Modulation of major histocompatibility complex protein expression by human gamma interferon mediated by cysteine proteinase-adhesin polyproteins of Porphyromonas gingivalis

Abstract

Cysteine proteinases have been emphasized in the virulence of Porphyromonas gingivalis in chronic periodontitis. These hydrolases may promote the degradation of extracellular matrix proteins and disrupt components of the immune system. In this study it was shown that purified Arg-gingipain and Lys-gingipain inhibited expression of class II major histocompatibility complex (MHC) proteins in response to the stimulation of endothelial cells with human gamma interferon (IFN-gamma). Treatment with the cysteine proteinases resulted in a rapid shift in the apparent molecular size of IFN-gamma from 17 to 15 kDa, as shown by Western blot analysis, a response which also occurred in the presence of serum. Further, glycosylated natural IFN-gamma from human leukocytes and unglycosylated recombinant IFN-gamma from Escherichia coli were both digested by the cysteine proteinases. Immunoblot analysis indicated that cleavage within the carboxyl terminus of recombinant IFN-gamma correlated with the loss of induction of MHC class II expression as monitored by analytical flow cytometry. No hydrolysis of MHC class II molecules or human IFN-gamma receptor by these proteinases was detected by Western blot analysis. These findings suggest that P. gingivalis cysteine proteinases may alter the cytokine network at the point of infection through the cleavage of IFN-gamma. Degradation of IFN-gamma could have important consequences for the recruitment and activation of leukocytes and therefore may contribute significantly to the destruction of the periodontal attachment.

FIG. 1

SDS-PAGE of purified RgpA and Kgp. Gingipains were isolated as described in Materials and Methods, denatured by boiling in SDS loading buffer, and then resolved by SDS-PAGE. Lane 1, RgpA; lane 2, Kgp.

FIG. 2

Time-dependent inhibition by RgpA of HLA-DR expression induced in HUVE cells. HUVE cells were seeded subconfluently at a density of 10⁵ cells/cm² and maintained for 4 days in supplemented medium containing 20% FCS as described in Materials and Methods. At the start of the incubation, rIFN-γ (3 nM) was added to some wells (b to e). RgpA (60 nM) was added either at the time of (c), 1 day after (d), or 2 days after (e) the addition of the rIFN-γ. Four days after the start of the incubation, cells were removed and analyzed for HLA-DR expression by flow cytometric analysis as described in Materials and Methods. The cell number (y axis) versus logarithm of fluorescence (x axis) is represented. The data are representative of four separate experiments.

FIG. 3

Inhibition by RgpA of HLA-DR expression induced in HUVE cells is thiol mediated. RgpA was preincubated with or without the thiol-protease inhibitor TLCK for 1 h at 37°C and then dialyzed exhaustively against PBS. rIFN-γ (3 nM) and the TLCK-treated RgpA (60 nM) were simultaneously added to HUVE cells seeded at a density of 10⁵ cells/cm² and then incubated for 4 days in supplemented medium containing 20% FCS as described in Materials and Methods. After 4 days of culture, cells were removed and analyzed for HLA-DR expression by flow cytometric analysis. The cell number (y axis) versus the logarithm of fluorescence (x axis) is represented. The data are representative of three separate experiments.

FIG. 4

Dose-dependent inhibition by RgpA and Kgp of HLA-DR expression induced in HUVE cells. HUVE cells were seeded subconfluently at a density of 10⁵ cells/cm² and maintained for 4 days in supplemented medium containing 20% FCS. At the start of the incubation, rIFN-γ (3 nM) and various concentrations of RgpA or Kgp were added simultaneously, and the cultures were then maintained for 4 days. After 4 days, cells were removed and analyzed for HLA-DR expression by flow cytometric analysis. Error bars show the means and standard errors of the means for three separate experiments, which were representative of multiple experiments. Symbols: ●, RgpA; ▴, Kgp.

FIG. 5

RgpA or Kgp do not cleave the HLA-DR molecule on HUVE cells. HUVE cells were seeded at a density of 10⁵ cells/cm² and incubated for 3 days in supplemented medium containing 3 nM rIFN-γ to induce HLA-DR expression. Cells were then washed and cultured for an additional 24 h in serum-containing supplemented medium with 60 nM RgpA or Kgp. The cells were subsequently removed, washed, and solubilized in SDS; the proteins were then resolved by SDS-PAGE and subjected to Western blot analysis. (A) Detection of the HLA-DR α chain. (B) Detection of the HLA-DR β chain. Lane 1, untreated HUVE cells; lane 2, RgpA treatment of HUVE cells; lane 3, Kgp treatment of HUVE cells. The data are representative of three separate experiments.

FIG. 6

Lack of RgpA or Kgp effect on the IFN-γ receptor molecule. Confluent HUVE cells seeded at a density of 10⁵ cells/cm² in supplemented medium containing 20% FCS were incubated for 1 or 4 days with 60 nM RgpA or Kgp. The cells were removed, washed, and solublized in SDS; the proteins were then resolved by SDS-PAGE and subjected to Western blot analysis. (A) Detection of the IFN-γ R α chain. Lanes 1 to 5 show HUVE cells treated as follows: lane 1, Kgp for 1 day; lane 2, RgpA for 1 day; lane 3, Kgp for 4 days; lane 4, RgpA for 4 days; lane 5, medium only. (B) Detection of the IFN-γ R β-chain. Lanes 1 to 3 show HUVE cells treated as follows: lane 1, Kgp for 1 day; lane 2, RgpA for 1 day; lane 3, medium only. The data are representative of three separate experiments.

FIG. 7

Time course of native IFN-γ degradation by RgpA and Kgp in serum. RgpA or Kgp (4.7 pM each) was preincubated for 15 min at 37°C with 5 mM l-cysteine. The activated gingipains were then mixed with whole bovine serum and combined with an equimolar ratio of native IFN-γ (4.7 pM in each reaction) for a final serum concentration of 20%. Digestions were incubated at 37°C for various times and then stopped in aliquots with TLCK (2 mM, final concentration). Aliquots were resolved by SDS-PAGE for Western blot analysis with polyclonal antibodies against IFN-γ as described in Materials and Methods. Control samples incubated without gingipains are labeled IFN-γ. (A) Digestion with RgpA. (B) Digestion with Kgp. The data are representative of three separate experiments.

FIG. 8

Time course of rIFN-γ degradation by RgpA and Kgp in the absence of serum. RgpA or Kgp was preincubated for 15 min at 37°C with 5 mM l-cysteine. The activated gingipains were then incubated with rIFN-γ at a final S:E ratio of 1,000:1 (15 pM rIFN-γ with 15 fM gingipains in each reaction). Digestions were incubated at 37°C for various times and then stopped in aliquots with TLCK (2 mM, final concentration). Aliquots were resolved by SDS-PAGE for Western blot analysis with rabbit anti-human IFN-γ polyclonal antibodies as described in Materials and Methods. Control samples incubated without gingipain are labeled IFN-γ. (A) Digestion with RgpA. (B) Digestion with Kgp. The data are representative of three separate experiments.

FIG. 9

Degradation of recombinant IFN-γ by RgpA or Kgp in the presence of leupeptin. RgpA or Kgp was preincubated with 5 mM l-cysteine for 15 min and then with or without 0.1 mM leupeptin for 15 min at 37°C. The gingipains were then combined with rIFN-γ for a final E:S ratio of 1:28 (0.54 pM gingipains with 15 pM rIFN-γ in each reaction). Digestions were incubated at 37°C for various times and then stopped in aliquots with TLCK (2 mM, final concentration). Aliquots were resolved by SDS-PAGE for Western blot analysis with rabbit anti-human IFN-γ polyclonal antibodies as described in Materials and Methods. (A) Digestion with RgpA. Lanes 1 and 2, cysteine (5 mM), at 10 min and 80 min respectively; lane 3, human rIFN-γ as a control; lanes 4 to 7, cysteine (5 mM) and leupeptin (0.1 mM) at 10, 20, 40, and 80 min, respectively. (B) Digestion with Kgp. Lanes 1 to 4, cysteine (5 mM) and leupeptin (0.1 mM) at 10, 20, 40, and 80 min, respectively; lane 5, human rIFN-γ as a control; lanes 6 and 7, cysteine (5 mM) at 10 and 80 min, respectively.

FIG. 10

Loss of COOH-terminal epitope resulting from digestion of rIFN-γ by RgpA or Kgp results in loss of HLA-DR induction. Western blots of rIFN-γ (4.7 pM in each reaction) incubated with RgpA (A) or Kgp (B) (0.67 pM each) for various times in the absence of serum or with RgpA in the presence of 20% FCS (C). Digestions were stopped in aliquots with TLCK (2 mM, final concentration), and the products were resolved by SDS-PAGE for Western blot analysis with polyclonal antibody corresponding to amino acids 148 to 166 mapping at the COOH terminus of the rIFN-γ. Control samples incubated without gingipain are labeled IFN-γ. (D and E) Flow cytometric analysis of HLA-DR expression induced by gingipain-treated rIFN-γ. Samples of rIFN-γ (D) or nIFN-γ (E) treated with RgpA or Kgp for 15 min in the absence of serum as described above were dialyzed against PBS and then incubated with HUVE cells seeded at a density of 10⁵ cells/cm² in supplemented medium containing 20% FCS for 4 days. The samples were then analyzed for HLA-DR expression by flow cytometric analysis. The cell number (y axis) versus the logarithm of fluorescence (x axis) is represented. The data are representative of three separate experiments.