High molecular weight gingipains from Porphyromonas gingivalis induce cytokine responses from human macrophage-like cells via a nonproteolytic mechanism

Abstract

Periodontal disease is an oral inflammatory disease affecting the supporting structures of teeth. Porphyromonas gingivalis, a major pathogenic agent for the disease, expresses a number of virulence factors, including cysteine proteases called the gingipains. The arginine- and lysine-specific gingipains, HRgpA and Kgp, respectively, are expressed as high molecular weight forms containing both catalytic and adhesin subunits. We examined the expression pattern of cytokines and their receptors in differentiated macrophages following exposure to active and inactive forms of the gingipains, using a cDNA array, quantitative PCR and ELISA analysis. Amongst other pro-inflammatory cytokines, results from the cDNA array suggested that interleukin-1beta, granulocyte-macrophage colony stimulatory factor and interferon-gamma were upregulated after exposure of the macrophages to the gingipains. Quantitative PCR analysis substantiated these observations and indicated that active or inactive forms of the high molecular weight gingipains were able to upregulate expression of transcripts for these cytokines. The strongly enhanced production of interleukin-1beta and granulocyte-macrophage colony stimulatory factor by differentiated macrophages in response to active or inactive forms of the high molecular weight gingipains was confirmed at the protein level by ELISA analysis. The results indicate that the adhesin subunits of the gingipains mediate strong upregulation of the expression of pro-inflammatory cytokines in macrophages.

Fig. 1

Analysis of IL-1β, IFN-γ, GM-CSF, IL-10 and IRF1 expression in THP-1 cells in response to stimulation with 2.5 nM active HRgpA (dark bars), 2.5 nM inactive HRgpA (grey bars) or 2.5 nM active Kgp (clear bars) for 1 h. cDNA synthesized from RNA extracted immediately (1 h) or 3, 6 or 24 h later from THP-1 cells challenged with each treatment was used as a template for qPCR reactions using IL-1β, IFN-γ, GM-CSF, IL-10, IRF1 and GAPDH primers. The cycle threshold values were used to calculate IL-1/β, IFN-γ, GM-CSF, IL-10 and IRF1 transcript levels in treated cultures normalized to GAPDH relative to the normalized level of the transcripts for the same molecules in untreated samples. Data are presented as means ± SEM (n = 3). *** p ≤ 0.001. Comparisons between 2 groups were made by unpaired Student's t tests.

Fig. 2

IL-1p and GM-CSF secretion in THP-1 cells stimulated with 2–200 nM active HRgpA (HA), inactive HRgpA (HI), active Kgp (KA), inactive Kgp (KI), active RgpB (RA), inactive RgpB (RI), 100 (xM PAR-1 AP (P1AP) or 100 (xM PAR-2 AP (P2AP), compared to a control (CTL). The conditioned medium was recovered at 18 h (dark bars) or 36 h (light bars) and analyzed for levels of IL-1p and GM-CSF and compared to controls. Data are presented as means ± SEM (n = 3). * p ≤ 0.05; ** p ≤ 0.01; *** p ≤ 0.001. Comparisons between 2 groups were made by unpaired Student's t tests.