Cytokine responses of human gingival fibroblasts to Actinobacillus actinomycetemcomitans cytolethal distending toxin

Cytokine responses of human gingival fibroblasts to Actinobacillus actinomycetemcomitans cytolethal distending toxin

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www.elsevier.com/locate/issn/10434666 Cytokine 30 (2005) 56e63

Cytokine responses of human gingival fibroblasts to Actinobacillus actinomycetemcomitans cytolethal distending toxin G.N. Belibasakisa,b,*, A. Johanssonc, Y. Wangd, C. Chend, T. Lagerga˚rde, S. Kalfasf, U.H. Lernerb a

Division of Oral Microbiology, Department of Odontology, S-901 87 Umea˚ University, Umea˚, Sweden Division of Oral Cell Biology, Department of Odontology, S-901 87 Umea˚ University, Umea˚, Sweden c Division of Periodontology, Department of Odontology, S-901 87 Umea˚ University, Umea˚, Sweden d Division of Primary Oral Health Care, University of Southern California School of Dentistry, Los Angeles, CA, USA e Department of Medical Microbiology and Immunology, University of Go¨teborg, Go¨teborg, Sweden f School of Dentistry, Aristotle University of Thessaloniki, Thessaloniki, Greece b

Received 25 October 2004; received in revised form 26 November 2004; accepted 29 November 2004

Abstract Actinobacillus actinomycetemcomitans is implicated in the pathogenesis of localized aggressive periodontitis, and has the capacity to express a cytolethal distending toxin (Cdt). Gingival fibroblasts (GF) are resident cells of the periodontium, which can express several osteolytic cytokines. The aims of this study were a) to investigate the role of Cdt in A. actinomycetemcomitans-induced expression of osteolytic cytokines and their cognate receptors in GF and b) to determine if the previously demonstrated induction of receptor activator of NFkB ligand (RANKL) by A. actinomycetemcomitans is mediated by these pro-inflammatory cytokines or by prostaglandin E2 (PGE2). A. actinomycetemcomitans clearly induced interleukin (IL)-6, IL-1b, and to a minimal extent, tumor necrosis factor (TNF)-a mRNA expression. At the protein level, IL-6 but not IL-1b or TNF-a expression was stimulated. The mRNA expression of the different receptor subtypes recognizing IL-6, IL-1b and TNF-a was not affected. A cdt-knockout strain of A. actinomycetemcomitans had similar effects on cytokine and cytokine receptor mRNA expression, compared to its parental wildtype strain. Purified Cdt stimulated IL-6, but not IL-1b or TNF-a protein biosynthesis. Antibodies neutralizing IL-6, IL-1 or TNF-a, and the PGE2 synthesis inhibitor indomethacin, did not affect A. actinomycetemcomitans-induced RANKL expression. In conclusion, a) A. actinomycetemcomitans induces IL-6 production in GF by a mechanism largely independent of its Cdt and b) A. actinomycetemcomitans-induced RANKL expression in GF occurs independently of IL-1, IL-6, TNF-a, or PGE2. Ó 2005 Elsevier Ltd. All rights reserved. Keywords: Actinobacillus actinomycetemcomitans; Cytokines; Cytolethal distending toxin; Gingival fibroblasts

1. Introduction Periodontitis is an inflammatory disease of infectious aetiology that leads to the destruction of tooth supporting tissues. The Gram-negative facultative * Corresponding author. Tel.: C46 90 785 6147; fax: C46 90 785 6129. E-mail address: [email protected] (G.N. Belibasakis). 1043-4666/$ - see front matter Ó 2005 Elsevier Ltd. All rights reserved. doi:10.1016/j.cyto.2004.11.008

anaerobe A. actinomycetemcomitans is associated with localized aggressive periodontitis, a form of periodontal disease occurring in young individuals and characterized by the rapid and severe loss of alveolar bone and connective tissue attachment. Bone loss is the end point of cellular mechanisms triggered to form and activate osteoclasts, the bone resorbing cells. Osteoclasts formation in inflammatory processes, such as marginal or apical periodontitis and rheumatoid arthritis, is suggested to occur via the enhanced expression of

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osteoclast-stimulating pro-inflammatory cytokines [1,2], including interleukin (IL) -1, IL-6 and tumor necrosis factor (TNF)-a, as well as prostanoids, including prostaglandin E2 (PGE2). Resident cells of the gingival connective tissue, namely the gingival fibroblasts (GF), together with infiltrating leukocytes, are considered a major source of these cytokines in periodontal disease [3]. Various A. actinomycetemcomitans components have been shown to stimulate the expression of cytokines and prostanoids by GF [4e14]. Interleukin-1, IL-6, TNF-a and PGE2 are all known to regulate the expression of receptor activator of NF-kB ligand (RANKL) and osteoprotegerin (OPG) in stromal cells and osteoblasts [15]. RANKL, a member of the TNF ligand superfamily, is expressed as a surfaceassociated ligand on osteoblasts in periosteal tissues, and stromal cells in haematopoetic tissues [16], as well as on T-lymphocytes [2,17]. It binds to its cognate receptor RANK on osteoclast progenitor cells [18], leading to their differentiation into multinucleated bone-resorbing osteoclasts [19,20]. This cellular interaction can be inhibited by OPG, a decoy receptor with homology to RANK, which is released by stromal cells and osteoblasts [21]. Previous reports have demonstrated a direct, or cytokine-mediated induction of RANKL in various host cells by lipopolysaccharide (LPS) from Treponema denticola, A. actinomycetemcomitans and Escherichia coli [22e25], by the gingipains of Porphyromonas gingivalis [26] and by the cytolethal distending toxin (Cdt) of A. actinomycetemcomitans [27]. The Cdt is the most recently described protein exotoxin of A. actinomycetemcomitans [28e30]. The Cdt family is known to induce growth arrest in various mammalian cells, by inflicting DNA strand-breaks and activation of DNA-damage checkpoint responses [31e33]. In terms of cytokine production, A. actinomycetemcomitans Cdt stimulates the synthesis of IL-1b, IL-6 and IL-8, but not TNF-a, macrophage colonystimulating factor (M-CSF), or IL-12, by human peripheral blood mononuclear cells [34]. In a recent study, we demonstrated that A. actinomycetemcomitans induces RANKL expression by human GF, an event attributed to its Cdt [27]. However, the involvement of Cdt in A. actinomycetemcomitans-induced expression of pro-inflammatory cytokines, or their cognate receptors in GF has not been investigated. Moreover, it is not known whether the A. actinomycetemcomitans-induced RANKL expression in GF is mediated by inflammatory cytokines known to stimulate RANKL. Therefore, the aims of this study were a) to investigate the role of Cdt in the A. actinomycetemcomitans-regulated expression of pro-inflammatory cytokines and their receptors (IL-1R I, IL-1R II, TNF-R I, TNF-R II, IL-6R) in GF, and b) to determine if the A. actinomycetemcomitans-induced RANKL expression in GF is mediated by IL-1, IL-6, TNF-a, or PGE2.

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2. Results Analyses using semi-quantitative RT-PCR showed that the D7SS wild-type strain enhanced mRNA expression of IL-6, IL-1b, and to a lesser extent of TNF-a (Fig. 1). Extract from the D7SS cdt-mutant strain induced mRNA expression of these cytokines to an extent similar to the wild-type extract. None of the two bacterial extracts affected M-CSF mRNA expression. The expression of the different receptor subtypes recognizing TNF-a, IL-1 and IL-6 was also investigated. As appears in Fig. 2, no differences could be observed in the mRNA expression of TNF-R I, TNF-R II, IL-1R I, or IL-6 R between the unchallenged controls and the cells challenged with either the D7SS wild-type strain, or the D7SS cdt-mutant strain. IL-1R II mRNA was not expressed by GF in any of the groups (data not shown). To investigate if the enhanced mRNA expression of IL-6, IL-1b and TNF-a induced by A. actinomycetemcomitans resulted in increased protein expression, the amount of cytokines produced by the cells was analysed by ELISA. Secreted IL-1b and TNF-a were not detectable in any of the groups, after 24 h of bacterial challenge, whereas IL-6 protein secreted from the cells to the culture media was detected in the unchallenged GF culture supernatants and was significantly upregulated in all bacterially challenged cultures (Table 1). The D7SS wild-type and cdt-mutant strains induced a 10-fold and 8.5-fold up-regulation of IL-6 protein,

Fig. 1. Semi-quantitative RT-PCR analysis of IL-6, TNF-a, IL-1b and M-CSF mRNA expression in GF challenged with 1% A. actinomycetemcomitans extract. GF were cultured in the absence (Con) or presence of D7SS wild-type (WT), or D7SS cdt-mutant (M) extract, for 24 h. Number of PCR cycles is shown.

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Fig. 2. Semi-quantitative RT-PCR analysis of IL-6R, TNF-RI, TNFRII and IL-1R I mRNA expression in GF challenged with 1% A. actinomycetemcomitans extract. GF were cultured in the absence (Con), or presence D7SS wild-type (WT), or D7SS cdt-mutant (M) extract, for 24 h. IL-R II was not expressed. Number of PCR cycles is shown.

a degree similar to the wild-type strain indicates that extract components other than Cdt are responsible for this enhancement. However, this does not exclude the possibility that Cdt alone may also have such a property [34]. To further investigate the role of Cdt in IL-6 induction by the bacterial extract, increasing concentrations of purified recombinant Cdt from H. ducreyi (95% homology to A. actinomycetemcomitans Cdt) were administered to GF, for 24 h. The mRNA expression of IL-6 remained unaffected irrespective of Cdt concentration (Fig. 3A). However, secreted IL-6 protein levels were up-regulated by increasing concentrations of purified Cdt, and there was a statistically significant 2.2-fold enhancement of secreted IL-6 by highest concentration of the toxin used (1000 ng/ml), compared to the un-challenged cells (Fig. 3B). Heat treatment of the toxin preparation (65  C for 30 min) abolished its capacity to stimulate IL-6 production (data not shown), excluding the possibility that contaminant LPS was responsible for this effect. IL-1b and TNF-a secreted protein levels were below detection limits, irrespective of challenge with increasing Cdt concentrations (data not shown).

respectively. The difference in IL-6 induction between the two bacterial extracts (w17%) did not prove to be statistically significant. No variations in cytokine production were observed among cell lines obtained from different individuals. Since IL-1b mRNA, and to a lesser extent TNF-a mRNA, were up-regulated, but neither IL-1b, nor TNF-a protein were detected in the culture supernatant, the possibility was investigated that induced IL-1b or TNF-a proteins remained intracellularly. However, the intracellular IL-1b and TNF-a protein levels were below detection limits (!0.01 ng/ml), in both control and Cdt-treated groups. The observation that D7SS cdt-mutant strain enhanced the expression of IL-6 mRNA and protein to

Table 1 Cytokine secretion in GF challenged with A. actinomycetemcomitans

IL-1b TNF-a IL-6

Control

D7SS cdt-mutant

D7SS wild-type

!0.01 !0.01 2.4G0.5

!0.01 !0.01 20.4G1.6*

!0.01 !0.01 24.0G1.2*

Values represent mean averageGSEM of secreted cytokine concentration (in ng/ml), from six experiments with one GF cell line. The cells were challenged for 24 h with 1% extract (D7SS wild-type, or D7SS cdt-mutant). Each experiment was performed in quadruplicate cultures. The asterisk indicates a statistically significant difference between the test group and the control group.

Fig. 3. GF were cultured in the absence (Con) or presence of increasing concentrations of H. ducreyi Cdt (in ng/ml), for 24 h. (A) Semiquantitative RT-PCR analysis of IL-6 mRNA expression (PCR cycles shown: IL-6: 23, GAPDH: 20). (B) Determination of IL-6 secretion in the culture media by ELISA. The values represent mean averageG SEM of secreted cytokine concentrations (in ng/ml), from one out of three representative experiments performed in quadruplicate cultures. The asterisk indicates a statistically significant difference between the compared groups.

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Fig. 4. Semi-quantitative RT-PCR analysis of RANKL mRNA expression in GF challenged with 1% D7SS wild-type extract. GF were cultured in culture medium (Con), or challenged with 1% D7SS wild-type extract alone (D7SS), or in combination with 100 ng/ml IL-6 antibody (IL-6 ab), 100 ng/ml TNF-a antibody (TNF-a ab), 100 ng/ml IL-1 receptor antagonist (IRAP), or pre-treated with 1 nmol/ml indomethacin for 3 h and then challenged with D7SS wild-type extract plus indomethacin, for 24 h. RT-PCR cycles: RANKL: 38, GAPDH: 20.

The next aim was to investigate if the previously demonstrated RANKL induction by A. actinomycetemcomitans [27] is mediated by IL-6, IL-1, or TNF-a, all known to be able to up-regulate RANKL in various cell types. For this purpose, antibodies neutralizing IL-6, or TNF-a, as well as recombinant IRAP were added to the cells, simultaneously exposed to the D7SS wild-type extract for 24 h. Neither the antibodies, nor IRAP, were able to inhibit RANKL mRNA expression induced by the bacterial extract (Fig. 4). To investigate the possible involvement of PGE2 in A. actinomycetemcomitans-induced RANKL expression, GF cultures were pre-treated for 3 h with indomethacin, and further challenged with D7SS wild-type extract for 24 h. As indicated by Fig. 4, indomethacin did not affect A. actinomycetemcomitans induced RANKL mRNA expression.

3. Discussion The present study demonstrates that A. actinomycetemcomitans induces IL-6 and IL-1b, and to a lesser extent, TNF-a mRNA expression in GF, but it does not regulate the expression of M-CSF. However, only IL-6 protein was increased, whereas the intracellular and secreted amounts of the other two cytokines were below detection levels. The capacity of A. actinomycetemcomitans to stimulate IL-6 protein production and the inability to induce IL-1b or TNF-a protein in GF is in accordance with previous studies [4e6,9]. However, at variance from the findings in GF, peripheral blood mononuclear cells have been shown to produce all three cytokines in response to an extract of A. actinomycetemcomitans surface associated components [7,8]. The different responses seen in GF and peripheral blood mononuclear cells are likely to be either related to the

different properties in the two cell types, or to heterogeneities in the various extract preparations used. In the present study, A. actinomycetemcomitans induced IL-1b mRNA expression, in contrast to previous reports demonstrating that IL-1b mRNA is not induced in GF challenged with A. actinomycetemcomitans surface components [6,9]. However, we were unable to show that the increased IL-1b mRNA expression was concomitant with IL-1b protein production. Gingival fibroblasts are not only able to secrete IL-1, TNF-a and IL-6 when stimulated properly, but also have the capacity to respond to several cytokines in either an autocrine or paracrine manner. Therefore, we investigated if A. actinomycetemcomitans regulates the expression of the cognate IL-1, TNF-a and IL-6 receptor subtypes. The IL-1 RI is the signal transducing receptor, whereas the IL-1 RII is not involved in signal transduction, but acts as a decoy receptor preventing binding of IL-1, to the type I receptor [35]. TNF-R1 is the functional receptor, whereas TNF-R2 contributes to the cells sensitivity to TNF-a [36]. The mRNA expression of the IL-1, IL-6 and TNF-a receptors was not regulated by the bacterial extract, indicating that the challenged cells did not become more responsive to the different cytokines. The expression of the c-fms receptor (cognate to M-CSF), was not studied since it is only expressed in cells of haematopoietic origin [19]. The Cdt is most well known for its capacity to induce growth arrest and eventually apoptosis in host cells [31,33]. However, interestingly, some reports have shown that it may as well have pro-inflammatory cykokine stimulating capacity [27,34,37e39]. In the present study, the A. actinomycetemcomitans D7SS cdtmutant strain exerted similar effect on IL-6, IL-1b and TNF-a mRNA expression, as its parental wild-type strain, indicating that the toxin is not involved in the transcriptional regulation of these cytokines. IL-6 secreted protein was significantly up-regulated in both

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A. actinomycetemcomitans challenged GF compared to the controls, but the difference between the two A. actinomycetemcomitans challenged groups (D7SS wild-type and D7SS cdt-mutant) was not statistically significant. This observation is in line with the previous observation that Campylobacter jejuni stimulates IL-1b, TNF-a and IL-6 protein production in a human monocytic cell line, most likely independent of its Cdt, as demonstrated by using a C. jejuni strain expressing a non-functional mutated form of Cdt [38]. It appears that cdt-gene deletion does not ablate the cytokine stimulatory capacity of the different Cdt-producing bacteria. However, this does not exclude the possibility that Cdt alone may also induce cytokine production. In the present study, when GF were exposed to purified Cdt, a concentration-dependent enhancement in the secretion of IL-6, but not IL-1b or TNF-a, was observed. However, the amount of IL-6 produced in response to purified Cdt was considerably less (2-fold stimulation by 1 mg/ml of Cdt; Fig. 3B) than that caused by the bacterial extracts (8e10-fold stimulation by 1% extract; Table 1), indicating that components other than Cdt present in the extract are the most potent inducers of IL-6 synthesis. The observation that Cdt alone can regulate IL-6 protein production is in accordance with a previous report demonstrating that purified recombinant A. actinomycetemcomitans Cdt stimulates IL-1b, IL-6, IL-8, but not TNF-a protein in human peripheral blood mononuclear cells [34]. The Cdts of C. jejuni and H. ducreyi have been shown to induce IL-8 production by intestinal epithelial [39] and dendritic cells [40], respectively, although the latter could inhibit cytokine production by already activated dendritic cells [40]. We have previously demonstrated that A. actinomycetemcomitans surface extracts induce RANKL expression in GF, and that the Cdt was responsible for this induction [27]. One of the purposes of this study was to investigate if RANKL induction by A. actinomycetemcomitans is mediated by IL-1, TNF-a, IL-6, or PGE2, all known to be able to induce RANKL expression in bone cells [15]. Antagonism of IL-1 receptor type I by IRAP, neutralization of TNF-a and IL-6 ligands by antibodies, or inhibition of PGE2 synthesis by indomethacin, did not abolish A. actinomycetemcomitans-induced RANKL expression in GF. Therefore, the observed RANKL induction is not an effect mediated by IL-1, IL-6, TNF-a, or PGE2. It has recently been demonstrated that E. coli LPS induces RANKL expression by periodontal ligament fibroblasts, in a manner dependent on prior induction of IL-1b and TNF-a [25]. Taken together, this information is intriguing since it indicates that although LPS-induced RANKL expression is mediated by pro-inflammatory cytokines, Cdt-induced RANKL expression occurs in a manner independent of the classical inflammatory mediators.

In conclusion, our results indicate that A. actinomycetemcomitans is able to induce IL-6 and RANKL expression in GF, two cytokines crucial for the induction of osteoclast formation and bone resorption. The Cdt is responsible for the induction of RANKL expression [27] and may also have a role in IL-6 production, although other A. actinomycetemcomitans components appear to be more crucial in this effect. Collectively, these findings suggest that the Cdt of A. actinomycetemcomitans has a unique property to induce the expression of RANKL in a manner independent from other inflammatory mediators, and may be involved in the pathological bone resorption occurring in localized aggressive periodontitis.

4. Materials and methods 4.1. Materials Dulbecco’s-Modified Eagle Medium (D-MEM), fetal bovine serum (FBS), penicillin/streptomycin, L-glutamine and trypsin were purchased from Gibco BRL; the DAPI-fluorescent staining kit for mycoplasma detection from Sigma, St. Louis, MO, USA; limulus ameboecyte lysate-based assay for LPS determination from CoamaticÒ Chromo-LAL, Chromogenix, Mo¨lndal, Sweden; TRIzol Reagent from Life Technologies, GibcoBRL; First Strand cDNA Synthesis Kit for RTPCR (AMV) and PCR Core Kit for cDNA amplification from Roche, Mannheim, Germany; ‘‘Hot-start’’ Taq DNA Polymerase Kit from Qiagen, Germany; DYEnamic ET terminator cycle sequencing kit from Amersham Biosciences, Amersham, UK; BD OptEIAÔ ELISA kits for human IL-6, IL-1b and TNF-a protein quantification from BD Biosciences Pharmingen, San Diego, CA, USA; IL-1 receptor antagonist (IRAP) and antibodies for human IL-6 and TNF-a from R & D Systems, Abington, UK. Indomethacin was a kind gift from Merck, Sharp & Dohme, Haarlem, The Netherlands. 4.2. Cell cultures Human GF primary cell lines were established as previously described [41]. The gingival tissue biopsies used were obtained from four healthy young individuals (age: 13 to 16 years), who were about to have their first pre-molars removed in the course of orthodontic treatment. Informed consent was given by all subjects and approval was granted by the Human Studies Ethical Committee of Umea˚ University, Sweden (x68/03, dnr 03e029). The cells were cultured in D-MEM supplemented with 10% FBS, 20 U/ml penicillin, 20 mg/ml streptomycin and 200 mM L-glutamine. All cell lines were confirmed to be free of mycoplasma infections by

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using DAPI-fluorescent staining. Cells used in all experiments were between passages 4 and 7. For the experiments, GF were seeded at a concentration of 25!103 cells/cm2 and allowed to attach for 24 h, maintaining a sub-confluent status. Thereafter, the cells were cultured in presence or absence of bacterial extracts, purified Cdt, antisera or test substances. 4.3. Preparation of the bacterial extract Outer membrane components from A. actinomycetemcomitans were extracted as previously described [41,42]. Briefly, bacteria were harvested from cultures of A. actinomycetemcomitans D7S-smooth wild-type strain and its derivative cdtABC-deletion mutant, grown on Bacteroides agar containing horse laked blood, for 2 days at 37  C in the presence of 5% CO2. The bacteria were suspended in phosphate-buffered (10 mM, pH 7.2) saline (PBS) containing 30% FBS, to a final concentration of 6!109 bacteria/ml. This suspension was gently agitated at 4  C for 1 h to extract bacterial components. Following centrifugation (4  C, 10,000!g, 15 min), the supernatant was aspirated and frozen at 80  C until use. Before the initiation of each experiment, this preparation was diluted in the cell-culture medium. The extract concentration used in each experiment is expressed as the percenatge of the above described stock-extract dilution (v/v) in the culture medium. The D7S-smooth cdtABC-deletion mutant was constructed, characterized and employed in functional assays as previously described [41,43,44]. In the present report, the D7S-smooth strain is designated as D7SS wild-type, while the D7S-smooth DcdtABC strain as D7SS cdt-mutant. The concentration of LPS in the bacterial extracts was determined by a limulus ameboecyte lysate-based assay. One percent of the extract from D7SS wild-type and D7SS cdt-mutant contained 0.73 and 0.72 mg/ml LPS, respectively. 4.4. Haemophilus ducreyi Cdt Recombinant Cdt holotoxin from H. ducreyi was purified as previously described [45]. The Cdts of H. ducreyi and A. actinomycetemcomitans exhibit 95% amino-acid sequence homology [28]. 4.5. RNA isolation, first-strand cDNA synthesis and semi-quantitative polymerase chain reactions (PCR) At the end of the experiments, cell cultures were rinsed with 3!1 ml PBS and total RNA was extracted from the cells with TRIzol reagent, following the manufacturer’s protocol. The RNA was quantified spectophotometrically and the integrity of the RNA preparations was examined by agarose gel electrophoresis. One microgram

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of total RNA was reverse-transcribed into singlestranded cDNA using oligo(dT) primers (25  C for 10 min, 42  C for 60 min and 99  C for 5 min). The cDNA was kept in a volume of 20 ml, at 80  C, until used for the PCR. Aliquots of 0.5 ml (Z25 ng) of the synthesized cDNA in a total volume of 25 ml ‘‘Hot-start’’ Taq DNA polymerase master-mix, were amplified in a thermal cycler (Mastergradient Cycler, Eppendorf, Germany). The primer concentration used was 0.4 mM, whereas that of MgCl2 was 1.5 mM. The conditions for PCR were: activation of the polymerase at 95  C for 15 min during the first cycle, followed by denaturation at 94  C for 35 s, annealing at variable temperatures (IL-6, TNF-R I and TNF-R II: 55  C, IL-1b: 56  C, GAPDH: 57  C, M-CSF: 58  C, IL-1R I and IL-1R II: 59  C, TNF-a: 60  C, RANKL: 66  C) for 35 s, followed by elongation at 72  C for 45 s. After completion of all cycles, an additional step of elongation at 72  C for 10 min followed. The expression of these genes was compared at the logarithmic increase phase of the PCR reaction. The PCR products were electrophoretically size-fractionated on 1.5% agarose gels and visualized using ethidium bromide. PCR of samples without the preceding reverse-transcription step did not result in any products, demonstrating that there was no genomic DNA amplification. The sequence of primers used, the GenBank accession numbers and the size of the PCR products are provided in Table 2. The identity of the PCR products was confirmed using a cycle sequencing kit. The nucleotide sequence was determined on an ABMI 377 XL DNA sequencer (PE Applied Biosystems, Foster City, CA, USA). 4.6. Protein analysis for cytokines Cytokine concentration was quantified by commercially available ELISA kits for IL-1b, TNF-a and IL-6. To determine the amount of secreted cytokines, cell culture supernatants were collected and analyzed. To determine the amount of intracellular cytokines cells were lysed in 0.1% Triton-X detergent and the cell lysates were collected. The detection limit of these cytokines was 0.01 ng/ml. 4.7. Treatment with TNF-a and IL-6 antibody, IRAP and indomethacin To investigate if the A. actinomycetemcomitansinduced RANKL expression in GF is mediated by TNF-a, IL-6, or IL-1, cells were challenged with D7SS wild-type strain extract, in the absence or presence of TNF-a antibody, IL-6 antibody, or IRAP, respectively. These antibodies and the receptor antagonist have been tested in functional assays and have been shown to specifically neutralize the different cytokines [46,47].

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Table 2 Primers used in RT-PCR Gene

Accession No.

Primer sequence

Product size (bp)

RANKL

AF019047

517

IL-6

NM000600

IL-1b

NM000576

TNF-a

NM000594

M-CSF

BC021117

IL-6R

NM000565

TNF-R1

BC010140

TNF-R2

NM001066

IL-1R I

AF531102

IL-1R II

X59770

GAPDH

NM002046

Fwd: 5#-CAGCACATCAGAGCAGAGAAAGC-3# Rev: 5#-CCCCAAAGTATGTTGCATCCTG-3# Fwd: 5#-CCAGGAGCCCAGCTATGAAC-3# Rev: 5#-TCAGCCATCTTTGGAAGGTTC-5# Fwd: 5#-GACCTTCCAGGAGAATGACC-3# Rev: 5#-GGCTTATCATCTTTCAACACG-3# Fwd: 5#-TCAGATCATCTTCTCGAACC-3# Rev: 5#-CAGATAGATGGGCTCATACC-3# Fwd: 5#-ACATAATGGAGGACACCATGC-3# Rev: 5#-ATGGCTTTGGGGTACAGG-3# Fwd: 5#-TCAGATATCGGGCTGAACG-3# Rev: 5#-AACAATGGCAATGCAGAGG-3# Fwd: 5#-ACCAAGTGCCACAAAGGAAC-3# Rev: 5#-CTGCAATTGAAGCACTGGAA-3# Fwd: 5#-TTCCGCTCTTCCAGTTGGACT-3# Rev: 5#-CACCAGGGGAAGAATCTGAG-3# Fwd: 5#-GTGAGCCCAGCTAATGAGACAAT-3# Rev: 5#-ACCCATTCCACTTCCAGTAAGC-3# Fwd: 5#-GGAACCGGCACACCCTTAA-3# Rev: 5#-CCGGGTAGGCGCTCTCTATG-3# Fwd: 5#-CAACTACATGGTTTACATGTTC-3# Rev: 5#-GCCAGTGGACTCCACGAC-3#

To investigate the role of PGE2, the cells were pretreated with indomethacin for 3 h, and then exposed to A. actinomycetemcomitans, plus indomethacin. We have previously shown that treatment of cells with indomethacin at the concentration used (106 M), abolishes the synthesis of PGE2 induced by a variety of cytokines and kinins [46e48]. 4.8. Statistical analysis The significance of differences was assessed by Oneway ANOVA and Bonferroni post-hoc test. P values of %0.05 indicated statistically significant difference. The data is expressed as meansGstandard error of means (SEM). Acknowledgements This study has been supported in part by the Swedish Research Council (project No. 7525), the Swedish Rheumatism Association, the Royal 80 Year Fund of King Gustav V, the County Council of Va¨sterbotten, and NIDCR grant RO1 DE12212 (C. Chen). References [1] Firestein GS. Nature 2003;423:356e61. [2] Taubman MA, Kawai T. Crit Rev Oral Biol Med 2001;12: 125e35. [3] AAP. J Periodontol 1999;70:457e70. [4] Dongari-Bagtzoglou AI, Ebersole JL. J Periodontal Res 1996;31:90e8.

306 332 358 318 265 262 346 108 72 163

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