INFECTION AND IMMUNITY, Mar. 2002, p. 1272–1278 0019-9567/02/$04.00⫹0 DOI: 10.1128/IAI.70.3.1272–1278.2002 Copyright © 2002, American Society for Microbiology. All Rights Reserved.
Vol. 70, No. 3
Human Gingival CD14⫹ Fibroblasts Primed with Gamma Interferon Increase Production of Interleukin-8 in Response to Lipopolysaccharide through Up-Regulation of Membrane CD14 and MyD88 mRNA Expression Riyoko Tamai,1 Tetsuya Sakuta,2 Kenji Matsushita,2 Mitsuo Torii,2 Osamu Takeuchi,3 Shizuo Akira,3 Sachiko Akashi,4 Terje Espevik,5 Shunji Sugawara,1 and Haruhiko Takada1* Department of Microbiology and Immunology, Tohoku University School of Dentistry, Sendai 980-8575,1 Department of Operative Dentistry and Endodontology, Kagoshima University Dental School, Kagoshima 890-8544,2 Department of Host Defense, Research Institute for Microbial Defenses, Osaka University, Osaka 565-0871,3 and Division of Infectious Genetics, Department of Microbiology and Immunology, The Institute of Medical Science, The University of Tokyo, Tokyo 108-8639,4 Japan, and Institute of Cancer Research and Molecular Biology, Norwegian University of Science and Technology, Trondheim, Norway5 Received 13 August 2001/Returned for modification 26 September 2001/Accepted 27 November 2001
Gamma interferon (IFN-␥)-primed human gingival fibroblasts (HGF) have been shown to produce higher levels of interleukin-8 (IL-8) upon stimulation with bacterial products and inflammatory cytokines than nonprimed controls. In this study, we examined whether priming of HGF with IFN-␥ up-regulates IL-8 production by the cells in response to purified lipopolysaccharide (LPS). The priming effect of IFN-␥ was clearly observed in the high-CD14-expressing (CD14high) HGF but not in the low-CD14-expressing (CD14low) HGF. The CD14high HGF were most effectively primed with IFN-␥ (1,000 IU/ml) for 72 h. To elucidate the mechanism of the priming effects of IFN-␥ for the LPS response by HGF, we examined whether IFN-␥ regulated expression of CD14, Toll-like receptor 2 (TLR2), TLR4, MD-2, and MyD88, all of which are molecules suggested to be associated with LPS signaling. In CD14high HGF, IFN-␥ markedly up-regulated CD14 and MyD88 but not TLR4 protein and MD-2 mRNA expression, while in CD14low HGF, IFN-␥ slightly increased MyD88 and scarcely affected CD14, TLR4 protein, and MD-2 mRNA levels. LPS-induced IL-8 production by IFN-␥-primed CD14high HGF was significantly inhibited by monoclonal antibodies (MAbs) against CD14 and TLR4, but not by an anti-TLR2 MAb. These findings suggested that IFN-␥ primed CD14high HGF to enhance production of IL-8 in response to LPS through augmentation of the CD14-TLR system, where the presence of membrane CD14 was indispensable for the response of HGF to LPS. ATCC 25611, and we designated it as Prevotella glycoprotein (PGP) (19). We assumed that unique bioactivities of BPB LPS, different from the LPS of Enterobacteriaceae, including those on HGF might be attributable to PGP or related materials, and not to LPS itself. Over the last decade, the LPS recognition and signaling system has been exhaustively studied (2, 22). Briefly, LPS is opsonized by LPS binding protein (LBP) in serum (46), and the LPS-LBP complex or LPS by itself is recognized by CD14 on cells (membrane CD14 [mCD14]) such as monocytes and macrophages (45), which associates with the cell surface by means of a glycosylphosphatidylinositol (16). In the case of cells lacking mCD14, such as epithelial and endothelial cells, soluble CD14 (sCD14) endowed the cells with responsiveness to LPS (10). mCD14 is not capable of generating a transmembrane signal, but LPS and CD14 (and perhaps also LBP) might form a complex with Toll-like receptor 4 (TLR4) (6, 18, 27, 28), which in turn signals through an adapter protein, MyD88 (20). MD-2, a secreted protein that might bind to the extracellular domain of TLR4, is also essential for LPS signaling (4, 33). Fibroblasts are generally devoid of mCD14. Hayashi et al. (14) reported that HGF, which lacked either mCD14 or CD14 mRNA expression, was activated by E. coli LPS in an sCD14dependent manner. On the other hand, Watanabe et al. (44)
Treatment of various cells such as human monocytes (15) and human endothelial cells (23) with human gamma interferon (IFN-␥) enhances the cytokine production by the cells in response to lipopolysaccharide (LPS). Human gingival fibroblasts (HGF) are the major constituents of periodontal tissue and produce various inflammatory cytokines such as interleukin-1 (IL-1), IL-6, and IL-8 upon stimulation with bacteria and their components (38). Previously, it was reported that HGF generated IL-1 and IL-6 activity (39) and enhanced expression of IL-1␣ and IL-8 mRNA (42) in response to the LPS fraction from oral black-pigmented bacteria (BPB) such as Porphyromonas gingivalis and Prevotella intermedia, but not LPS from Enterobacteriaceae such as Salmonella strains and Escherichia coli. Furthermore, we demonstrated a priming effect of IFN-␥ to enhance IL-8 mRNA expression and IL-8 production in response to P. intermedia LPS, but not to Salmonella LPS, in HGF cultures (30, 42). In the course of our studies of unique bioactivities of BPB LPS different from those of Enterobacteriaceae LPS, we isolated an immunobiologically active glycoprotein from the hot-phenol-water extract of P. intermedia
* Corresponding author. Mailing address: Department of Microbiology and Immunology, Tohoku University School of Dentistry, 4-1 Seiryo-machi, Aoba-ku, Sendai 980-8575, Japan. Phone: 81 22 717 8305. Fax: 81 22 717 8309. E-mail:
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reported that HGF responded to P. gingivalis LPS in an mCD14-dependent manner. Then, Sugawara et al. (36) demonstrated heterogeneous mCD14 expression in the HGF from 10 donors, and showed that high-CD14-expressing (CD14high), but not low-CD14-expressing (CD14low), HGF produced IL-8 in response to LPS from Enterobacteriaceae and a synthetic E. coli-type lipid A in an mCD14-dependent manner. Sugawara et al. (36) also reported that human IFN-␥ up-regulated mCD14 expression and CD14 mRNA expression in CD14high HGF, but not in CD14low HGF. In this study, we examined the possible priming effect of human IFN-␥ for IL-8 production by CD14high HGF in response to LPS from Enterobacteriaceae, with special reference to a possible augmentation of signaling via the CD14-TLR4 system. MATERIALS AND METHODS Reagents. Ultrapurified LPS prepared from Salmonella enterica serovar Abortus-equi (Novo-pyrexal) (12) was supplied by C. Galanas (Max Plank Institut für Immunbiologie, Freiburg, Germany). Human natural IFN-␥ was provided by the Hayashibara Bioscience Institute (Okayama, Japan). Alpha minimal essential medium (␣-MEM) and fetal bovine serum (FBS) were purchased from Gibco BRL Life Technologies (Auckland, New Zealand). Anti-human TLR4 monoclonal antibody (MAb) HTA125 (mouse immunoglobulin G2a [IgG2a]), HTA1216 (mouse IgG1), and anti-human TLR2 TL2.1 (mouse IgG2a) were prepared as described previously (3, 9, 33). Anti-CD14 MAb MY4 (mouse IgG2b) and isotype-matched control mouse MAb IgG2a and IgG2b were purchased from Coulter Co. (Miami, Fla.). Unless otherwise indicated, other reagents were purchased from Sigma Chemical Co. (St. Louis, Mo.). Cell culture. HGF were prepared from the explants of normal gingival tissues of 8- to 18-year-old patients with the informed consent of their parents or guardians. The explants were cut into pieces and cultured in tissue culture dishes 100 mm in diameter (Falcon; BD Labware, Lincoln Park, N.J.) in ␣-MEM supplemented with 10% FBS, L-glutamine, and kanamycin (0.2 mg/ml) with a medium change every 3 days for 10 to 15 days until confluent cell monolayers were formed. After three to four subcultures by trypsinization, homogeneous, slim, spindle-shaped cells grown in characteristic swirls were obtained. The cells were used as confluent monolayers at subculture levels 5 through 8. As described above, HGF expresses CD14 heterogeneously (36). In this study, we mainly used two representative HGF: CD14high HGF from donor 1 (male, 12 years old) (56.1% CD14-positive cells) and CD14low HGF from donor 2 (male, 10 years old) (19.1% CD14-positive cells) (36). The experimental procedures were approved by the Ethical Review Board of Tohoku School of Dentistry. cDNA probes. Plasmids containing human MyD88 cDNA and CD14 cDNA were prepared as described previously (1, 35). A human glyceraldehyde-3-phosphate dehydrogenase (GAPDH) cDNA clone (43) was provided by I. Sakiyama (Chiba Cancer Center Research Institute and Hospital, Chiba, Japan). For Northern blotting, inserts were excised with restriction enzymes, then purified from vector sequences by agarose gel electrophoresis. Detection of IL-8 by ELISA. IFN-␥-primed or nonprimed confluent HGF in 96-well flat-bottom plates in 200 l of ␣-MEM with 1% FBS were washed three times with phosphate-buffered saline and stimulated with LPS (0.1 g/ml in most experiments) for 48 h. After incubation, the culture supernatants were collected and the levels of IL-8 in the supernatants were determined with a human IL-8 enzyme-linked immunosorbent assay (ELISA) kit (OptEIA Set; PharMingen, San Diego, Calif.). The IL-8 assays were performed according to the instructions of the manufacturer. The concentrations of IL-8 in the supernatants were determined using the SoftMAX data analysis program (Molecular Devices Corp., Menlo Park, Calif.). Flow cytometry. For immunofluorescent staining, HGF were collected by trypsinization, washed in PBS, and used for staining. For analysis of the expression of CD14, the cells were stained with fluorescein isothiocyanate-labeled anti-CD14 MAb MY4 at 4°C for 20 min. For analysis of the expression of TLR4, the cells were stained with anti-TLR4 MAb HTA1216 or control mouse antibody (Ab) at 4°C for 20 min, followed by fluorescein isothiocyanate-conjugated goat anti-mouse IgG (Biosource International Inc., Camarillo, Calif.) at 4°C for a further 20 min. Flow cytometric analyses were performed with a fluorescenceactivated cell sorter (FACScan; Becton Dickinson, Mountain View, Calif.).
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Northern blotting. Total cellular RNA was extracted from 2.5 ⫻ 106 cells (one plate 100 mm in diameter) by Isogen (Nippon Genes, Tokyo, Japan) according to the manufacturer’s instructions. Total cellular RNA was resolved by electrophoresis through a 1.2% agarose gel containing formaldehyde (0.66 mol/liter) and transferred onto nylon membranes (Zeta-Probe; Bio-Rad Laboratories, Richmond, Calif.). The membranes were then baked and prehybridized for 18 h at 42°C with 50% formamide, 1% sodium dodecyl sulfate (SDS), 4⫻ SSPE (1⫻ SSPE is 0.18 M NaCl plus 0.015 M sodium citrate), 0.5% nonfat powdered milk, and 0.5 g of denatured salmon sperm DNA per ml. Hybridization was performed for 16 h at 42°C in the same reagent used for prehybridization containing 106 cpm of 32P-labeled cDNA probe per ml. Probes were labeled using random primers, Klenow fragment (Roche Diagnostics, Mannheim, Germany), and [␣-32P]dCTP (NEN, Wilmington, Del.) according to the method of Feinberg and Vogelstein (8). After hybridization, the membranes were washed with 2⫻ SSC (1⫻ SSC is 0.15 M NaCl plus 0.015 M sodium citrate), 2⫻ SSC–0.1% SDS, 0.5⫻ SSC–0.1% SDS, and 0.1⫻ SSC–0.1% SDS at room temperature for 15 min. The membranes were dried and exposed to an Imaging Plate (Fuji Photo Film Co., Tokyo, Japan). Blots were further quantified using a Bio-Imaging Analyzer (BAS 1500 Mac; Fuji Photo Film Co.). The results are expressed as relative mRNA accumulation compared with GAPDH mRNA as an internal standard. Data analysis. All experiments in this study were performed at least twice to confirm the reproducibility of the results, and representative results are shown. In ELISAs, experimental values are presented as means ⫾ standard deviations (SD) of triplicate assays. The statistical significance of differences between the two means were evaluated by one-way analysis of variance, using the Bonferroni or Dunn method, and P values less than 0.05 were considered significant.
RESULTS Priming of CD14high HGF with IFN-␥ up-regulates IL-8 production by the cells in response to LPS. We first examined whether IFN-␥ priming modulates IL-8 production by HGF in response to LPS. Based on a previous report (30) and preliminary experiments, CD14high and CD14low HGF were treated with 1,000 U of natural human IFN-␥/ml for 72 h, and then cells were stimulated with 0.001 to 10 g of LPS/ml for 48 h. As shown in Fig. 1, CD14high HGF showed significantly increased IL-8 production in response to LPS. In the cells, IFN-␥ priming clearly up-regulated IL-8 production in response to LPS; i.e., IFN-␥-primed HGF released significantly higher levels of IL-8 than nonprimed HGF in response to the respective concentration of LPS. The highest level of IL-8 production was induced by stimulation with 0.1 g of LPS/ml. In contrast, no such priming effect was observed in CD14low HGF; IL-8 was scarcely induced by LPS, irrespective of IFN-␥-priming. Next, we examined the priming effects of IFN-␥ for various periods on IL-8 production in response to LPS in CD14high HGF. As expected, priming with IFN-␥ for 72 h was most effective for up-regulating IL-8 production by CD14high HGF in response to LPS (Fig. 2); CD14high HGF primed with IFN-␥ for 72 h produced about twofold-higher levels of IL-8 than the nonprimed cells on stimulation with LPS for 48 h. We also examined priming effects of various concentrations of IFN-␥ on the IL-8 production in response to LPS in CD14high HGF. IL-8 production was up-regulated in a dose-dependent manner from 1 to 1,000 U of IFN-␥/ml and then decreased at 10,000 U/ml (Fig. 3). As expected, 1,000 U of IFN-␥/ml was most effective for up-regulating IL-8 production by CD14high HGF in response to LPS. IFN-␥ enhanced mCD14 and CD14 mRNA expression by CD14high HGF. As mentioned above, Sugawara et al. (36) demonstrated by flow cytometry and RT-PCR, respectively, that IFN-␥ up-regulated mCD14 and CD14 mRNA expression of CD14high HGF. We confirmed that IFN-␥ enhanced
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FIG. 1. Enhanced production of IL-8 by IFN-␥-primed CD14high HGF on stimulation with LPS. CD14high and CD14low HGF were incubated with (closed bars) or without (open bars) IFN-␥ (1,000 IU/ml) for 72 h, washed three times, and then stimulated with LPS at the indicated concentration for 48 h. After incubation, the IL-8 concentration in the culture supernatants was determined by ELISA. An additional experiment gave results similar to those shown here. Each assay was carried out in triplicate. Error bars indicate SD. ⴱ and ⴱⴱ, P ⬍ 0.05 and P ⬍ 0.01, respectively, versus respective control culture without IFN-␥-priming.
mCD14 expression at 3 days in culture (Fig. 4A). We also found that CD14 mRNA expression by CD14high HGF reached the peak at 48 h (Fig. 4B). In CD14low HGF, neither mCD14 nor CD14 mRNA expression was ever detected after treatment with IFN-␥ in CD14low HGF (data not shown). IFN-␥ did not influence TLR4 expression in CD14high and
FIG. 2. Priming effects of IFN-␥ for various periods on the IL-8 production by CD14high HGF in response to LPS. CD14high HGF were incubated with or without IFN-␥ (1,000 U/ml) for the indicated periods and then stimulated with LPS (0.1 g/ml) for 48 h. After incubation, the IL-8 concentration in the culture supernatants was determined by ELISA. Each assay was carried out in triplicate. Error bars indicate SD. Two additional experiments gave results similar to those shown here. ⴱ and ⴱⴱ, P ⬍ 0.05 and P ⬍ 0.01, respectively, versus respective control culture stimulated with LPS without IFN-␥-priming (medium3LPS).
CD14low HGF. Next, to elucidate whether IFN-␥ also regulates expression of TLR-related molecules in HGF, we examined expression of TLR4 by CD14high and CD14low HGF. Both HGF expressed the same level of TLR4 protein (Fig. 5). We also examined mRNA for MD-2, a TLR4-associated molecule. MD-2 mRNA expression by CD14high and CD14low HGF was not regulated by IFN-␥ (data not shown). IFN-␥ up-regulates MyD88 mRNA expression by HGF. Then, we examined MyD88 mRNA expression by Northern blotting. IFN-␥ markedly up-regulated MyD88 mRNA expression by CD14high HGF (Fig. 6A) and slightly up-regulated that
FIG. 3. Priming effects of various concentrations of IFN-␥ on the IL-8 production by CD14high HGF in response to LPS. CD14high HGF were incubated with the indicated concentrations of IFN-␥ for 72 h, followed by stimulation with LPS (0.1 g/ml) for 48 h. After incubation, the IL-8 concentration in the culture supernatants was determined by ELISA. Each assay was carried out in triplicate. Error bars indicate SD. Two additional experiments gave results similar to those shown here. ⴱ and ⴱⴱ, P ⬍ 0.05 and P ⬍ 0.01, respectively, versus the control culture stimulated with LPS without IFN-␥-priming (0 U/ml).
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FIG. 4. IFN-␥ up-regulated mCD14 expression and CD14 mRNA expression in CD14high HGF. (A) mCD14 expression determined by flow cytometry. CD14high HGF were incubated with or without IFN-␥ (1,000 IU/ml) for 3 days. Cells were then collected and stained with anti-CD14 MAb (MY4) (CD14 [solid line]) or the second Ab alone (control [dotted line]). (B) CD14 mRNA expression determined by Northern blotting. CD14high HGF were incubated with or without IFN-␥ (1,000 IU/ml) for the indicated periods. Total cellular RNA (25 g per lane) was subjected to electrophoresis in agarose-formaldehyde gels, blotted onto nylon membranes, and then hybridized with the 32P-labeled CD14 probe as described in Materials and Methods. Blots were further quantified using a Bio-Imaging Analyzer. The results are expressed as relative mRNA accumulation compared with GAPDH mRNA as an internal standard. Similar results were obtained in two independent experiments.
by CD14low HGF (Fig. 6B). The peak of effect was observed at 24 h after stimulation with IFN-␥ in both cell lines. These results strongly suggested that the priming effects of IFN-␥ for LPS response in CD14high HGF was caused by modulation of the CD14-TLR system, i.e., up-regulation of CD14 and MyD88 expression, and that the presence of mCD14
is indispensable for the response of HGF to LPS, irrespective of IFN-␥ treatment. Anti-CD14 and anti-TLR4 MAb inhibited IL-8 production by IFN-␥-primed and nonprimed CD14high HGF in response to LPS. Finally, we examined whether IFN-␥-primed HGF as well as nonprimed HGF produced IL-8 through the CD14 and TLR4 pathway. We carried out inhibition experiments using anti-CD14 MAb MY4, anti-TLR4 MAb HTA125, and antiTLR2 MAb TL2.1. Figure 7 shows that both anti-CD14 and anti-TLR4 MAbs significantly inhibited IL-8 production by IFN-␥-primed as well as nonprimed CD14high HGF, whereas anti-TLR2 MAb and control Ab (IgG2b matched to MY4) were devoid of inhibitory activities in this respect. It should be noted that another control MAb (IgG2a, isotype-matched to HTA125 and TL2.1) was also inactive in a separate experiment (data not shown). DISCUSSION
FIG. 5. IFN-␥ did not influence TLR4 expression in either CD14high or CD14low HGF. TLR4 expression was determined by flow cytometry. CD14high (A) and CD14low (B) HGF were incubated with (upper panels) or without (lower panels) IFN-␥ (1,000 IU/ml) for 3 days. Cells were then collected and stained with anti-TLR4 MAb (HTA1216) (TLR4 [solid line]) or the second Ab alone (control [dotted line]). An additional experiment gave results similar to those shown here.
In this study, we found that IFN-␥ primed CD14high HGF, but not CD14low HGF, to enhance production of IL-8 in response to LPS (Fig. 1). In agreement with a previous study (36), IFN-␥ up-regulated CD14 expression of CD14high HGF, while this was not the case in CD14low HGF, probably resulting in more augmentation of LPS-induced IL-8 production by CD14high HGF than that produced by CD14low HGF. Although TLR4 proteins were detected in CD14low as well as CD14high HGF (Fig. 5), IFN-␥-priming did not up-regulate IL-8 production by CD14low HGF in response to LPS. These findings suggested that mCD14 is required for the priming effect of IFN-␥ on HGF response to LPS. sCD14 might also participate in the response of HGF to LPS (14), but the mechanism did not work in our assay system using medium contain-
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FIG. 6. IFN-␥ up-regulated MyD88 mRNA expression by CD14high HGF. CD14high (A) and CD14low (B) HGF were incubated with or without IFN-␥ (1,000 IU/ml) for the indicated periods. Procedures for Northern blotting are described in the legend to Fig. 4. Three independent experiments gave results similar to those shown here.
ing 1% FBS. In this context, sCD14 is also produced at lower levels by CD14low HGF than by CD14high HGF on stimulation with IFN-␥ or LPS (36). We showed that IFN-␥ up-regulated CD14 and MyD88 mRNA expression in CD14high HGF, with especially high degrees of up-regulation for CD14 and MyD88 mRNA expression (Fig. 4 and 6). Figure 7 shows that anti-CD14 MAb MY4 and anti-TLR4 MAb HTA125 inhibited IFN-␥-enhanced IL-8
Fig. 7. Anti-CD14 and anti-TLR4 MAb inhibited IL-8 production by IFN-␥-primed and nonprimed CD14high HGF in response to LPS. CD14high HGF were preincubated with (closed bars) or without (open bars) IFN-␥ (1,000 IU/ml) for 72 h. After three washes, they were incubated with or without MAb against CD14 (MY4), TLR4 (HTA125), TLR2 (TL2.1), or control Ab (IgG2b) for 30 min and then stimulated with LPS (0.1 g/ml) for 48 h. After incubation, the IL-8 concentration in the culture supernatants was determined by ELISA. Each assay was carried out in triplicate. Error bars indicate SD. An additional experiment gave results similar to those shown here. Symbols: ⴱⴱ, P ⬍ 0.01 versus respective control culture stimulated with LPS alone (nonpriming); ##, P ⬍ 0.01 versus respective control culture stimulated with LPS after IFN-␥-priming.
production in response to LPS by CD14high HGF, whereas anti-TLR2 MAb did not. Therefore, IFN-␥-enhanced CD14 expression might be responsible for the IFN-␥-up-regulated IL-8 production in response to LPS in CD14high HGF. On the other hand, MyD88 is a critical molecule for responses to LPS and other bacterial components so far examined (20, 41). As mentioned above, LPS activates cells via the TLR4–MyD88– NF-B pathway (2, 22), and NF-B activation is required for IL-8 production (17). Recently, it was revealed that PGP activates cells via CD14 and TLR2 (37). Furthermore, we observed that the IFN-␥-primed CD14high HGF produced higher IL-8 upon stimulation with a P. gingivalis LPS fraction, and the activity was completely inhibited by anti-CD14 MAb and only partially inhibited by either anti-TLR2 or anti-TLR4 MAb, possibly because of contamination of the LPS fraction with PGP-like material (R. Tamai and H. Takada, unpublished data). It is reasonable that MyD88 mRNA expression in CD14high HGF reached the peak 24 h after stimulation with IFN-␥ (Fig. 6), as the most-effective priming with IFN-␥ was observed at 72 h (Fig. 2). Rehli et al. (29) reported that IFN consensus sequence binding protein regulates expression of the human TLR4 gene in THP-1 cells. IFN consensus sequence binding protein is a member of the IFN regulatory factor (IRF) family of transcription factors, which is regulated by IFN-␥ (7). Thus, we expected that IFN-␥ would enhance TLR4 expression, but it did not (Fig. 5). The genomic IL-8 sequence has NF-B and AP-1 sites and an IRF-1 binding site, and the latter is required for LPS-enhanced production of IL-8 (24, 25). IFN-␥ can also activate IRF-1 and cause its binding to DNA (5, 26, 31, 32). Thus, IRF-1 might be also responsible for up-regulation of LPS-induced IL-8 mRNA expression by IFN-␥. In the course of investigation of whether Th1-type or Th2type responses are dominantly associated with periodontitis, IFN-␥ mRNA-expressing mononuclear cells, mainly CD4⫹ T cells, were consistently detected in gingival tissues from pa-
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tients with periodontitis (11, 40). However, T cells in gingival tissues expressed IFN-␥ mRNA at lower levels than those in peripheral blood (47). In this context, Lundqvist et al. (21) reported that CD4⫹ ␥␦ T cells specifically expressed IFN-␥. Furthermore, numbers of IFN-␥-positive cells were generally higher than those of either IL-4- or IL-10-positive cells in P. gingivalis-specific T-cell lines established from peripheral blood cells (13), although a tendency toward Th2 dominance associated with periodontitis has also been reported (13, 34). These findings suggested that gingival fibroblasts are primed with IFN-␥ in inflamed lesions of periodontitis. In conclusion, IFN-␥ primed CD14high HGF to enhance production of IL-8 in response to LPS, probably through upregulation of CD14 and MyD88. The up-regulation of CD14 and MyD88 by IFN-␥ might also induce priming effect on HGF to various bacterial components as well as LPS. By these positive regulatory mechanisms for IFN-␥, HGF might play important roles in the development of chronic inflammatory lesions in periodontal tissues. Furthermore, investigations of the mechanism of elevated IFN-␥ production in periodontal tissues are currently in progress in our laboratory.
12. 13. 14.
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ACKNOWLEDGMENTS 20.
We thank K. Miyake (The University of Tokyo, Tokyo, Japan) for generously supplying anti-TLR4 MAb HTA125 and HTA1216. We also thank D. Mrozek (Medical English Service, Kyoto, Japan) for reviewing the manuscript. This work was supported in part by Grants-in-Aid for Scientific Research from the Ministry of Education, Sports, Science, and Culture, Japan (no. 10470378) and from the Japan Society for the Promotion of Science (no. 12470380).
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