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Previous Article | Next Article 
Infection and Immunity, October 2004, p. 5910-5918, Vol. 72, No. 10
0019-9567/04/$08.00+0 DOI: 10.1128/IAI.72.10.5910-5918.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.
Department of Infectious Diseases,1 Department of Periodontology, Charité, CVK,2 Robert Koch-Institut, Berlin, Germany,3 Department of Oral Biology, Boston University Goldman School of Dental Medicine, Boston, Massachusetts,4 Department of Oral Biology, State University of New York, Buffalo, New York5
Received 19 February 2004/ Returned for modification 5 May 2004/ Accepted 14 July 2004
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ABSTRACT |
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B
, followed by nuclear translocation of NF-B. In addition, both strains induced enhanced expression of endothelial adhesion molecules E-selectin and intracellular adhesion molecule 1 (ICAM-1). Target cell activation was independent of bacterial fimbriae expression since the fimA knockout strain A7436 
fimA induced the same level of ICAM-1 as the corresponding wild type (A7436-WT). Thus, two P. gingivalis strains, ATCC 53799 and DSMZ 20709, infect endothelial cells and trigger signaling cascades leading to endothelial activation, which in turn may result in or promote severe local and systemic inflammation. |
INTRODUCTION |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Therefore, the first goal of our study was to assess the capability of two different P. gingivalis strains to infect or invade human umbilical vein endothelial cells (HUVEC). Expression of the endothelial adhesion molecules E-selectin and ICAM-1 was used to demonstrate subsequent target cell activation. Due to the different virulence capacities of the known strains, we used the most virulent strain (ATCC 53977), according to the study of Ebersole et al. (14), and a less virulent strain, DSMZ 20709 (equivalent to strain ATCC 33277, used by Ebersole et al.).
Mitogen-activated protein kinase (MAPK)-related signal transduction pathways are among the most widespread mechanisms of eukaryotic cell regulation (activation, stress response, differentiation, and growth). MAPK-dependent activation of transcription factors is considered to be a prerequisite for altered gene expression in stimulated target cells (for a review, see references 4 and 32). Specifically, p38 MAPK is strongly activated during inflammatory reactions and appears to be of specific importance during LPS-mediated signal transduction (22, 48).
The expression of inflammatory mediators such as cytokines or adhesion molecules relies on the activation of cytosolic transcription factors. Among the primary transcription factors, NF-B plays a central role in the regulation of these proinflammatory molecules (5, 50) Nuclear translocation of NF-B induced by proinflammatory stimuli (cytokines, bacteria, or bacterial toxins) is regulated by phosphorylation and degradation of its naturally occurring inhibitor IB (16, 28, 35). Little is known about signal transduction pathways activated in target cells upon infection with P. gingivalis. Therefore, the second objective of the present study was to investigate P. gingivalis-dependent activation of host cell signal transduction pathways in human endothelial cells. In our study we demonstrated that both P. gingivalis strains induced a phosphorylation of p38 MAPK, degradation of IB, and translocation and activation of endothelial cell NF-B, with subsequently increased transcription and translation of E-selectin and ICAM-1.
(Part of this research was conducted by Clemens Walter in partial fulfillment of the requirements for an MD degree from Charité, Universität Medizin Berlin.
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MATERIALS AND METHODS |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Preparation of HUVEC. HUVEC were isolated from human umbilical cord veins and identified according to a previously described method (26). Briefly, cells obtained from collagenase digestion were washed, resuspended in MCDB-131 medium-10% fetal calf serum, and seeded into gelatin-coated 6-, 24-, or 96-well plates. Only confluent monolayers of primary cultures were used for all experiments.
Bacterial strains.
P. gingivalis ATCC 53977 was from the laboratory of H. K. Kuramitsu (State University at Buffalo, Buffalo, N.Y.), P. gingivalis DSMZ 20709 was obtained from the Deutsche Sammlung von Mikroorganismen und Zellkulturen (DSMZ; Braunschweig, Germany). Strains were characterized and tested for purity by HAIN life science (Nehren, Germany) and by means of a microbiological test kit ID 32A (BioMerieux, Marcy l'Etoile, France). Wild-type A7436 (A7436-WT) and the corresponding fimA knockout mutant A7436 fimA were provided by J. Hayashi and H. K. Kuramitsu (unpublished data). All stocks were grown in brain heart infusion broth containing cysteine (0.1%), yeast (0.5%), vitamin K (0.1%), and hemin (0.25%) in an anaerobic chamber (Thermo Life Science) in an atmosphere of 85% N2, 5% H2, and 10% CO2 for 3 to 5 days, and erythromycin (10 µg/ml) was added to the A7436 fimA culture. Aliquots were stored at –70°C. An aliquot of the initial stock solution was used for each experiment without subculturing.
P. gingivalis infection assay. Thawed aliquots of both strains were grown to mid-logarithmic phase, centrifuged (1,500 x g for 5 min at 37°C) and resuspended in HUVEC media. A multiplicity of infection (MOI; as determined spectrophotometrically by species-specific curves) of 0.01 to 100 was used to infect HUVEC. Bacteria were centrifuged onto HUVEC monolayers in order to enhance the rate of infection (350 x g for 5 min). After incubation at 37°C for 1.5 h, the supernatant was replaced with fresh medium containing gentamicin (0.5 mg/ml) and metronidazole (0.1 mg/ml) in order to kill remaining extracellular (EC) bacteria. Plates were processed for experiments at the times indicated.
Scanning electron microscopy. HUVEC were grown on glass slides in 24-well plates, infected at an MOI of 100 as described above, and fixed with 8% Karnowsky's solution (2% glutaraldehyde and 4% paraformaldehyde [PFA] in PBS) for 24 h at 4°C. After washing with HEPES buffer, samples were dehydrated with graded ethanol, critical-point dried with CO2, sputter coated with 10 nm of gold, and examined with a scanning electron microscope with 5 kV in the secondary mode (LEO 1530 Field emission scanning electron microscope; LEO Elektronenmikroskopie GmbH, Oberkochen, Germany).
Antibiotic protection and invasion assays. Invasion of endothelial cell monolayers by P. gingivalis strains was quantified by determining the number of CFU recovered following antibiotic treatment by using an antibiotic protection assay as recently described by Deshpande et al. and Madianos et al. (10, 34). Briefly, confluent HUVEC monolayers in 6-well plates were infected at an MOI of 50 (final concentration, 5 x 107 bacteria) and incubated for 1.5 or 4 h (at 37°C in 5% CO2). After 1.5 h, unattached bacteria were removed by washing the monolayer twice with sterile PBS. Half of the monolayers were lysed with sterile H2O (30 min). Lysates were diluted and plated on Columbia agar plates containing vitamin K (0.1%) and hemin (0.25%) and incubated for 7 days under strictly anaerobic conditions. On the other half of the monolayers, external adherent cells were killed by incubating the infected monolayers with fresh medium containing gentamicin (0.5 mg/ml) and metronidazole (0.1 mg/ml). This concentration of antibiotics was sufficient to completely kill 108 bacteria per ml in 1 h (data not shown). The antibiotic did not affect the morphology or viability of the endothelial cells as detected by lactate dehydrogenase release. Controls for antibiotic killing of P. gingivalis were included in all experiments. After 4 h the remaining HUVEC were also lysed, and lysates were diluted and plated on agar plates (see above). CFU of invasive organisms were then enumerated. Invasion was expressed as the percentage of the initial inoculum recovered after antibiotic treatment and endothelial cell lysis. Following this protocol, we recovered different fractions of bacteria; the first fraction, isolated after 1.5 h, represented all bacteria of the initial inoculum that adhered to and/or invaded the endothelial cells (total, or EC bacteria plus intracellular [IC] bacteria). The second fraction (after 4 h), recovered from cells in parallel wells to which antibiotic was added, represent only those bacteria that were internalized (IC bacteria). According to the method published by Dorn et al. (11) EC P. gingivalis organisms that only adhered but did not invade endothelial cells were calculated by subtracting IC bacteria (isolated after 4 h of incubation) from the amount of bacteria we were able to isolate after 1.5 h of incubation (total, or first fraction). Control experiments confirmed the viability of P. gingivalis after exposure to water for 30 min or to HUVEC media for 1.5 h or after incubation in air for up to 5 h (data not shown). The numbers of CFU of at least five independent experiments were counted.
Cell surface ELISA. Expression of different adhesion molecules on monolayers of endothelial cells preincubated with P. gingivalis was determined by cell surface enzyme-linked immunosorbent assay (ELISA) as described previously (31). Briefly, confluent pretreated HUVEC monolayers in 96-well flat-bottom microtiter plates were washed and fixed with 2% PFA for 10 min. Human immunoglobulin (Ig) was used to reduce nonspecific binding and primary mouse antibodies were added for 25 min. Thereafter, cells were washed three times and exposed to a horseradish peroxidase-conjugated rabbit anti-mouse Ig antibody for 20 min. After the cells were washed, ABTS [2,2'-azinobis(3-ethylbenzthiazolinesulfonic acid)] (Sigma, München, Germany) was added for 10 min. Data are indicated as optical densities at 405 nm.
Western blotting.
For determination of p38 MAPK phosphorylation in HUVEC, cells were starved for 12 h in serum-free medium, stimulated as indicated, washed twice in ice-cold HEPES buffer, pH 7.4, containing 100 mM sodium fluoride, 2 mM sodium vanadate, and 15 mM sodium pyrophosphate. Cells were then harvested on ice by scraping with lysis buffer containing 1 mM EDTA, 1% Triton X-100, 1 mM phenylmethylsulfonyl fluoride, and leupeptin, pepstatin, and antipain at a concentration of 2 µg/ml each. After removal of the cell debris by centrifugation, cell lysates were subjected to sodium dodecyl sulfate-12.5% polyacrylamide gel electrophoresis and blotted on Hybond-ECL membranes (Amersham Biosciences, Freiburg, Germany). Each lane contains 80 µg of protein (ascertained by Bradford assay). Immunodetection of phosphorylated p38 MAPK was carried out with phospho-specific p38 MAPK antibodies (Cell Signaling, Frankfurt, Germany). Degradation of IB was analyzed in HUVEC lysates by using a rabbit polyclonal antibody (Santa Cruz Biotechnologies, Santa Cruz, Calif.) as described previously (22). In all experiments, ERK2 kinase (Santa Cruz Biotechnologies) was detected simultaneously to confirm equal protein loads. Proteins were visualized by incubation with secondary IRDye 800- or Cy5.5-labeled antibodies (23) by using an Odyssey infrared imaging system (LICOR Inc., Bad Homburg, Germany).
Phospho-p38 MAPK ELISA. Cell culture, stimulation, and protein extraction were performed as described above, and commercial immunoassay kits (P-p38 MAPK; BioSource Europe SA, Nivelles, Belgium) were used. Assays were carried out according to the manufacturer's instructions. Assay standards or cell lysates were incubated for 2 h at room temperature in anti-P-p38-coated 8-well flat-bottom microtiter strips and then aspirated and washed four times. Detection antibody was added to each well and incubated for 1 h. After washing, wells were exposed to horseradish peroxidase-conjugated anti-rabbit antibody for 30 min. Afterwards, stabilized chromogen was added for 30 min. Absorption was measured at 450 nm after the addition of stop solution. The results were normalized for protein concentrations.
Confocal laser scanning microscopy analysis of NF-B nuclear translocation.
After stimulation of HUVEC grown on Thermanox slides (Falcon Culture Slide; Becton Dickinson, Rutherford, N.J.) with 107 bacteria/ml (MOI, 100) for 15, 45, 90, and 120 min, cells were fixed with 3% PFA for 20 min and permeabilized with 1% Triton for 15 min. To reduce nonspecific background staining, cells were treated with 5% goat serum for 30 min in PBS before the addition of antibodies. For NF-B p65 staining, the primary antibody (polyclonal rabbit anti-human NF-B p65; Santa Cruz Biotechnology) was incubated overnight at 4°C. Bound antibodies were detected with Alexa Fluor 488-conjugated goat anti-rabbit monoclonal antibody (4°C, overnight) (Molecular Probes, Eugene, Oreg.). After each step cells were washed three times with sterile PBS. Coverslips were sealed, and cells were analyzed with the use of a Pascal 5 confocal laser scanning microscope (Zeiss, Jena, Germany).
Statistical methods. A one-way analysis of variance was used for data shown in the figures (see Fig. 2 to 5). A P value of <0.05 was considered to be significant.
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RESULTS |
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EC bacteria are bacteria that only adhered to HUVEC and were calculated by subtraction of the amount of IC bacteria from the total amount of P. gingivalis. About 34% of adhering bacteria of strain ATCC 53977 invaded endothelial cells. For strain DSMZ 20709, we demonstrated an invasion rate of 5.4% of adhering bacteria.
P. gingivalis-induced expression of E-selectin and ICAM-1 in human endothelial cells. Endothelial adhesion molecule expression was determined by cell surface ELISA. Both strains induced a time- and dose-dependent increase in the endothelial cell adhesion molecules E-selectin and ICAM-1. E-selectin expression in P. gingivalis-stimulated endothelial cells peaked at 5 h and declined to almost baseline after 12 to 24 h (Fig. 3A and B). Even concentrations as low as 103 bacteria/ml were able to induce E-selectin responses (Fig. 3A). ICAM-1 surface expression in P. gingivalis-stimulated HUVEC cells increased at 4 to 8 h postinfection, peaked at 12 h, and remained elevated up to 24 h (Fig. 3C and D) and longer (data not shown). At an MOI of 10 or 1, ICAM-1 expression was more pronounced in ATCC 53977-stimulated HUVEC than in DSMZ 20709-treated endothelial cells.
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fimA and compared it to its corresponding wild-type A7436-WT strain as well as to strains ATCC 53977 and DSMZ 20709. Cell surface analysis for ICAM-1 expression demonstrated that P. gingivalis strain A7436-WT was equipotent to strain ATCC 53977 (Fig. 4). Interestingly, strain A7436 fimA induced the same level of ICAM-1 expression as the corresponding wild-type strain (A7436-WT).
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) was used as a positive control for inducing p38 MAPK expression.
P. gingivalis-induced IB degradation and NF-B translocation.
Recent publications have demonstrated the importance of NF-B for the regulation of the transcriptional activities of the E-selectin and ICAM-1 genes (5, 7, 50). Western blot analysis with strain ATCC 53977 demonstrated a time-dependent degradation of IB, with maximal effects 2 h postinfection (Fig. 6). NF-B translocation in P. gingivalis-infected endothelial cells was also demonstrated by immunofluorescence microscopy. Both strains induced a nuclear translocation of NF-B starting at 45 (ATCC 53977) to 90 min (DSMZ 20709) postinfection. The signal remained elevated up to 120 min with both strains (Fig. 7) and decreased to almost baseline levels after 180 min (data not shown).
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DISCUSSION |
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B—that were activated after stimulation of endothelial cells with both strains. These observations add important new properties to this bacteria regarding its capacity to initiate a cascade of events leading to endothelial cell activation. The potential of different P. gingivalis strains to induce adhesion molecule expression on different target cells is somewhat controversial and has been discussed previously (9, 24). Results may vary due to the use of different strains, the growth conditions of these strains, and the target cells used in different in vitro systems (29). In our model of cell activation we used (i) primary liquid broth-cultured bacteria in the mid-log phase of growth made from frozen bacterial stocks and (ii) freshly isolated human endothelial cells. The approach with primary isolated endothelial cells seemed especially important since different studies have demonstrated that these cells lose several of their properties during subculture (2, 27).
Several groups have recently described different virulence capacities of selected P. gingivalis strains (36, 43). Since these strains have not been characterized in detail, however, it still remains unclear which mechanisms are responsible for the variability in infection. Diverse rates of bacterial adherence and target cell infection, expression of different virulence factors, and various levels of host cell sensitivity may influence the severity of an infection with P. gingivalis (15, 29).
It has been established that the invasion efficiencies of different P. gingivalis strains usually are very low, ranging from 0.04 to 0.3% (10). In our model both strains were able to invade endothelial cells but with striking differences relative to infectivity. While strain ATCC 53977 infected HUVEC at a rate recently described for many other P. gingivalis strains (10), we could demonstrate only a very low invasion efficiency of 0.007% for strain DSMZ 20709. However, these data were consistent with studies published by Eick et al. and Deshpande et al. (10, 15). Interestingly, both strains were able to activate endothelial signal transduction pathways (phosphorylation of p38 MAPK and translocation of NF-B) and to induce the expression of adhesion molecules in a similar manner. Although there were some differences in the intensity of the cellular responses (early decrease of p38 MAPK phosphorylation, delayed translocation of NF-B, and reduced expression of E-selectin and ICAM-1 with strain DSMZ 20709), the overall effects were the same with both strains, indicating that infectivity is an important virulence factor but not the only one. In our in vitro model with isolated endothelial cells, both strains (even the less invasive strain DSMZ 20709), therefore, turned out to be equally virulent, inducing similar levels of target cell activation. These results are in contrast to the in vivo study of Ebersole et al., which used a mouse abscess model and analyzed lesion size and lethality to identify several more or less virulent strains. In their model, strain ATCC 53977 appeared to be the most virulent (14). Little is known about the expression of different virulence factors in strains DSMZ 20709 and ATCC 53977, especially in regard to factors mediating adhesion or the invasion of target cells. Several studies have suggested that fimbriae, proteases, or ceramides are key factors during the process of target cell infection and activation (29, 39, 51). In our model of target cell activation, expression of fimbriae (FimA) appeared not to be required for P. gingivalis-mediated ICAM-1 expression; the fimA knockout strain A7436 fimA induced the same level of ICAM-1 expression as the corresponding wild type (A7436-WT). These results appear to be in contrast to the study by Khglatian et al. (29), but this might be due to the different mutants applied in both studies. The mutant used by Khglatian (DPG3) is apparently pleiotropic, since it also is affected in expression of the proteases arginine-specific gingipain, Rgp, and lysine-specific-gingipain Kgp as well as some adhesins (6). In contrast, the A7436 fimA mutant expresses normal levels of the proteases (J. Hayashi and H. K. Kuramitsu, unpublished results).
Different groups have identified the importance of p38 MAPK in the signal transduction of P. gingivalis-stimulated monocytes (8). Ogawa et al. demonstrated that fimbriae extracted from P. gingivalis were able to stimulate p38 MAPK phosphorylation in human monocytes (41). These data were supported by Darveau et al., who used P. gingivalis LPS with human monocytes. However, in that study, LPS failed to induce phosphorylation of p38 MAPK in endothelial cells (8). By using viable bacteria in our model, we were able to demonstrate by Western blotting and ELISA that ATCC 53977 as well as DSMZ 20709 were able to stimulate the phosphorylation of p38 MAPK.
The expression of inflammatory mediators like cytokines or adhesion molecules depends on the activation of transcription factors. Among the primary transcription factors, NF-B plays a central role in the regulation of these proinflammatory molecules (5, 50). While Watanabe et al. failed to measure a NF-B activation in gingival epithelial cells with strain ATCC 33277 (52), we could clearly demonstrate that both strains DSMZ 20709 and ATCC 53977 were able to activate degradation of IB and subsequent nuclear translocation of NF-B in endothelial cells.
The interpretation of our study is limited to cultured human large vessel endothelial cells. For an exact analysis of P. gingivalis-related alterations of endothelial function in clinical disorders, it would be desirable to also study human small vessel endothelial cells especially of the periodontium. However, the isolation and culture of these cells in sufficient quantities are difficult, and therefore the applicability of the data presented to human disease must be verified in further studies.
In conclusion, we present evidence that two different strains of P. gingivalis, ATCC 53977 and DSMZ 20709, can infect HUVEC and activate different signal transduction pathways; p38 MAPK phosphorylation, degradation of IB, and activation and translocation of NF-B occurred within minutes. Within hours, increased surface expression of E-selectin and ICAM-1 was observed. This effect was apparently independent of fimA expression in at least strain A7436. Taken together, the data presented suggest that P. gingivalis infection triggers a cascade of events that could lead to endothelial damage as well as local and systemic inflammation.
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ACKNOWLEDGMENTS |
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We gratefully acknowledge the excellent technical assistance of Kerstin Möhr and Verena Kanitz.
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FOOTNOTES |
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