both PAR-1 and PAR-2 throughout the gingival epithelium; some examples of positive cells are indicated by small arrowheads (A and B). Macrophages and mast cells scattered in the subepithelial connective tissue were also positive for PAR-1 and PAR-2 (A and B, large arrowheads). Bar (panel E), 25 μm.

FIG. 3

The [Ca²⁺]_i response of KB cells to different concentrations of RgpB, trypsin, and thrombin. Each data point represents the mean from two traces similar to those shown in the figure.

FIG. 4

The [Ca²⁺]_i responses in KB cells to 100 μM RAP (A), 100 μM TRAP (B), 750 μM TRAP-4 (C), and 20 nM RgpB inactivated with antipain, followed by 100 μM trypsin to demonstrate that the cells were capable of mobilizing [Ca²⁺]_i, or 20 nM RgpB alone (D).

FIG. 5

The [Ca²⁺]_i responses in KB cells to 100 nM trypsin followed by 100 nM thrombin (A), 100 nM thrombin followed by 100 nM trypsin (B), 20 nM RgpB followed by 100 nM thrombin (C), 100 nM thrombin followed by 20 nM RgpB (D), 2 μM antipain followed by 100 nM thrombin (E), 20 nM RgpB followed by 2 μM antipain and then 100 nM thrombin (F), 100 nM trypsin followed by 20 nM RgpB (G), and 20 nM RgpB followed by 100 nM trypsin (H).

FIG. 6

Calcium mobilization in N1LF PAR-1 cells (A and B) or CHO PAR-2 cells (C and D) treated with 20 nM thrombin (A), 18 nM RgpB (B), 1 nM trypsin (C), or 1 nM RgpB (D). The traces are representative of three observed.

FIG. 7

Measurement of IL-6 concentrations by ELISA in culture supernatants from KB cells following treatment for 1 h (A through C) or 15 min (D) with 100 μM TRAP or RAP (A), 2.5 nM thrombin or trypsin (B), 2.5 nM RgpB or 25 nM RgpB (C), or 2.5 nM RgpB or 2.5 nMRgpB inactivated with antipain (D). The results shown in panels A, B, and D represent the mean ± standard error of the mean obtained from three experiments and those shown in panel C are results obtained from five experiments. All results were significantly different (P < 0.001).