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,
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Departments of
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Oral Biology,
Periodontics,
Biochemistry, and
Medicine/Dermatology, University of Washington, Seattle, WA 98195
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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-defensin (hBD) family are part of the innate immune responses that play a role in mucosal defense. hBDs are made in epithelia including oral epithelium where the bacterial load is particularly great. hBD-2 and hBD-3 are up-regulated in response to bacterial stimuli. Previous studies show that hBD-2 expression in human gingival epithelial cells (GEC) is stimulated by both nonpathogenic and pathogenic bacteria, including Porphyromonas gingivalis, a Gram-negative pathogen associated with periodontitis. Present evidence suggests that hBD-2 expression in GEC uses several signaling pathways, including an NF-
B-mediated pathway but without apparent LPS-TLR4 signaling. Protease-activated receptors (PAR) are G-protein-coupled receptors that mediate cellular responses to extracellular proteinases. P. gingivalis secretes multiple proteases that contribute to its virulence mechanisms. To determine whether PAR signaling is used in hBD-2 induction, GEC were stimulated with wild-type P. gingivalis or mutants lacking one or more proteases. hBD-2 mRNA expression was reduced in GEC stimulated with single protease mutants (11–67% compared with wild type), strongly reduced in double mutants (0.1–16%), and restored to wild-type levels (93%) in mutant with restored protease activity. Stimulation by wild type was partially blocked by inhibitors of phospholipase C, a main signaling pathway for PARs. Expression of hBD-3 was unaffected. Peptide agonist of PAR-2, but not PAR-1 activator, also induced hBD-2 in GEC. Thus, P. gingivalis proteases are directly involved in regulation of hBD-2 in cultured GEC, and this induction partially uses the PAR-2 receptor and signaling pathway. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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- and -defensins and a cathelicidin family member, LL-37 (4, 5, 6). The -defensins were first identified in bovine tracheal epithelial cells and subsequently found in many human epithelia including kidney and urinary tract, oral mucosa, and skin (7, 8, 9, 10). Human -defensin-1 (hBD-1)3 is expressed constitutively in epithelial tissues, whereas hBD-2 and hBD-3 are expressed when epithelia are stimulated with both commensal and pathogenic bacteria, Candida albicans, IL-1, or TNF- (11, 12, 13, 14). Although hBD-2 is induced and expressed only in inflamed sites in most tissues, in the oral epithelium it is expressed in normal uninflamed gingival tissue as well, presumably because of the high level of exposure of this tissue to commensal organisms (15). Our earlier studies suggested that the regulation of expression of hBD-2 in oral and skin keratinocytes by commensal bacteria involved MAPK and calcium (Ca2+)-mediated signaling pathways, whereas regulation by pathogenic bacteria involved both MAPK and NF-B signaling pathways (16, 17, 18). However, TLR, in particular TLR-4, are thought not to be involved in hBD-2 up-regulation in gingival epithelial cells (GEC), because LPS from several Gram-negative organisms was a poor stimulant of hBD-2 in cultured GEC even when human serum was added as a source of LPS-binding protein or soluble CD14 (13). Protease-activated receptors (PARs) are seven-transmembrane domain, G-protein-coupled receptors that mediate cellular responses to extracellular proteinases (19). They are activated by proteolytic cleavage of N-terminal domain, generating a new N-terminal receptor that serves as a tethered ligand that binds to one of the extracellular loops of the receptor (19, 20). PARs are widely expressed, and there have been four PARs identified so far. PAR-1 is expressed by platelets, fibroblasts, endothelial cells, and neurons, whereas PAR-2 is expressed by epithelial cells, endothelial cells, smooth muscle cells, T cells, neutrophils, and neurons (21, 22, 23, 24). PAR-1, -3, and -4 are activated by thrombin, and PAR-2 is activated by trypsin and a number of trypsin-like serine proteases, including Porphyromonas gingivalis proteases (19, 25).
P. gingivalis is a Gram-negative bacteria strongly associated with severe adult periodontitis. Part of the notable virulence factors of P. gingivalis are fimbriae and proteases. The fimA gene encodes fimbriae used in the adherence of P. gingivalis to human gingival fibroblasts and epithelial cells, thus playing a part in the inflammatory response of GEC (26). P. gingivalis proteases, arginine- and lysine-specific gingipains (RgpA and RgpB, and Kgp, respectively), are known to be involved in the degradation of the adherens junctions of epithelial cells, thus a possible GEC invasion mechanism (27). This enzymatic activity may confer P. gingivalis its unique additional mechanisms of interaction with GEC. We reported earlier that P. gingivalis crude cell wall extract, treated with protease inhibitors, did not induce hBD-2 in cultured GEC (13). We also observed that, whereas P. gingivalis cells washed in PBS failed to induce hBD-2 in GEC, whole-cell P. gingivalis and the cell-free supernatant of P. gingivalis culture induced hBD-2 expression, suggesting a possible role of P. gingivalis-secreted protease in hBD-2 induction (our unpublished observations). Lourbakos et al. (25) showed that one or more of the P. gingivalis gingipains stimulated PARs in gingival tissues as part of the proinflammatory response. Therefore, it was of interest for us to examine the role of P. gingivalis proteases in hBD-2 induction and additional signaling mechanisms involved in the induction. The goal of this study was to determine the role of P. gingivalis proteases in the hBD-2 induction and to test whether PARs are used in P. gingivalis-induced hBD-2 expression in cultured GEC. We used various P. gingivalis protease mutants, which were previously characterized to show decreased protease activities and decreased virulence (28, 29, 30), to examine the role of proteases in the epithelial cell response to P. gingivalis. We show evidence that the P. gingivalis proteases play a crucial role in hBD-2 induction and that part of the pathway used by P. gingivalis proteases in hBD-2 induction is via PAR-2.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Healthy gingival samples were obtained from subjects undergoing third-molar extraction at Department of Oral Surgery, School of Dentistry, University of Washington. Tissue was cut into small pieces (2 x 2 mm) and treated with 6 mg/ml Dispase (BD Biosciences, Franklin Lakes, NJ) overnight at 4°C to separate the epithelium from the underlying fibrous connective tissue. The epithelium readily lifted off and was incubated at 37°C in 5 ml of trypsin-EDTA (0.05% trypsin, 0.53 mM EDTA) for 10 min. Subsequently, isolated GEC were grown to 80% confluence in keratinocyte basal medium supplemented with keratinocyte growth medium bullet kit (Cambrex, Walkersville, MD). P. gingivalis mutant strains were generous gifts from various sources. The mutant strains used in this study, their genotypes, and their sources are listed in Table I. P. gingivalis wild-type and mutant strains were cultured in anaerobic conditions (85% N2, 10% H2, 5% CO2) at 37°C in trypticase soy broth (BBL, Sparks, MD) supplemented with 1 g of yeast extract, 5 mg of hemin, and 1 mg of menadione per liter. Appropriate antibiotics previously described were added to each culture of mutant P. gingivalis strains (28, 29, 30). Bacterial numbers were determined by density in a GENios Multidetection Reader (Phenix, Hayward, CA).
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Total RNA was extracted from keratinocytes using the RNeasy Mini kit (Qiagen, Valencia, CA) according to the manufacturer’s suggestion. Reverse transcription was performed with 1 µg of total RNA, 1x reverse transcriptase (RT) buffer, 250 nM oligo(dT) primer, 10 mM dNTP mix, 50 U of RT, and 13 U of RNase inhibitor. Initial denaturation of secondary RNA structure was conducted at 72°C for 2 min, followed by annealing of the primer and template at 42°C for 1 h. The temperature was subsequently raised to 95°C for 10 min to inactivate RT. Controls without RT were included in each experiment. Amplification of the resulting cDNA was conducted with each 50 µl of PCR mixture containing 3 µl of cDNA, 1x PCR buffer, 1.5 mM MgCl2, 10 mM dNTP mix, 250 nM each of sense and antisense primers, and 2.5 U of TaqDNA polymerase. The housekeeping gene ribosomal phosphoprotein (RPO) was used as a control to determine the total RNA level. The PCR conditions were denaturation at 94°C for 30 s, annealing at 57°C for 30 s, and elongation at 72°C for 2 min for 35 cycles for hBD-2, and 25 cycles for RPO. The PCR conditions for IL-8 were denaturation at 94°C for 45 s, annealing at 60°C for 1 min, and elongation at 72°C for 2 min for 28 cycles. The PCR conditions for hBD-3 were denaturation at 94°C for 45 s, annealing at 62°C for 45 s, and elongation at 72°C for 45 s for 35 cycles. The oligonucleotides for hBD-2, hBD-3, IL-8, and RPO have been described previously (8, 31).
Conditions for real-time PCR
The resulting cDNA was analyzed using the iCycler (Bio-Rad, Hercules, CA) and Brilliant SYBR Green PCR kit (Stratagene, La Jolla, CA) according to the manufacturer’s suggestion. SYBR Green mix contained 100 mM KCl, 40 mM Tris-HCl (pH 8.4), 0.4 mM each dNTP, 50 U/ml iTaq DNA polymerase, 6 mM MgCl2, SYBR Green I, and 20 nM fluorescein. The reaction was set up in a 96-well plate, with each well containing 25 µl of SYBR Green mix, 5 µl of cDNA, and 250 nM primers. The amplification conditions were initial denaturation at 95°C for 15 min followed by 40 cycles of denaturation at 95°C for 15 s, annealing at 57°C for 15 s, and elongation at 72°C for 30 s. Melt-curve analysis was performed to confirm that the detected signal is that of SYBR Green binding to the expected amplification product and not to the possible primer-dimers. The amplified product was run on an agarose gel to confirm that there were no spurious products amplified during the cycles. Real-time PCR amplification was performed in duplicate, and an average was calculated. An amplification plot from a series of dilutions of hBD-2 or RPO plasmids was used to obtain a linear correlation between threshold cycle (Ct), at which fluorescence is determined to be statistically significant above background, and the log amount of template present. The starting quantity of the samples was normalized against the housekeeping control gene named RPO (16).
PAR activators and inhibitors used
Thrombin (Calbiochem, San Diego, CA) was selected as the PAR-1 activator, and a peptide agonist, SLIGRL-NH2, was custom synthesized (Tocris Cookson, Ellesville, MO) as the PAR-2 activator. Scrambled peptide LRGILS-NH2 (Tocris Cookson) was used as a control. Thrombin was tested at 100, 200, and 400 nM for induction of hBD-2. PAR-2 peptide agonist and control peptide were tested at 5, 10, and 20 µM for hBD-2 induction. The inhibitor of phospholipase C (PLC) (U73122; Calbiochem), a main signaling pathway for G-protein-coupled receptors, was tested at 100 and 400 nM, and a mock inhibitor (U73343; Calbiochem) was used as a control. Both inhibitors did not induce hBD-2 in GEC at the concentrations tested.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Various P. gingivalis mutant strains lacking one or a combination of proteases and the corresponding wild-type strain were tested for their ability to induce hBD-2 in cultured GEC. hBD-2 mRNA expression levels were reduced for all GEC stimulated with P. gingivalis protease mutants, compared with GEC stimulated with corresponding wild type (Fig. 1). The arginine-specific single protease mutant, V2546 (rgpA–), gave a reduced hBD-2 expression level compared with lysine-specific gingipain mutants V2543 and V2577 (kgp–) (Fig. 1A). This result indicates that among the individual proteases, an arginine-specific gingipain may be a more potent inducer of hBD-2 than lysine-specific gingipain. In addition, the mutant lacking both rgpA and kgp (V2383) showed an even more reduced hBD-2 expression level than the mutants lacking either rgpA (V2546) or kgp (V2543), suggesting a possible synergistic effect between arginine-specific and lysine-specific proteases in hBD-2 induction (Fig. 1A). Between the two Kgp-deficient mutant strains, a mutant constructed using allelic exchange (V2577) gave a slightly stronger hBD-2 induction level than the naturally occurring variant (V2543) (Fig. 1A). Each mutant was tested at least three times, using GEC from three different subjects, and the results were consistent.
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To test additional markers for innate immunity in response to various protease mutant strains, the expression of hBD-3 and IL-8 was examined. Unlike hBD-2, hBD-3 expression is often observed in unstimulated epithelial cells, but like hBD-2, it can be induced. IL-8 is a chemokine that directs polymorphonuclear neutrophil migration to the site of infection and a major factor in innate immune responses. Fig. 1A shows that the expression of hBD-3 was consistently present in all GEC tested, including unstimulated cells. The IL-8 expression was strongly induced by wild-type P. gingivalis 33277, and decreased in the mutants, with the mutants KDP111 (rgpB–) and KDP112 (rgpA–/rgpB–) showing little IL-8 induction (Fig. 1A). IL-8 expression was also reduced in the GEC stimulated with V2383 (rgpA–/kgp–), compared with the GEC stimulated with other P. gingivalis mutants corresponding to the wild-type W83 (Fig. 1A). This parallels the reduced hBD-2 induction seen in the same cell line (Fig. 1A).
Quantitative analysis of hBD-2 expression by real-time PCR
To confirm the RT-PCR data with quantitative analysis, real-time PCR was performed. End-point values obtained by RT-PCR are only semiquantitative, because they can be influenced by limiting reagent, small differences in reaction components, or cycling parameters. In the real-time PCR, data are taken during the exponential phase of PCR amplification; thus, factors like limiting reagents do not influence the results, providing a more reliable quantitative analysis. The samples were run in duplicate, and the mean starting quantity of hBD-2 in nanograms was normalized against the housekeeping gene control RPO. GEC from at least two different subjects were tested for real-time PCR analysis. The real-time PCR data show significant reduction of hBD-2 induction by P. gingivalis protease mutants compared with the corresponding wild type, in accordance with the RT-PCR data (Fig. 1, B and C). Compared with the hBD-2 expression level induced by the wild-type strain 33277, the mutants showed the following expression levels: KDP112 (rgpA–/rgpB–), 0.12%; KDP111 (rgpB–), 11.14%; and unstimulated, 0.64%. Similar to the results obtained with RT-PCR, the mutant lacking two proteases (KDP112) showed the lowest hBD-2 mRNA starting quantity (Fig. 1B). This confirms that mutants lacking two proteases show significantly reduced hBD-2 induction levels when compared with single protease mutants. Real-time PCR data also confirmed the arginine-specific gingipain, rgpA–, is more influential in inducing hBD-2 than the lysine-specific gingipain, kgp–. Compared with the wild-type strain W83, the mutant strains showed the following hBD-2 expression levels: V2383 (rgpA–/kgp), 16.3%; V2546 (rgpA–), 21.9%; V2543 (kgp–), 37.9%; V2577 (kgp–), 67%; and unstimulated, 0.15%. These results also support the RT-PCR data (Fig. 1A) by showing single protease mutants induce more hBD-2 expression level than a double protease mutant. The level of hBD-2 induced by P. gingivalis wild-type W83 was similar to the level induced by TNF-, a known inducer of hBD-2, whereas the induction by IL-1, a keratinocyte stimulus, was slightly less (Fig. 1C). V2602, a P. gingivalis strain with kgp gene restored into V2543, induced hBD-2 at similar level (93%) as the wild-type W83, and the difference was not statistically significant (Fig. 1C). Our data suggest that the P. gingivalis proteases play a crucial role in the hBD-2 induction.
hBD-2 induction by P. gingivalis fimA– mutant strain
It has been previously reported that rgpA– mutants showed morphologically different fimbriae (30, 32). Because rgpA– mutant strains showed reduced ability to induce hBD-2, we also included a fimA– mutant strain to test for its ability to induce hBD-2 expression. The fimA– mutant (DPG3) induced an hBD-2 expression level similar to the level observed when the GEC were stimulated with the corresponding wild type (Fig. 2). This indicates that P. gingivalis fimbriae may not be involved in hBD-2 induction in GEC.
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To determine the involvement of PAR signaling in the stimulation of hBD-2 expression, GEC were stimulated with various concentrations of PAR-1 activator thrombin, or with PAR-2 activator peptide agonist SLIGRL-NH2 for various time points. Thrombin was a relatively poor stimulant of hBD-2 between 100 and 400 nM (Fig. 3A). No hBD-2 induction was seen in GEC stimulated with thrombin at 6, 24, and 48 h poststimulation (data not shown). However, PAR-2 peptide agonist was an excellent stimulant of hBD-2. hBD-2 expression was detectable after overnight stimulation with SLIGRL-NH2 at 5 µM, and strong hBD-2 expression was observed at the concentrations of 10 and 20 µM (Fig. 3B). GEC stimulated with 10 µM SLIGRL-NH2 showed slight hBD-2 expression at 6 h; the expression peaked at 24 h, and then decreased at 48 h (data not shown). The scrambled peptide LRGILS-NH2 was used as a control, and no hBD-2 induction was observed (Fig. 3B). The level of IL-8 was increased in all GEC stimulated with thrombin or peptide agonist SLIGRL-NH2, confirming that the GEC responded to the stimulation with thrombin or peptide agonist, but that the hBD-2 response was mediated by PAR-2 (data not shown).
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When GEC were treated with an inhibitor of PLC, a main signaling pathway for G-protein-coupled receptors, 1 h before the exposure to PAR activators, partial decrease in hBD-2 expression was observed (Fig. 4). At the inhibitor concentration of 400 nM, the cells treated with PLC inhibitor (U73122) showed decreased hBD-2 expression, whereas the cells treated with mock inhibitor (U73343) showed the same hBD-2 induction level as the control (Fig. 4). However, GEC treated with 100 nM of U73122 or of U73343 showed little decrease in hBD-2 induction when subsequently stimulated with PAR-2 peptide agonist (Fig. 4). The involvement of PAR-2 in hBD-2 stimulation was further analyzed by specific inhibitors of PLC in combination with P. gingivalis. When GEC were treated with 400 nM U73122 1 h before the stimulation with P. gingivalis wild-type strain American Type Culture Collection (ATCC) 33277 for 18 h, partial decrease in hBD-2 expression was observed (Fig. 4). On the contrary, the cells treated with 400 nM U73343, the mock inhibitor, and subsequently stimulated with wild-type P. gingivalis, showed no decrease in hBD-2 induction level (Fig. 4). Real-time PCR analysis in Fig. 4B shows the decreased level of hBD-2 induction when the cells were treated with 400 nM U73122 before stimulation. Therefore, the data suggest that one of the pathways used by P. gingivalis in inducing hBD-2 is via PAR-2 signaling pathway.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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B signaling pathways in hBD-2 induction (16, 17). Ca2+ flux is also involved in hBD-2 up-regulation in GEC, and Ca2+-dependent host signaling system is thought to be involved in P. gingivalis invasion of GEC (18, 35). But cell surface receptors responsible for GEC recognition of bacterial signals stimulating hBD-2 have not been identified. PARs are G-protein-coupled transmembrane receptors that are implicated in inflammatory responses and are expressed on many cell types, including GEC (25, 36). Each of the three main Gram-negative periopathogens, P. gingivalis, Tannerella forsythensis (formerly Bacteroides forsythus), and Treponema denticola, produces proteases as virulence factors (37, 38, 39). However, a specific pathway or a combination of pathways that proteases may take in inducing an innate immune response is not clear at this time. Previous research from our laboratory showed that whole-cell P. gingivalis and cell-free supernatant from P. gingivalis induced hBD-2 in GEC, whereas washed P. gingivalis did not induce hBD-2 (Ref.40 and unpublished data). This led to the hypothesis that components P. gingivalis secreted into the medium may be responsible for hBD-2 induction in cultured GEC. Our present study confirms that P. gingivalis proteases play a critical role in the regulation of hBD-2 in GEC, and that this regulation is in part done through PAR-2 signaling. Our data is in agreement with an earlier study that suggests P. gingivalis proteases may stimulate PARs as part of the proinflammatory action on the periodontal tissues (25). This PAR mechanism is also consistent with the importance of intracellular Ca2+ flux, an effect of this G-protein-coupled mechanism, in hBD-2 induction (20).
Our results with protease mutants indicate that the type of protease influences the degree of hBD-2 expression in GEC. The arginine-specific gingipains, RgpA and RgpB, are more potent inducers of hBD-2 than the lysine-specific gingipain, Kgp. The specific activating cleavage site of human PAR-2 may explain this preference. The potential sites of cleavage within the human PAR-2 include arginine and lysine, with the cleavage at the arginine site leading to tethered ligand domain and receptor activation (41). Thus, the arginine-specific P. gingivalis protease may cleave and subsequently activate PAR-2, leading to hBD-2 expression in GEC. Mutants lacking two proteases, rgpA–/rgpB– and rgpA/kgp–, showed the least hBD-2 expression when compared with single protease mutants (Fig. 1). The reason for this observation may be related to both the type of proteases and the amount of protease secreted. The rgpB– mutant strain (KDP111) has 52% reduction in Rgp activity, whereas the amount of Kgp activity is the same as that of the wild-type strain 33277 (30). Yet this strain is a poor inducer of hBD-2. Similarly, rgpA–/B– mutant strain KDP112 showed no Rgp activity and reduced Kgp activity (54%) (30), and yielded essentially no hBD-2 induction.
Arginine-specific protease genes rgpA and rgpB are highly homologous and differ only in a small segment of adhesion domain in the C-terminal sequence (30). Previous studies showed that rgpA– mutants, but not rgpB– mutants, had morphologically different fimbriae (30, 32). Because fimbriae is one of the virulence factors of P. gingivalis and because arginine-specific protease activity was shown to modulate adhesion in P. gingivalis (30), we compared the level of hBD-2 induction between fimA– mutant and rgpA– mutants. In contrast to the mutant strains lacking rgpA, the fimA– mutant strain did not show any decreased ability to induce hBD-2, suggesting that fimbriae-associated adhesion is not directly involved in hBD-2 expression in GEC. Earlier studies reported P. gingivalis fimbriae used TLR-2 in IL-6 and IL-8 induction in human monocytes and GEC, respectively (42, 43). Our work suggests that hBD-2 induction by P. gingivalis in GEC is mediated via PAR-2, but also suggests there may be additional pathways, including pattern recognition receptors, involved in this process (16). A recent study reports the presence of a PAR-like receptor on the hemocyte surface of the horseshoe crab and suggests its role as a pattern recognition receptor for LPS in innate immunity for this organism (44). We are currently investigating the involvement of other cell surface receptors in hBD-2 induction.
The regulation of hBD-3 expression differs from that of hBD-2. hBD-3 mRNA expression was present in both unstimulated and stimulated cells in our study and was not responsive to either P. gingivalis or PAR activators. hBD-3 transcripts are widely expressed in noninflamed as well as inflamed oral tissues, suggesting an important role of hBD-3 as well as hBD-2 in innate host defense in the oral cavity (45). However, the two inducible -defensins (hBD-2 and hBD-3) may be regulated differently in the innate immune response to pathogens and to commensals.
Regulation of IL-8 mRNA expression in GEC closely paralleled the findings with hBD-2, both with respect to P. gingivalis strains and the PAR-2 peptide agonist. IL-8 was also up-regulated by thrombin, the PAR-1 activator. These results suggest that both of these markers of innate immune responses can be triggered by PAR-2. There are differing previous reports on the effects of P. gingivalis on IL-8 expression by GEC at the mRNA and protein levels, ranging from inhibition of IL-8 accumulation in GEC by P. gingivalis 33277 (46, 47), to up-regulation (48, 49). There is also evidence for intracellular accumulation of IL-8 protein in GEC incubated with P. gingivalis (Ref.50 , and R. P. Darveau, unpublished observations). The reason for this discrepancy is not understood. The IL-8 mRNA may be expressed in the presence of P. gingivalis, although the protein may be degraded after secretion. Alternatively, difference in detection procedures, sampling preparations, variation in the number of bacteria used, or the multiplicity of infection (MOI) relative to the epithelial cells may all contribute to results that vary between investigators. We used an MOI of 100:1, considerably lower than the MOI of 500:1 or 1000:1 used by others (46, 49). Use of washed P. gingivalis by these investigators would also be expected to give very different results, because the proteases that stimulate via PAR-2 are secreted into the bacterial medium.
We have identified PAR-2 as a receptor used by P. gingivalis proteases in hBD-2 induction in GEC and have also demonstrated hBD-2 induction by PAR-2 peptide agonist. We project that the further investigations in the role of PARs in GEC in response to pathogenic bacteria such as P. gingivalis and others will open new avenues in the investigation of signaling pathways for hBD-2 as a component of innate immunity and may lead to development of better preventive therapies for mucosal infection.
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Acknowledgments |
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Footnotes |
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1 This work was funded by National Institute of Dental and Craniofacial Research Grants R01 DE 013573, R21 DE 0159972, and P60 DE 013061. 
2 Address correspondence and reprint requests to Dr. Whasun O. Chung, Department of Oral Biology, School of Dentistry, University of Washington, Box 357132, Seattle, WA 98195-7132. E-mail address: sochung{at}u.washington.edu
3 Abbreviations used in this paper: hBD, human -defensin; GEC, gingival epithelial cell; PAR, protease-activated receptor; RT, reverse transcriptase; RPO, ribosomal phosphoprotein; PLC, phospholipase C; MOI, multiplicity of infection.
Received for publication April 2, 2004. Accepted for publication August 17, 2004.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-defensin 1 in cultured gingival epithelial cells and gingival tissue. Infect. Immun. 66:4222.-defensin-1: an antimicrobial peptide of urogenital tissues. J. Clin. Invest. 101:1633.[Medline]
-defensin hBD-1 in human secretory glands and epithelial cells. FEBS Lett. 396:319.[Medline]
-defensin antimicrobial peptides by the oral mucosa and salivary glands. Infect. Immun. 67:2740.-defensins hBD-1 and hBD-2 in intestinal epithelium. J. Immunol. 163:6718.-defensin 2 by Fusobacterium nucleatum in oral epithelial cells: multiple signaling pathways and role of commensal bacteria in innate immunity and the epithelial barrier. Infect. Immun. 68:2907., and IL-1, but not IL-6, induce human -defensin-2 in respiratory epithelia. Am. J. Respir. Cell Mol. Biol. 22:714.-defensin-2 in gingival epithelial cells: the involvement of mitogen-activated protein kinase pathways, but not the NF-B transcription factor family. J. Immunol. 168:316.-defensin-2 expression in oral epithelial cells. J. Dent. Res. 82:877.-defensin-3, a novel human inducible peptide antibiotic. J. Biol. Chem. 276:5707.-defensin gene expression in tracheal epithelial cells. Infect. Immun. 68:113.-defensin-3 is widely expressed in oral tissues. Eur. J. Oral Sci. 110:121.[Medline]
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K. Ouhara, H. Komatsuzawa, H. Shiba, Y. Uchida, T. Kawai, K. Sayama, K. Hashimoto, M. A. Taubman, H. Kurihara, and M. Sugai Actinobacillus actinomycetemcomitans Outer Membrane Protein 100 Triggers Innate Immunity and Production of {beta}-Defensin and the 18-Kilodalton Cationic Antimicrobial Protein through the Fibronectin-Integrin Pathway in Human Gingival Epithelial Cells Infect. Immun., September 1, 2006; 74(9): 5211 - 5220. [Abstract] [Full Text] [PDF]
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E. Andrian, D. Grenier, and M. Rouabhia Porphyromonas gingivalis-Epithelial Cell Interactions in Periodontitis Journal of Dental Research, May 1, 2006; 85(5): 392 - 403. [Abstract] [Full Text] [PDF]
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 M. Holzhausen, L. C. Spolidorio, R. P. Ellen, M.-C. Jobin, M. Steinhoff, P. Andrade-Gordon, and N. Vergnolle Protease-Activated Receptor-2 Activation: A Major Role in the Pathogenesis of Porphyromonas gingivalis Infection Am. J. Pathol., April 1, 2006; 168(4): 1189 - 1199. [Abstract] [Full Text] [PDF]
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Y.-T.A. Teng Protective and Destructive Immunity in the Periodontium: Part 1--Innate and Humoral Immunity and the Periodontium Journal of Dental Research, March 1, 2006; 85(3): 198 - 208. [Abstract] [Full Text] [PDF]
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 G. R. Martin and J. L. Wallace Gastrointestinal Inflammation: A Central Component of Mucosal Defense and Repair Exp Biol Med, February 1, 2006; 231(2): 130 - 137. [Abstract] [Full Text] [PDF]
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