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Infection and Immunity, October 2002, p. 5695-5705, Vol. 70, No. 10
0019-9567/02/$04.00+0     DOI: 10.1128/IAI.70.10.5695-5705.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.

Modulation of an Interleukin-12 and Gamma Interferon Synergistic Feedback Regulatory Cycle of T-Cell and Monocyte Cocultures by Porphyromonas gingivalis Lipopolysaccharide in the Absence or Presence of Cysteine Proteinases

Peter L. W. Yun,1* Arthur A. DeCarlo,2 Charles Collyer,3 and Neil Hunter1

Institute of Dental Research, Centre for Oral Health, Westmead Hospital, Wentworthville, Sydney, New South Wales 2145,1 School of Molecular & Microbial BioSciences, University of Sydney, Sydney, New South Wales 2006, Australia,3 NSU Dental, Nova Southeastern University, Fort Lauderdale, Florida2

Received 10 April 2002/ Returned for modification 20 June 2002/ Accepted 22 July 2002


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ABSTRACT
 
Interleukin 12 (IL-12) is an efficient inducer and enhancer of gamma interferon (IFN-{gamma}) production by both resting and activated T cells. There is evidence that human monocytes exposed to IFN-{gamma} have enhanced ability to produce IL-12 when stimulated with lipopolysaccharide (LPS). In this study, it was demonstrated that LPS from the oral periodontal pathogen Porphyromonas gingivalis stimulated monocytes primed with IFN-{gamma} to release IL-12, thereby enhancing IFN-{gamma} accumulation in T-cell populations. P. gingivalis LPS was shown to enhance IL-12 induction of IFN-{gamma} in T cells in a manner independent from TNF-{alpha} contribution. The levels of T-cell IL-12 receptors were not affected by P. gingivalis LPS and played only a minor role in the magnitude of the IFN-{gamma} response. These data suggest that LPS from P. gingivalis establishes an activation loop with IL-12 and IFN-{gamma} with potential to augment the production of inflammatory cytokines in relation to the immunopathology of periodontitis. We previously reported that the major cysteine proteinases (gingipains) copurifying with LPS in this organism were responsible for reduced IFN-{gamma} accumulation in the presence of IL-12. However, the addition of the gingipains in the presence of LPS resulted in partial restoration of the IFN-{gamma} levels. In the destructive periodontitis lesion, release of gingipains from the outer membrane (OM) of P. gingivalis could lead to the downregulation of Th1 responses, while gingipain associated with LPS in the OM or in OM vesicles released from the organism could have net stimulatory effects.


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INTRODUCTION
 
Periodontal diseases are considered immunopathological inflammatory responses associated with the presence of pathogenic microorganisms, particularly the gram-negative anaerobic bacterium Porphyromonas gingivalis. Proteinases from the organism have been reported to exhibit enzymatic activity against a broad range of host proteins (7, 9, 17, 50, 51), with the majority of this attributed to cysteine proteinases referred to as gingipains that cleave substrates after arginine (RgpA and RgpB) (30) or lysine (Kgp) residues (29). The proteinases RgpA and Kgp can be purified from the cell surface of P. gingivalis ATCC 33277 as catalytic domains noncovalently linked with C-terminal adhesin or hemagglutinin domains. Henderson et al. identified the gingipain-associated hemagglutinin 2 domain as a lipid A binding peptide (11). The contribution of lipopolysaccharide (LPS) from P. gingivalis as a virulence factor in periodontitis remains to be fully evaluated.

LPS from P. gingivalis differs biochemically from classical LPS derived from enterobacteria by lacking heptose and 2-keto-3-deoxyoctonate (22). Bramanti et al. reported that rhamnose represented the dominant sugar of LPS from P. gingivalis strain ATCC 33277 (3). P. gingivalis LPS and its lipid A, the endotoxic and bioactive center of LPS, induce relatively weak production of proinflammatory cytokines, such as tumor necrosis factor alpha (TNF-{alpha}) and interleukin-1 beta (IL-1ß), in human peripheral blood monocytes compared to those stimulated by standard LPS preparations from enterobacteria (28, 37).

CD14 as a glycosylphosphatidylinositol-anchored membrane protein or major receptor for LPS, which lacks transmembrane and cytoplasmic domains (44-46), was not thought to participate directly in signaling. In this context, the human homologues of Drosophila Toll or Toll-like receptors (TLRs) of the IL-1/Toll receptor family (13, 15) have been demonstrated to transduce intracellular signaling by LPS stimulation, and TLR4 has been shown to be resistant to gingipain treatment (36).

IL-12 is the major factor for the efficient induction and enhancement of gamma interferon (IFN-{gamma}) production by both resting and activated T cells of the Th1 phenotype (21, 42, 49). The induction of IL-12 production by LPS of gram-negative bacteria has been described (5, 41), and although IFN-{gamma} is not an absolute requirement for IL-12 production, this feedback mechanism is particularly powerful and is probably required for optimal production of IL-12 by monocytes (16, 49). However, there is little information regarding the enhancement of IL-12 production by IFN-{gamma}-primed monocytes stimulated by P. gingivalis LPS or the effect of LPS from this pathogen on T-cell populations. Further, little information is available to indicate the potential of P. gingivalis LPS to modulate T-cell IL-12 receptor expression.

A role for vascular endothelial cells in the priming of CD4+ T cells for costimulatory effects of IL-12 has been reported (20). In this context, the leukocyte function-associated antigen 3 has been identified in human umbilical vein endothelial (HUVE) cells as the major ligand for CD2 expressed on all T cells (4), and this interaction has been shown to augment the effects of IL-12 on monocyte costimulation of T-cell IFN-{gamma} production. The presence of IFN-{gamma} favors the development of a Th1 cytokine pattern, characterized by the production of IFN-{gamma} and IL-2, and induction of cell-mediated immunity, while Th2 responses, characterized by the production of IL-4, IL-5, IL-6, and IL-10 and humoral immune responses, are suppressed (25, 26, 32). In the context of periodontal disease, a higher level of expression of IFN-{gamma} mRNA in inflamed gingival tissue has been reported (31, 35, 39). Other studies reported that P. gingivalis LPS can induce IFN-{gamma} production in cultured human peripheral blood mononuclear cells (14, 28).

Since gingipains are colocated in the outer membrane with LPS (11), it is likely that LPS and gingipains act together in vivo. Hence, the aim of the work in this report was to clarify the combined effects of P. gingivalis LPS with gingipains on T-cell coculture responses. Specifically, this study investigates whether CD4+ T cells cocultured with monocytes can produce functional IFN-{gamma} in the presence of P. gingivalis LPS and gingipains.

In this report, we demonstrate (i) that P. gingivalis LPS enhances IFN-{gamma} accumulation in T-cell populations synergistically with IL-12, an effect dependent on the presence of monocytes; (ii) that P. gingivalis LPS synergistically enhances IFN-{gamma} stimulation of monocytes to secrete IL-12; (iii) that levels of IL-12R are not affected by LPS; (iv) that catalytically active RgpA and Kgp reduced IFN-{gamma} accumulation in the presence of P. gingivalis LPS and IL-12; and (v) that addition of higher levels of P. gingivalis LPS resulted in partial restoration of the IL-12 effect on IFN-{gamma} accumulation.


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MATERIALS AND METHODS
 
Chemicals and reagents. Ficoll-Hypaque was purchased from Pharmacia (Uppsala, Sweden). Collagenase type 1A, endothelial cell growth factor, L-cysteine, mitomycin-C, phytohemagglutinin (PHA), propanol, L-rhamnose, sodium dodecyl sulfate (SDS), N-{alpha}-tosyl-L-lysyl chloromethyl ketone (TLCK), Trizma base, Tris-HCl, trypsin, and Tween 20 were purchased from Sigma (St. Louis, Mo.). Trypticase soy broth was purchased from Difco (Detroit, Mich.). RPMI and M199 medium were obtained from ICN Biochemicals (Irvine, Calif.). 3-[(3-cholamidopropyl) dimethylammonio]-1-propanesulfonate was purchased from Calbiochem (La Jolla, Calif.). Phosphate-buffered saline (PBS) and Trypticase soy broth were purchased from Oxoid (Basingstoke, United Kingdom). Dynabeads were purchased from Dynal, (Lake Success, N.Y.). DNase/RNase and proteinase K were purchased from Roche (Castle Hill, New South Wales). All reagents for electrophoresis and Western blotting were from Bio-Rad (Richmond, Calif.).

Recombinant cytokines and antibodies. Recombinant interleukin-12 expressed in a eukaryotic baculovirus system was obtained from R&D Systems (Minneapolis, Minn.). Recombinant human IFN-{gamma} and recombinant human TNF-{alpha} were purchased from Endogen (Cambridge, Mass.). Mouse monoclonal and goat polyclonal antibodies specific for human IL-12 were purchased from R&D Systems. Monoclonal and polyclonal rabbit anti-human IFN-{gamma} antibodies were purchased from Endogen. Monoclonal antibody (MAb) specific for human CD56 was purchased from Dako (Glostrup, Denmark). MAb specific for human CD25 was obtained from PharMingen (San Diego, Calif.). Rabbit polyclonal immunoglobulin G (IgG) antibody specific for IL-12R was purchased from Santa Cruz Biotechnology (Santa Cruz, Calif.). Goat polyclonal IgG antibody specific for its ability to neutralize the bioactivity of human TNF-{alpha} was purchased from R&D Systems.

LPS, RgpA, and Kgp preparations and reagents. Salmonella enterica serovar Typhimurium LPS purified by the hot phenol-water extraction method was purchased from Sigma Chemical Co. Sydney, Australia, and used as a comparative control for P. gingivalis LPS activities. P. gingivalis ATCC 33277 was grown in enriched Trypticase soy broth under anaerobic conditions for 48 h (51). The bacteria at a density of 1.5 g/cm3 were suspended in saline, stirred for 1 h at 4°C and washed three times with pyrogen-free water, and lyophilized. P. gingivalis LPS was extracted from lyophilized cells by the hot phenol-water method (47, 48). Briefly, bacteria suspended in sterile saline were mixed with an equal volume of water-saturated butanol for 15 min at 4°C. The aqueous phases were pooled, centrifuged, and dialyzed for 48 h against pyrogen-free water at 4°C and then lyophilized. The lyophilized extracts were suspended in pyrogen-free water and mixed with an equal volume of phenol for 10 min at 68°C. The phenol phases were separated by centrifugation at 35,000 x g, and the aqueous phases were pooled, dialyzed, and then lyophilized. The crude extract was purified by repeated ultracentrifugation (100,000 x g, 2 h) followed by treatment with DNase/RNase and proteinase K. The P. gingivalis LPS was resuspended in pyrogen-free water and purified by ultracentrifugation at 100,000 x g for 1 h at 4°C. The P. gingivalis LPS preparation (5 µg/lane) was analyzed by 12% SDS-polyacrylamide gel electrophoresis (18) and visualized by silver staining (23, 43), showing a ladder pattern typical of LPS (data not shown). The P. gingivalis LPS was negative for Coomassie blue staining, indicating the purity of the preparation. Further, the P. gingivalis LPS contained 0.43% protein by weight as determined by the Bradford assay using bovine serum albumin as a standard. In the context of this study, the maximum possible contamination of the LPS preparation by gingipain components was 0.03 nM per 1,000 ng of LPS.

Gingipain-R and gingipain-K proteinase-adhesin complexes were purified as previously described (51).

Monosaccharide analysis. Rhamnose in the P. gingivalis LPS preparations was quantitated by the Dische-Shettles cysteine sulfuric acid reaction for methyl pentoses as previously described (8). Briefly, the reaction was carried out in acid-resistant capped bottles on P. gingivalis LPS, gingipain, and standards containing 2 to 20 µg of rhamnose in 1 ml of water. The ratios of P. gingivalis LPS in the gingipain preparations were then determined. The P. gingivalis LPS preparation contained 40% rhamnose by weight in agreement with the figure of 36% detected previously for this strain of P. gingivalis (3). The gingipain preparations contained an average of 4.4% rhamnose, indicating the presence of 11% LPS by weight.

Preparation of HUVE cells and CD4+ T cells. HUVE cells were isolated and cultured as previously described (40). Cells used in these experiments were confluent and at passage levels 4 through 6. HUVE cells were identified by reaction with Ulex agglutinin (Dako). Human peripheral blood mononuclear cells (PBMC) were separated from healthy volunteers (Blood Bank, Red Cross Transfusion Service, New South Wales, Australia) using Ficoll-Hypaque gradients (2).

CD4+ T cells were obtained from PBMC by positive selection using magnetic beads coated with anti-CD4 antibody (Dynal Inc., Lake Success, N.Y.) (4, 51). The purity of the CD4+ T cells isolated by this method was approximately 99% with 0.4 to 0.6% CD4+ CD14+ monocytes and 0.5 to 1% CD56+ NK cells as analyzed by flow-cytometric analysis (see below); the T cells were not activated, as analyzed by the lack of major histocompatibility class II antigen and CD25 expression. CD4+ T cells were then cultured in complete medium (RPMI 1640 containing 10% fetal calf serum, 1% penicillin-streptomycin, 2 mM glutamine, and 50 µM 2-mercaptoethanol) in a humidified atmosphere with 5% CO2 at 37°C for 48 h. T-cell populations were used within 4 h of isolation.

Depletion of adherent monocytes. CD4+-T-cell preparations at a concentration of 106/ml were depleted of adherent cells in culture dishes (60 by 15 mm) (Sarstedt, Sydney, Australia). After 3 h of depletion for the removal of adherent cells at 37°C, the percentages of CD4+ CD14+ monocytes remaining in the CD4+ T cell populations were analyzed by flow cytometry.

Isolation of human peripheral blood monocytes. Fresh monocytes were isolated from the blood of healthy donors by the adherence method (34). Briefly, PBMC were separated from healthy volunteers (Blood bank, Red Cross Transfusion Service, Sydney, New South Wales, Australia) using Ficoll-Hypaque gradients (2). The mononuclear cell fraction was collected, washed twice, and resuspended in RPMI medium. The cells were then seeded in 24-well culture plates (Sarstedt) (105/well) and incubated for 2 h at 37°C in RPMI medium. Following the removal of nonadherent cells and rinsing in medium, the adherent cells contained >99% CD14+ monocytes as assessed by flow-cytometric analysis of cells harvested as described previously (10). Monocyte cultures were stained for NK cells using monoclonal anti-CD56 and a peroxidase detection system. At least 200 cells/culture were assessed in culture slides, with <1% of cells determined to be CD56 positive.

Detection of CD4+ CD14+ monocytes and CD56+ NK cells by flow cytometry. For the identification of CD14+ monocytes in CD4+ T-cell preparations, flow-cytometric analysis was performed as previously described (50). Briefly, enriched CD4+ T-cell populations were labeled with a saturating 1:50 concentration of primary goat anti-human CD14 polyclonal antibody or goat serum (Dako), stained with a 1:50 concentration of fluorescein isothiocyanate (FITC)-conjugated rabbit anti-goat secondary antibody (Dako), and quantitated using a FACScan analyzer (Becton Dickinson, Franklin Lakes, N.J.). Incubations were for 45 min at 4°C. Volume gates were set to include the entire monocyte population. For the identification of NK cell populations, cells were stained with primary mouse anti-CD56 monoclonal antibody or isotype-matched control antibody, followed by FITC-conjugated rabbit anti-mouse secondary antibody (Dako).

IFN-{gamma} assays with LPS. An accepted coculture system where HUVE cells provide the accessory molecules for the CD4+ T cells to enable response to IL-12 was performed to induce T-cell secretion of IFN-{gamma} (4, 50). All T-cell cocultures were prepared as follows: HUVE cells at a density of 105 cells/cm2 in M199-supplemented media containing 20% FCS were grown to confluence in 96-well cell culture plates (Sarstedt) and were treated with mitomycin C (50 µg/ml), an inhibitor of cell proliferation, for 1 h and washed three times in M199 medium before coculture. CD4+ T cells (2 x 104 per well) were added to each well in a final volume of 200 µl of RPMI medium. PHA was then added at a final concentration of 1 µg/ml to provide a general cellular activation level. The T-cell cocultures were incubated for 48 h at 37°C in the presence of IL-12 (10 pg/ml) and LPS (100 ng/ml to 10 µg/ml) isolated from P. gingivalis ATCC 33277 or Salmonella serovar Typhimurium. The IFN-{gamma} released into the supernatant (100 µl per well) by CD4+ T cells was quantified by enzyme-linked immunosorbent assay (ELISA).

Anti-TNF-{alpha} MAb on CD4+ T-cell cocultures treated with LPS. The effect of TNF-{alpha} production by monocytes on IL-12 (10 pg/ml) induced T-cell IFN-{gamma} release in the presence of LPSs from P. gingivalis or Salmonella serovar Typhimurium (10 pg/ml to 10 µg/ml) was assessed by addition of a neutralizing anti-TNF-{alpha} polyclonal antibody (100 ng/ml or 2.5 µg/ml per well). The effect of TNF-{alpha} on T-cell IFN-{gamma} release in the absence or presence of P. gingivalis LPS was also evaluated. IFN-{gamma} release by T cells was quantified by ELISA after 48 h of culture at 37°C.

IL-12 bioassay. Peripheral blood monocytes were stimulated with 10 or 100 ng or 1 or 10 µg of P. gingivalis LPS or Salmonella serovar Typhimurium LPS/ml in the presence of IFN-{gamma} (25 ng/ml), and the IL-12 concentration in the supernatant (100 µl per well) was measured by ELISA 48 h later.

Accumulated IFN-{gamma} production after gingipain-P. gingivalis LPS cotreatment. CD4+-T-cell (2 x 104/well) cocultures with PHA (1 µg/ml) in each well were adjusted to a final volume of 200 µl of RPMI medium as described. RgpA or Kgp at various concentrations (0.4 to 13.3 nM) was treated with 2.5 mM L-cysteine for 15 min at room temperature. Activated gingipains were then incubated with or without the thiol-protease inhibitor TLCK (final concentration, 2 mM) for 1 h at 37°C, and the TLCK-treated gingipains were dialyzed against sterile PBS. In some of the experimental conditions, RgpA and Kgp were heat treated at 80°C for 60 min. Interleukin-12 (10 pg/ml) was added simultaneously to the wells with gingipain-LPS, and the cultures were incubated for 48 h. Cell coculture supernatants (100 µl each) were taken and assayed for IFN-{gamma} by ELISA after 48 h of culture.

Cytokine ELISA. IFN-{gamma} and IL-12 were measured by ELISA. Briefly, a murine MAb to either IFN-{gamma} or IL-12 with 100 µl per well at a concentration of 10 µg/ml was used as a capture antibody to coat 96-well flat-bottom ELISA plates (Sarstedt, Sydney, Australia) overnight at room temperature. Blocking was performed with 0.1% Tween 20 in PBS for 2 h at room temperature. Subsequently, undiluted coculture supernatants or standards were added to wells overnight at room temperature, and secondary polyclonal anti-IFN-{gamma} or anti-IL-12 (both subunits) antibody conjugated with alkaline phosphatase (Dako) at a 1:1,000 dilution was added to wells for 3 h at room temperature. For the determination of IFN-{gamma} COOH-terminal activity (51), polyclonal goat anti-IFN-{gamma} antibody which recognizes the epitope corresponding to amino acids 148 to 166 mapping at the carboxy terminus of the IFN-{gamma} precursor was used as a capture antibody (Santa Cruz), and rabbit polyclonal anti-IFN-{gamma} alkaline phosphatase-conjugated antibody (Dako) was used as a secondary antibody. In between each step, the plates were washed in PBS with 0.1% Tween. ELISA was developed using 4-nitrophenyl phosphatase substrate (Boehringer Mannheim, Mannheim, Germany). Plates were read at 405 nm in a Titertek ELISA plate reader (ICN, Sydney, Australia). Optical densities of standard cytokines were plotted against the dilution factors, and the cytokine concentration of each sample was determined.

Statistics. Data are presented as means ± standard errors. Statistical analysis was performed by Student's t test using SigmaStat software (Jandel Corp.), and P values less than 0.05 were considered significant.


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RESULTS
 
Effect of P. gingivalis LPS or Salmonella serovar Typhimurium LPS on T-cell IFN-{gamma} production. PHA-activated populations of T cells produced relatively little IFN-{gamma}, while addition of IL-12 to the wells greatly enhanced (five- to eight-fold) the accumulation of IFN-{gamma} secreted by the T cells (Fig. 1A) (50). The addition of P. gingivalis LPS to the T-cell cultures significantly increased the level of IFN-{gamma} accumulation within the supernatant in a dose-dependent manner if IL-12 (10 pg/ml) was present (Fig. 1B). Increases in IFN-{gamma} accumulation induced by P. gingivalis LPS in the absence of exogenous IL-12 were not significant in these assays. LPS from Salmonella serovar Typhimurium stimulated IFN-{gamma} accumulation in the presence of IL-12 with potency equivalent to that with P. gingivalis LPS (Fig. 1B).



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  		FIG. 1. Dose-dependent release of IFN-{gamma} by CD4+ T cells cocultured with human monocytes stimulated with IL-12 and LPS isolated from P. gingivalis or Salmonella serovar Typhimurium. (A) Freshly isolated human CD4+ T cells were cultured with a range of concentrations of IL-12 in the absence or presence of HUVE cells in medium containing PHA (1 µg/ml). Culture supernatants were taken after 48 h, and the IFN-{gamma} concentration was assessed by ELISA. The values are mean cytokine levels ± the standard error of the mean of triplicate samples and are representative of three separate experiments. (B) CD4+-T-cell cocultures were established as described in the presence of increasing doses of LPS, isolated from P. gingivalis (P.g.) or Salmonella serovar Typhimurium (S.t.). IL-12 was added to cultures at a concentration of 10 pg/ml. Culture supernatants were collected, and the production of IFN-{gamma} was assessed at 48 h by ELISA. The values are mean cytokine levels ± the standard error of the mean of triplicate samples and are representative of three separate experiments. The difference between the P. gingivalis LPS-treated samples and Salmonella serovar Typhimurium LPS-treated samples was significant by Student's t test (*, P < 0.05).

Depletion by adherence of the remaining monocyte population in these T-cell cultures by 33% ± 15% demonstrated that this primary immunomodulatory effect was significantly dependent on a highly adherent monocyte population contribution within the coculture (P < 0.001 at 2.5 µg of P. gingivalis LPS/ml and 10 ng of IL-12/ml) (Fig. 2B).



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  		FIG. 2. Critical contribution of adherent CD4+ CD14+ monocytes to the IL-12-induced T-cell IFN-{gamma} production with LPS isolated from P. gingivalis or Salmonella serovar Typhimurium. (A) CD4+-T-cell cocultures were established with P. gingivalis (P.g.) LPS (up to 2.5 µg/ml) and IL-12 (10 pg/ml) either before (mo. +ve) or after (mo. -ve) 3 h of depletion for the removal of adherent CD4+ CD14+ monocytes. Supernatants were assessed at 48 h for IFN-{gamma} by ELISA. The values are mean cytokine levels ± the standard error of the mean of triplicate samples and are representative of three separate experiments. (B) Mean number (± standard error of the mean) of CD14+ monocytes lost by adherence from CD4+ T cells (~70,000 cells) was determined by flow-cytometric analysis as described in Materials and Methods.

Pretreatment of the endothelial cells with mitomycin C (50 µg/ml) 1 h before the addition of the T-cell population had no significant effect on the accumulation of IFN-{gamma} in the assay (data not shown).

On determining the relative roles of IL-12 and LPS in these assays, we found that P. gingivalis LPS had little effect on IFN-{gamma} accumulation in the absence of IL-12 (Fig. 2A). These LPS-driven levels were seven- to eightfold lower than levels which accumulated in combination with IL-12 and were lower than levels induced by IL-12 alone (Fig. 1A). The data indicate a synergistic effect of P. gingivalis LPS and IL-12 in T-cell cultures.

Role of IFN-{gamma} priming for IL-12 production in response to P. gingivalis and Salmonella serovar Typhimurium LPS. IL-12 levels in monocyte cultures were moderately stimulated by IFN-{gamma} (6 pg/ml in 48 h) although not at all by P. gingivalis LPS alone (Fig. 3A). LPS from P. gingivalis was, however, synergistic with IFN-{gamma} in a dose-dependent manner for IL-12 secretion and accumulation in the monocyte cultures. Similar but more potent stimulation with a preparation of Salmonella serovar Typhimurium LPS confirmed that the LPS effect was not specific for P. gingivalis LPS (Fig. 3B).



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  		FIG. 3. P. gingivalis LPS-induced IL-12 production and Salmonella serovar Typhimurium LPS-induced IL-12 production are enhanced by IFN-{gamma} priming. Peripheral blood monocytes (mo.) seeded on plastic at a density of 105/well were stimulated with P. gingivalis (P.g.) LPS (A) or Salmonella serovar Typhimurium (S.t.) LPS (B) in the absence or presence of IFN-{gamma} (IFN-g) (25 ng/ml), and IL-12 was measured by ELISA at 48 h of culture. The values are mean IL-12 levels ± the standard error of the mean of triplicate samples and are representative of three separate experiments.

Taken together, these data demonstrated that P. gingivalis LPS or Salmonella serovar Typhimurium LPS acts synergistically with IFN-{gamma} in up-regulating monocyte IL-12 production and that in turn, LPS from these microorganisms acts with IL-12 in up-regulation of T-cell IFN-{gamma}, establishing an activation loop with IL-12 and IFN-{gamma} as soluble mediators.

Partial inhibitory effects of anti-TNF-{alpha} MAb on IFN-{gamma} release from LPS-stimulated T-cell cultures. Since P. gingivalis LPS was known to be a weak inducer of TNF-{alpha} which can synergize with the IFN-{gamma} biological activity, the contribution of TNF-{alpha} was measured using a neutralizing antibody strategy (28, 38). Neutralizing antibody to TNF-{alpha} at concentrations as high as 2.5 µg/ml had little effect (10% ± 7%) on IL-12-enhanced LPS stimulation of IFN-{gamma} in the T-cell cocultures, indicating that TNF-{alpha} contributed insignificantly to the LPS induction of IFN-{gamma} in T cells (Fig. 4A and B). Moreover, the addition of TNF-{alpha} at concentrations up to 10 ng/ml in the presence or absence of P. gingivalis LPS did not significantly induce the IFN-{gamma} production in T cells (Fig. 4C). The results also show that P. gingivalis LPS at various concentrations (10 pg/ml to 10 µg/ml) in the presence of TNF-{alpha} at a concentration of 1 ng/ml did not significantly activate T-cell IFN-{gamma} production (Fig. 4D).



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  		FIG. 4. Effect of anti-TNF-{alpha} MAb on P. gingivalis LPS-induced or Salmonella Typhimurium LPS- and IL-12-induced CD4+-T-cell IFN-{gamma} production. The effect of TNF-{alpha} production by monocytes on IL-12 (10 pg/ml)-induced T-cell IFN-{gamma} production in the presence of LPS from P. gingivalis (P.g.) was assessed by a neutralizing anti-TNF-{alpha} MAb at 100 ng/ml (A) or at 2.5 µg/ml (B) per well. (C) T cells were cultured with various concentrations of TNF-{alpha}. (D) T cells were cultured with TNF-{alpha} (1 ng/ml) and P. gingivalis LPS at various concentrations. IFN-{gamma} production was quantified by ELISA after 48 h at 37°C. The values are mean cytokine levels ± the standard error of the mean of triplicate samples and are representative of three separate experiments.

P. gingivalis LPS does not affect the PHA-induced upregulation of IL-12R on CD4+ T cells. Determination of IL-12 as a critical monocyte-derived cofactor in IFN-{gamma} secretion by T cells prompted investigation into the regulation of IL-12R during these experiments. Increases in T-cell IL-12R, as measured by flow cytometry, indicated that expression of IL-12R was necessary for IL-12 enhancement of IFN-{gamma} secretion in T-cell cultures and was up-regulated in all PHA-activated cultures (Fig. 5A and B). Similar levels of IL-12R were expressed in response to IL-12 and IL-12 combined with P. gingivalis LPS in the stimulated T cells. These data suggest that the expression levels of IL-12R do not account for the increased induction of IFN-{gamma} in T cells by P. gingivalis LPS in the presence of IL-12.



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  		FIG. 5. Upregulation of IFN-{gamma} production is unrelated to induction of T-cell IL-12 receptor expression by P. gingivalis LPS. (A) CD4+-T-cell cocultures were established without PHA (unst.) in the absence or presence of P. gingivalis (P.g.) LPS (200 ng/ml); or with PHA (st.) (2 µg/ml) and in the absence or presence of P. gingivalis (P.g.) LPS (200 ng/ml) alone, IL-12 (10 pg/ml) alone, or P.g. LPS plus IL-12 for 48 h. Conditioned T cells from the cocultures were recovered with immunomagnetic beads specific for CD4+ T cells after 48 h. Recovered T cells were washed and then incubated with primary rabbit anti-human IL-12R polyclonal antibody, followed by the addition of FITC-conjugated mouse anti-rabbit IgG1 (Dako), and quantitated as described above. Error bars show the means and standard errors derived by pooling data from three independent experiments. (B) Results are parallel to studies of Fig. 5A. Supernatants (100 µl each) were assessed at 48 h and analyzed for IFN-{gamma} by ELISA. EC, endothelial cells. Error bars show the means and standard errors derived by pooling data from three independent experiments. The difference between the unstimulated or stimulated samples with or without P.g. LPS, IL-12, and P.g. LPS plus IL-12 was significant by Student's t test (**, P < 0.01; ***, P < 0.001).

Effect of purified RgpA and Kgp on P. gingivalis LPS-induced IFN-{gamma} production. We previously reported the ability of active gingipains treated with polymyxin B to reduce IL-12-enhanced T-cell IFN-{gamma} production in a dose-dependent manner (50). The biological activities of the gingipains were characterized as previously described (51). Based on the activity of P. gingivalis LPS (Fig. 1), RgpA and Kgp preparations used in this study contained 10% LPS by weight (Fig. 6), which correlates to the levels estimated by chemical analysis (percent rhamnose). Cochallenge with P. gingivalis LPS and RgpA or Kgp also had little effect on IFN-{gamma} production in the absence of IL-12 (Fig. 6A and B). In the present study, both catalytically active RgpA and Kgp at concentrations up to 1 nM, with no pretreatment with polymyxin B, were associated with reduced IFN-{gamma} accumulation in the presence of P. gingivalis LPS and IL-12 (Fig. 6A and B). The addition of higher concentrations of RgpA or Kgp (up to 13.3 nM) resulted in partial restoration of the IL-12 effect of IFN-{gamma} accumulation in the presence of higher levels of P. gingivalis LPS (up to 200 ng/ml) (Fig. 6A and B). Proteolytic activities of RgpA or Kgp were blocked when the gingipains were heat treated (Fig. 6C and D) or TLCK treated (Fig. 6E), and this, in each case, restored the enhancement of IFN-{gamma} levels in the presence of P. gingivalis LPS and IL-12 (Fig. 6C and D). No significant differences were found between the RgpA or Kgp preparations in this IFN-{gamma} assay.




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  		FIG. 6. Antagonistic effect of purified RgpA or Kgp together with P. gingivalis LPS on IL-12-induced monocyte/CD4+ T cell IFN-{gamma} production. RgpA (A) or Kgp (B) (0.4 to 13.3 nM) (13.3 nM of RgpA or Kgp is equivalent to 2 µg/ml RgpA or Kgp) was preincubated for 15 min at 37°C with 2.5 mM L-cysteine. In both panels C and D, RgpA or Kgp was heat treated (H.T.) at 80°C for 60 min before addition to the culture. (E) Activated RgpA or Kgp (0.4 to 13.3 nM) was incubated with the thiol-protease inhibitor TLCK (final concentration, 2 mM) for 1 h at 37°C, and the TLCK-treated gingipains were dialyzed against sterile PBS. CD4+-T-cell cocultures were activated by PHA at a concentration of 1 µg/ml in the presence of CD4+ CD14+ monocytes and HUVE cells. Interleukin-12 (10 pg/ml) was added to the wells simultaneously with the gingipain and LPS, and the cultures were incubated for 48 h. The production of IFN-{gamma} was measured by ELISA after 48 h. Error bars show the means and standard errors derived by pooling data from three independent experiments.

Partial degradation of IFN-{gamma} COOH-terminal epitope by RgpA or Kgp in the presence of P. gingivalis LPS. Previously, we reported that the immunomodulatory activities of human IFN-{gamma} could be drastically reduced by exposure to the gingipains of P. gingivalis even in the presence of serum (51). The removal of the amino acids at the COOH-terminal end of IFN-{gamma} reduces IFN-{gamma} specific activity (19). To determine whether the biologically active form of IFN-{gamma} was produced in the T-cell cultures, ELISA plates were coated with affinity-purified antibody to the carboxyl terminus of IFN-{gamma}, and the captured active form of IFN-{gamma} was detected by polyclonal antibody to IFN-{gamma}. In the presence of P. gingivalis LPS and either RgpA or Kgp, IL-12-induced IFN-{gamma} production was decreased in a limited dose-dependent manner by the addition of active gingipains (Fig. 7A and B). A plateau of inhibition of 30% IFN-{gamma} activity was reached at an approximately 13.3 nM concentration of RgpA or Kgp. Nevertheless, the level of active IFN-{gamma} produced in the presence of gingipain, LPS, and IL-12 remained greater than in the absence of IL-12 (P < 0.001 at 13.3 nM gingipains and 200 ng of P. gingivalis LPS/ml).



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  		FIG. 7. Partial degradation of the IFN-{gamma} COOH terminus by RgpA or Kgp in the presence of P. gingivalis LPS. RgpA (A) or Kgp (B) (0.4 to 13.3 nM) was preincubated for 15 min at 37°C with 2.5 mM L-cysteine. In both panels C and D, activated RgpA or Kgp (0.4 to 13.3 nM) was incubated with thiol-protease inhibitor TLCK (final concentration, 2 mM) for 1 h at 37°C, and the TLCK-treated gingipains were dialyzed against sterile PBS. CD4+-T-cell cocultures were activated by PHA at a concentration of 1 µg/ml. Interleukin-12 (10 pg/ml) was then added simultaneously to the wells with gingipain and LPS, and the cultures were incubated for 48 h. After 48 h, culture supernatants were harvested and functional COOH-terminal IFN-{gamma} was measured by ELISA as described in Materials and Methods. Error bars show the means and standard errors derived by pooling data from three independent experiments.

To confirm that the cleavage at the COOH terminal end of IFN-{gamma} was due to the enzymatic activity of gingipains, the protease inhibitor TLCK, which is effective for both RgpA and Kgp, was used. TLCK-inhibited gingipains were incubated together with IL-12 and LPS under the same conditions (Fig. 7C and D). In the presence of TLCK, all of the IFN-{gamma} recovered after 48 h was biologically active by this criterion.


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DISCUSSION
 
IL-12 produced by monocytes plays a key role in the differentiation and induction of CD4+ Th1-cell IFN-{gamma} production. IFN-{gamma} is critical for the development of type 1 T-cell responses, while this cytokine exerts a slight suppressive effect on IL-2- and IL-4-mediated proliferation of Th2 clones but not Th1 clones (25, 26). IFN-{gamma}-activated phagocytic cells are also primed to produce much higher levels of IL-12 in a positive feedback mechanism (16).

Using enriched CD4+-T-cell and HUVE-cell cocultures, P. gingivalis LPS in the presence of IL-12 significantly increased IFN-{gamma} production. Since the trace population of NK cells present in the T-cell preparations does not express either T-cell-receptor antigens or the LPS receptor CD14, it is unlikely that these cells provided the necessary costimulatory ligand (CD2) for the HUVE cells (leukocyte function-associated antigen 3) (4) or responded to the P. gingivalis LPS in IL-12-induced IFN-{gamma} production.

Using the depletion method, we demonstrated that trace monocytes remaining in the CD4+-T-cell cocultures contributed significantly to IFN-{gamma} production via the enhanced production of IL-12 in the presence of the P. gingivalis LPS or Salmonella serovar Typhimurium LPS. The fact that P. gingivalis LPS synergistically enhances IFN-{gamma} stimulation of monocytes to secrete IL-12 suggests that P. gingivalis LPS may enhance the development of Th1 cell responses in the periodontal lesion through a positive feedback cycle. We postulate that sufficient quantities of IFN-{gamma} are produced by T cells in a mixed mononuclear cell environment to enhance the effects of P. gingivalis LPS on the production of IL-12 by monocytes. The cooperative activity of proinflammatory agents demonstrates a positive feedback mechanism, which might enhance local monocyte/T cell responses in inflammatory periodontal lesions. These data suggest an activation loop which can be initiated by LPS and results in accumulation of both IFN-{gamma} and IL-12 in mixed T-cell cultures.

P. gingivalis strain-dependent activation of monocyte TNF-{alpha} secretion by LPS has been reported (33). In this study, blocking TNF-{alpha} activity in the cultures or adding TNF-{alpha} did not significantly affect CD4+-T-cell IFN-{gamma} production, suggesting that LPS extracted from P. gingivalis ATCC 33277 enhanced the IFN-{gamma} production by some mechanism other than the induction of monocyte TNF-{alpha} secretion. Other factors, such as IL-18 or IFN-{alpha}, might contribute to the high levels of IFN-{gamma} observed in response to IL-12 and P. gingivalis LPS or Salmonella serovar Typhimurium LPS in the cocultures, although these have not been measured yet.

Results from the present study show that the expression of IL-12R on CD4+ T cells was unchanged by the addition of P. gingivalis LPS in the presence of monocytes, which excludes the possibility that P. gingivalis LPS enhances IFN-{gamma} production by preferentially inducing CD4+-T-cell IL-12R expression.

Ogawa et al. reported that the predominant immunoglobulin subclass response against carbohydrate antigens of P. gingivalis LPS is IgG2, which dominates in periodontitis subjects with progressive lesions (27). Also, Morris et al. have demonstrated that the administration of IL-12 in vivo increases serum IgG2a concentration in mice, and the effect was IFN-{gamma} dependent (24). Our results suggest that IL-12 produced by monocytes might augment IgG2 production through the additive effect of P. gingivalis LPS and IFN-{gamma}. Despite the presence of bacterial virulence factors, much of the damage in periodontal disease may actually be the result of the host immune response to bacteria. Within the lesion, increased levels of IFN-{gamma} could stimulate macrophages to produce inflammatory mediators, such as IL-1 and TNF-{alpha}, with subsequent activation of osteoclasts to mediate destruction of supporting bone.

Sugawara et al. (36) reported the ability of the gingipains to cleave monocyte CD14 preferentially, resulting in down-regulation of the LPS-induced TNF-{alpha} production by the cells. In the present study, the addition of low levels of the gingipains, which cleave both exogenous IL-12 (50) and CD14 on monocytes, could lead to the dip in response to low levels of P. gingivalis LPS (30 ng/ml) and gingipain. However, in the presence of IL-12 and various concentrations of gingipains, the effective presence of higher levels of P. gingivalis LPS (50 to 200 ng/ml) in T-cell cultures restored IFN-{gamma} production in a dose-dependent manner. Results from the present study are not in contradiction with the current literature but suggest a mechanism similar to that reported by Sugawara et al. High concentrations of LPS are known to activate cells in a CD14-independent mechanism (44). Although both IL-12 and CD14 were unlikely to be functionally active in the presence of the gingipains, it remains unclear whether other signaling molecules, such as TLR2 (12), are as resistant to the gingipains as TLR4 (36) and signal independently of CD14 at high concentrations of P. gingivalis LPS.

IFN-{gamma} is a highly structured molecule able to resist proteolytic digestion except for a small exposed C-terminal sequence of 11 amino acids (19, 51). The carboxyl terminus of human IFN-{gamma} is known to contribute significantly to functional activity and formation of the receptor-binding site of the molecule. Results in the present study demonstrated that increased levels of gingipains correlated well with the slightly decreased functional IFN-{gamma} production detected under experimental conditions. Gingipains potentially cleave IFN-{gamma} at the carboxyl terminus at an early stage of release from the T cells.

P. gingivalis ATCC 33277 retains gingipains at the cell surface, but domain combinations are released during extended culture (6). W50 is intermediate in behavior (1), while in strain H66, gingipains are readily recovered from culture supernatant (30). Therefore, these reports suggest a range of effective presentations of gingipains and LPS within the local tissue environment. Taken together, our results suggest that a shifting balance could be expected whereby release of gingipain independent of LPS could lead to the downregulation of Th1 responses, while gingipain and LPS could have a net stimulatory effect. This is supported by the current studies, which demonstrate that P. gingivalis may also significantly disturb the Th1 pathway through very efficient proteolytic processing and inactivation of IL-12 (50) and IFN-{gamma} (51) by RgpA and Kgp. Dysregulation of cytokine networking systems may suppress the immune response to bacterial antigens, whereas the profound inflammatory response to P. gingivalis LPS or outer membrane proteins might bring about tissue destruction in periodontal disease.


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ACKNOWLEDGMENTS
 
This study was supported by a grant from the National Health and Medical Research Council of Australia.


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FOOTNOTES
 
* Corresponding author. Mailing address: Institute of Dental Research, Centre for Oral Health, Westmead Hospital, P.O. Box 533, Wentworthville, Sydney, NSW 2145, Australia. Phone: 61-2-98458764. Fax: 61-2-98457599. E-mail: plwyun{at}yahoo.com. Back

Editor: E. I. Tuomanen


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REFERENCES
 
    1
  1. Aduse-Opoku, J., J. Muir, J. M. Slaney, M. Rangarajan, and M. A. Curtis. 1995. Characterization, genetic analysis, and expression of a protease antigen (PrpRI) of Porphyromonas gingivalis W50. Infect. Immun. 63:4744-4754.[Abstract]
    2. 2
  2. Bernzweig, E., J. B. Payne, R. A. Reinhardt, J. K. Dyer, and K. D. Patil. 1998. Nicotine and smokeless tobacco effects on gingival and peripheral blood mononuclear cells. J. Clin. Periodontol. 25:246-252.[CrossRef][Medline]
    3. 3
  3. Bramanti, T. E., G. G. Wong, S. T. Weintraub, and S. C. Holt. 1989. Chemical characterization and biologic properties of lipopolysaccharide from Bacteroides gingivalis strains W50, W83, and ATCC 33277. Oral Microbiol. Immunol. 4:183-192.[Medline]
    4. 4
  4. Briscoe, D. M., L. E. Henault, C. Geehan, S. I. Alexander, and A. H. Lichtman. 1997. Human endothelial cell costimulation of T cell IFN-{gamma} production. J. Immunol. 159:3247-3256.[Abstract]
    5. 5
  5. D'Andrea, A., M. Rengaraju, N. M. Valiante, J. Chehimi, M. Kubin, M. AsteAmezaga, S. H. Chan, M. Kobayashi, D. Young, and E. Nickbarg. 1992. Production of natural killer cell stimulatory factor (NKSF/IL-12) by peripheral blood mononuclear cells. J. Exp. Med. 176:1387-1398.[Abstract/Free Full Text]
    6. 6
  6. DeCarlo, A. A., and G. J. Harber. 1997. Hemagglutinin activity and heterogeneity of related Porphyromonas gingivalis proteinases. Oral Microbiol. Immunol. 12:47-56.[Medline]
    7. 7
  7. DeCarlo, A. A., L. J. Windsor, M. K. Bodden, G. J. Harber, B. Birkedal-Hansen, and H. Birkedal-Hansen. 1997. Activation and novel processing of matrix metalloproteinases by a thiol-proteinase from the oral anaerobe Porphyromonas gingivalis. J. Dent. Res. 76:1260-1270.[Abstract/Free Full Text]
    8. 8
  8. Dische, Z., and L. B. Shettles. 1948. A specific color reaction for methyl pentoses and spectrophotometer micromethod for determination. J. Biol. Chem. 175:595-603.[Free Full Text]
    9. 9
  9. Genco, C. A., J. Potemp, J. Mikolajczyk-Pawlinska, and J. Travis. 1999. Role of gingipains R in the pathogenesis of Porphyromonas gingivalis-mediated periodontal disease. Clin. Infect. Dis. 28:456-656.[Medline]
    10. 10
  10. Hedberg, N. M., N. Hunter, C. Soussou, and A. E. Knop. 1993. Production of an early release, M(r) 36,000 monokine by stimulated rat macrophages. Immunology 80:367-372.[Medline]
    11. 11
  11. Henderson, B., L. Sharp, S. Poole, K. Reddi, J. Fletcher, S. Nair, M. Wilson, M. Curtis, and P. Tabona. 1998. A lipid A-associated protein of Porphyromonas gingivalis, derived from the haemagglutinating domain of the R1 protease gene family, is a potent stimulator of interleukin 6 synthesis. Microbiology 144:3019-3026.[Abstract/Free Full Text]
    12. 12
  12. Hirschfeld, M., J. J. Weis, V. Toshchakov, C. A. Salkowski, M. J. Cody, D. C. Ward, N. Qureshi, S. M. Michalek, and S. N. Vogel. 2001. Signaling by Toll-like receptor 2 and 4 agonists results in differential gene expression in murine macrophages. Infect. Immun. 69:1477-1482.[Abstract/Free Full Text]
    13. 13
  13. Hoffmann, J. A., F. C. Kafatos, C. A. Janeway, and R. A. B. Ezekowitz. 1999. Phylogenetic perspectives in innate immunity. Science 284:1313-1318.[Abstract/Free Full Text]
    14. 14
  14. Kobayashi, M., L. Fitz, M. Ryan, R. M. Hewick, S. C. Clark, S. Chan, R. Loudon, F. Sherman, B. Perussia, and G. Trinchieri. 1989. Identification and purification of natural killer cell stimulatory factor (NKSF), a cytokine with multiple biologic effects on human lymphocytes. J. Exp. Med. 170:827-845.[Abstract/Free Full Text]
    15. 15
  15. Kopp, E. B., and R. Medzhitov. 1999. The Toll-receptor family and control of innate immunity. Curr. Opin. Immunol. 11:13-18.[CrossRef][Medline]
    16. 16
  16. Kubin, M., J. M. Chow, and G. Trinchieri. 1994. Differential regulation of interleukin-12 (IL-12), tumor necrosis factor-alpha, and IL-1-beta production in human myeloid leukemia cell lines and peripheral blood mononuclear cells. Blood 83:1847-1855.[Abstract/Free Full Text]
    17. 17
  17. Kuramitsu, H. K. 1998. Proteases of Porphyromonas gingivalis: what don't they do? Oral Microbiol. Immunol. 13:263-270.[Medline]
    18. 18
  18. Laemmli, U. K. 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680-685.[CrossRef][Medline]
    19. 19
  19. Leinikki, P. O., J. Calderon, M. H. Luquette, and R. D. Schreiber. 1987. Reduced receptor binding by a human interferon-{gamma} fragment lacking 11 carboxyl-terminal amino acids. J. Immunol. 139:3360-3366.[Abstract]
    20. 20
  20. Ma, W., and J. S. Pober. 1998. Human endothelial cells effectively costimulate cytokine production by, but not differentiation of, naive CD4+ T cells. J. Immunol. 161:2158-2167.[Abstract/Free Full Text]
    21. 21
  21. Magram, J., S. E. Connaughton, R. R. Warrier, D. M. Carvajal, C. Y. Wu, J. Ferrante, C. Stewart, U. Sarmiento, D. A. Faherty, and M. K. Gately. 1996. IL-12 deficient mice are defective in IFN gamma production and type 1 cytokine responses. Immunity 4:471-481.[CrossRef][Medline]
    22. 22
  22. Mansheim, B. J., A. B. Onderdonk, and D. L. Kasper. 1978. Immunochemical and biologic studies of the lipopolysaccharide of Bacteroides melaninogenicus subspecies asaccharolyticus. J. Immunol. 120:72-78.[Abstract/Free Full Text]
    23. 23
  23. Merril, C. R., D. Goldman, and M. L. Van Keuren. 1982. Simplified silver protein detection and image enhancement methods in polyacrylamide gels. Electrophoresis 3:14-23.
    24. 24
  24. Morris, S. C., K. B. Madden, J. J. Adamovicz, W. C. Gause, B. R. Hubbard, M. K. Gately, and F. D. Finkelman. 1994. Effects of IL-12 on in vivo cytokine gene expression and Ig isotype selection. J. Immunol. 152:1047-1056.[Abstract]
    25. 25
  25. Mosmann, T. R., and R. L. Coffman. 1989. Th1 and Th2 cells: different patterns of lymphokine secretion lead to different functional properties. Annu. Rev. Immunol. 7:145-173.[CrossRef][Medline]
    26. 26
  26. Mosmann, T. R., and S. Sad. 1996. The expanding universe of T-cell subsets: Th1, Th2 and more. Immunol. Today 17:138-146.[CrossRef][Medline]
    27. 27
  27. Ogawa, T., Y. Kusumoto, S. Hamada, J. R. McGhee, and H. Kiyono. 1990. Bacteriodes gingivalis-specific serum IgG and IgA subclass antibodies in periodontal diseases. Clin. Exp. Immunol. 82:318-325.[Medline]
    28. 28
  28. Ogawa, T., H. Uchida, and K. Amino. 1994. Immunobiological activities of chemically defined lipid A from lipopolysaccharides of Porphyromonas gingivalis. Microbiology 140:1209-1216.[Abstract/Free Full Text]
    29. 29
  29. Pavloff, N., P. A. Pemberton, J. Potempa, W. C. A. Chen, N. R. Pike, V. Prochazka, M. C. Kiefer, J. Travis, and P. Barr. 1997. Molecular cloning and characterization of Porphyromonas gingivalis Lys-gingipain. A new member of an emerging family of pathogenic bacterial cysteine proteinases. J. Biol. Chem. 272:1595-1600.[Abstract/Free Full Text]
    30. 30
  30. Pavloff, N., J. Potempa, R. N. Pike, V. Prochazka, M. C. Kiefer, J. Travis, and P. J. Barr. 1995. Molecular cloning and structural characterization of the Arg-gingipain proteinase of Porphyromonas gingivalis: biosynthesis as a proteinase-adhesin polyprotein. J. Biol. Chem. 270:1007-1010.[Abstract/Free Full Text]
    31. 31
  31. Prabhu, A., B. S. Michalowicz, and A. Mathur. 1996. Detection of local and systemic cytokines in adult periodontitis. J. Periodontol. 67:515-522.[Medline]
    32. 32
  32. Romagnani, S. 1994. Lymphokine production by human T cells in disease states. Annu. Rev. Immunol. 12:227-258.[CrossRef][Medline]
    33. 33
  33. Shapira, L., C. Champagne, T. E. Van Dyky, and S. Amar. 1998. Strain-dependent activation of monocytes and inflammatory macrophages by lipopolysaccharide of Porphyromonas gingivalis. Infect. Immun. 66:2736-2742.[Abstract/Free Full Text]
    34. 34
  34. Shapira, L., W. A. Soskolne, M. N. Sela, S. Offenbacher, and V. Barak. 1994. The secretion of PGE2, IL-1 beta, IL-6 and TNF alpha by adherent mononuclear cells from early-onset periodontitis patients. J. Periodontol. 65:139-146.[Medline]
    35. 35
  35. Shimabukuro, Y., S. Murakami, and H. Okada. 1996. Antigen-presenting-cell function of interferon gamma-treated human gingival fibroblasts. J. Periodontal Res. 31:217-228.[CrossRef][Medline]
    36. 36
  36. Sugawara, S., E. Nemoto, H. Tada, K. Miyake, T. Imamura, and H. Takada. 2000. Proteolysis of human monocyte CD14 by cysteine proteinases (gingipains) from Porphyromonas gingivalis leading to lipopolysaccharide hyporesponsiveness. J. Immunol. 165:411-418.[Abstract/Free Full Text]
    37. 37
  37. Tanamoto, K., S. Azumi, Y. Haishima, H. Kumada, and T. Umemoto. 1997. Endotoxic properties of free lipid A from Porphyromonas gingivalis. Microbiology 143:63-71.[Abstract/Free Full Text]
    38. 38
  38. Tanamoto, K., S. Azumi, Y. Haishima, H. Kumada, and T. Umemoto. 1997. The lipid A moiety of Porphyromonas gingivalis lipopolysaccharide specifically mediates the activation of C3H/HeJ mice. J. Immunol. 158:4430-4436.[Abstract]
    39. 39
  39. Taubman, M., J. W. Eastcott, H. Shimauchi, O. Takeichi, and D. J. Smith. 1994. Modulatory role of T lymphocytes in periodontal inflammation, p. 147-159. In R. Genco, S. Hamada, T. Lehner, J. McGhee, and S. Mergenhagen, Molecular pathogenesis of periodontal diseases. ASM Press, Washington, D.C.
    40. 40
  40. Thornton, S. C., S. N. Mueller, and E. M. Levine. 1983. Human endothelial cells: use of heparin in cloning and long-term serial cultivation. Science 222:623-625.[Abstract/Free Full Text]
    41. 41
  41. Trinchieri, G. 1997. Cytokines acting on or secreted by macrophages during intracellular infection (IL-10, IL-12, IFN-{gamma}). Curr. Opin. Immunol. 9:17-23.[CrossRef][Medline]
    42. 42
  42. Trinchieri, G., and P. Scott. 1999. Interleukin-12: basic principles and clinical applications. Curr. Top. Microbiol. Immunol. 238:59-78.
    43. 43
  43. Tsai, C. M., and C. E. Frasch. 1982. A sensitive silver stain for detecting lipopolysaccharides in polyacrylamide gels. Anal. Biochem. 119:115-119.[CrossRef][Medline]
    44. 44
  44. Ulevitch, R. J., and P. S. Tobias. 1995. Receptor-dependent mechanisms of cell stimulation by bacterial endotoxin. Annu. Rev. Immunol. 13:437-457.[CrossRef][Medline]
    45. 45
  45. Ulevitch, R. J., and P. S. Tobias. 1994. Recognition of endotoxin by cells leading to transmembrane signaling. Curr. Opin. Immunol. 6:125-130.[CrossRef][Medline]
    46. 46
  46. Viriyakosol, S., and T. Kirkland. 1995. Knowledge of cellular receptors for bacterial endotoxin. Clin. Infect. Dis. 21:S190-S195.
    47. 47
  47. Westphal, O., and K. Jann. 1965. Bacterial lipopolysaccharides. Extraction with phenol-water and further applications of the procedure. Methods Carbohydr. Chem. 5:83-91.
    48. 48
  48. Wober, W., and P. Alaupovic. 1971. Studies on the protein moiety of endotoxin from Gram-negative bacteria. Characterization of the protein moiety isolated by phenol treatment of endotoxin from Serratia marcescens 08 and Escherichia coli 0 141:K85(B). Eur. J. Biochem. 19:340-356.[Medline]
    49. 49
  49. Wolf, S. F., P. A. Temple, M. Kobayashi, D. Young, M. Dicig, L. Lowe, R. Dziabo, L. Fitz, C. Ferenz, R. M. Hewick, K. Kelleher, S. H. Chan, G. Trinchieri, and B. Perussia. 1991. Cloning of cDNA for natural killer cell stimulation factor, a heterodimeric cytokine with multiple biological effects on T and natural killer cells. J. Immunol. 146:3074-3081.[Abstract]
    50. 50
  50. Yun, L. W. P., A. A. DeCarlo, C. Collyer, and N. Hunter. 2001. Hydrolysis of interleukin-12 by Porphyromonas gingivalis major cysteine proteinases may affect local interferon-gamma accumulation and the Th1 or Th2 T-cell phenotype in periodontitis. Infect. Immun. 69:5650-5660.[Abstract/Free Full Text]
    51. 51
  51. Yun, L. W. P., A. A. DeCarlo, and N. Hunter. 1999. Modulation of major histocompatibility complex protein expression by human gamma interferon mediated by cysteine proteinase-adhesin polyproteins of Porphyromonas gingivalis. Infect. Immun. 67:2986-2995.[Abstract/Free Full Text]

Infection and Immunity, October 2002, p. 5695-5705, Vol. 70, No. 10
0019-9567/02/$04.00+0     DOI: 10.1128/IAI.70.10.5695-5705.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.



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