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Caries-related Bacteria and Cytokines Induce CXCL10 in Dental Pulp
T. Adachi,
T. Nakanishi*,
H. Yumoto,
K. Hirao,
K. Takahashi,
K. Mukai,
H. Nakae and
T. Matsuo
Department of Conservative Dentistry, Institute of Health Biosciences, The University of Tokushima Graduate School, 3-18-15 Kuramoto-cho, Tokushima 770-8504, Japan
Correspondence: * corresponding author, tadashi{at}dent.tokushima-u.ac.jp
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ABSTRACT
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Marked infiltration of inflammatory cells, such as activated T-cells, is observed in the progression of pulpitis; however, little is known about the mechanism of their recruitment into pulpal lesions. It has been recently demonstrated that CXC chemokine ligand 10 (CXCL10) chemoattracts CXC chemokine receptor 3 (CXCR3)-positive activated T-cells. We therefore examined whether CXCL10 is involved in the pathogenesis of pulpitis. CXCL10 mRNA expression levels in clinically inflamed dental pulp were higher than those in healthy dental pulp. Immunostaining results revealed that CXCL10 was detected in macrophages, endothelial cells, and fibroblasts in inflamed dental pulp, and that CXCR3 expression was observed mainly on T-cells. Moreover, cultured dental pulp fibroblasts produced CXCL10 after stimulation with live caries-related bacteria, peptidoglycans, and pro-inflammatory cytokines. In contrast, heat-killed bacteria did not induce CXCL10 secretion. These findings suggest that CXCL10-CXCR3 may play an important role in the pulpal immune response to caries-related bacterial invasion. Abbreviations: CXCL10, CXC chemokine ligand 10; CXCR3, CXC chemokine receptor 3; IFN, interferon; FBS, fetal bovine serum; LTA, lipoteichoic acid; PGN, peptidoglycan; IL, interleukin; TNF, tumor necrosis factor; PBS, phosphate-buffered saline; ELISA, enzyme-linked immunosorbent assay; CCL, C-C chemokine ligand; TLR, Toll-like receptor; NOD, nucleotide oligomerization domain; HDPF, human dental pulp fibroblasts.
Key Words: CXCL10 • pulpitis • dental pulp fibroblast
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INTRODUCTION
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Pulpitis is characterized as the immune response that is mainly triggered by the invasion of caries-related micro-organisms into dentinal tubules and pulp. It is known that T-cells are most likely the predominant lymphocyte population in inflamed dental pulp tissue (Hahn et al., 1989; Izumi et al., 1995). Chemokines are responsible for the recruitment and subsequent activation of particular leukocytes, such as activated T-cells, into inflamed tissues via specific chemokine receptors expressed on the cells (Sallusto et al., 2000); however, little is known about the role of chemokines in the accumulation of lymphocytes into the dental pulp lesion.
Non-ELR (absence of an NH2-terminal sequence Glu-Leu-Arg) CXC chemokines are interferon (IFN)-inducible and potently chemoattract activated T-cells (Luster, 1998). These chemokines signal through a common receptor, CXC chemokine receptor 3 (CXCR3), predominantly expressed on memory/activated T-cells (Loetscher et al., 1996). CXC chemokine ligand 10 (CXCL10)/IFN- -inducible protein-10 is a member of the non-ELR CXC chemokine family, and it can be induced in a variety of cell types, including macrophages stimulated with IFN- (Loetscher et al., 1996). In pulpitis, the selective accumulation of lymphocyte subsets into inflamed dental pulp may be regulated by CXCL10-CXCR3 interactions.
In the present study, we examined the mRNA expression of CXCL10 in human dental pulp tissues by RT-PCR, the distribution of CXCL10- and CXCR3-expressing cells in dental pulp tissues by immunohistochemistry, and also the expression of CXCL10 in human dental pulp fibroblasts (HDPF) following exposure to caries-related bacteria, bacterial components, and various cytokines in vitro, to elucidate whether CXCL10 may contribute to the pathogenesis of pulpitis.
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MATERIALS & METHODS
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Dental Pulp Tissue Samples
Inflamed dental pulp tissue samples from 14 individuals (from 23- to 50-year-old patients) with irreversible pulpitis caused by carious exposure of the pulp, and showing spontaneous pain and/or lingering pain in response to cold and/or heat stimulus, and clinically healthy dental pulp tissue samples from 11 individuals (from 18- to 30-year-old patients) were obtained under informed consent at Tokushima University Hospital. The medical histories of all persons in this study were non-contributory. Inflamed pulps for RNA extraction were obtained from teeth undergoing pulpectomy (n = 9), and inflamed pulps for immunohistochemical analysis were obtained from carious third molars (n = 5). Healthy pulps for RNA extraction (n = 4), cell culture (n = 3), and immunohistochemical analysis (n = 4) were obtained from non-carious teeth extracted for orthodontic reasons. The study was performed with the approval and compliance of the Tokushima University Ethical Committee.
Cell Culture
Human dental pulp tissues were obtained from extracted non-carious premolars as described above. We established HDPF cultures from minced pieces of pulp tissues by explanting them into 35-mm culture dishes containing Dulbecco’s modified Eagle’s medium (DMEM) (Gibco, Grand Island, NY, USA), supplemented with 10% fetal bovine serum (FBS) (JRH Bioscience, Lenexa, KS, USA), 1 mM sodium pyruvate (Gibco), and 50 IU/mL penicillin/50 3g/mL streptomycin (Gibco) at 37°C in a humidified atmosphere of 5% CO2 (Sundqvist et al., 1994). Confluent primary cultures were harvested and subcultured. Morphologically fibroblastic cells obtained by this method were used as HDPF for experiments at passages 4 to 10.
Bacteria
The strains used in this study were kindly provided by the following colleagues: Streptococcus mutans MT8148 (Dr. T. Ooshima, Osaka University, Osaka, Japan), and Lactobacillus plantarum ATCC8014 (Dr. K. Fukui, Tokushima University, Tokushima, Japan). S. mutans were grown in Brain-Heart Infusion broth (Difco Laboratories, Detroit, MI, USA) at 37°C for 8 hrs, and L. plantarum were grown in Lactobacilli Inoculum broth (Nissui Pharmaceutical Co., Tokyo, Japan) at 37°C for 12 hrs. The bacterial concentrations were determined spectrophotometrically with a standard curve. Bacteria grown in liquid media were harvested in the stationary phase by centrifugation at 7000 g for 7 min and washed 3 times; the number of bacteria was then adjusted by dilution with DMEM devoid of antibiotics. We then boiled them at 100°C for 10 min to obtain heat-killed bacteria.
Stimulation Experiments
HDPF were seeded in wells of 24-well tissue culture plates and incubated until a confluent monolayer developed (5 x 104 cells/well). The media were then replaced with DMEM containing 1% FBS and antibiotics. After 24 hrs, the cells were treated with lipoteichoic acid (LTA) derived from Staphylococcus aureus (Sigma-Aldrich, Walkersville, MD, USA), peptidoglycan (PGN) derived from S. aureus (Sigma-Aldrich), IFN- (PeproTech, London, UK), interleukin (IL)-1β (R&D Systems, Minneapolis, MN, USA), IL-4 (Boehringer Mannheim, Mannheim, Germany), and tumor necrosis factor- (TNF-) (Upstate Biotechnology, Lake Placid, NY, USA) in fresh media for designated times. In the case of bacterial stimulant, live or heat-killed bacteria were added to the cells at the ratio (bacteria:cells) of 10:1 to 100:1 in DMEM without antibiotics for 12 hrs or 24 hrs, respectively. After incubation, we collected cell culture supernatants and used them to determine CXCL10 concentration. Attached cells were used for RNA extraction.
RNA Extraction and RT-PCR
Total RNA was extracted from dental pulp tissues or cultured cells with NucleoSpin RNA II (Macherey-Nagel Ltd., Banbury, UK), according to the manufacturer’s instructions. RT-PCR was performed with a Reverse-iT RTase Blend kit (Abgene, Epsom, UK). The following primers were used to amplify a fragment of CXCL10: 5'-TGACTCTAAGTGGCATTCAAGG-3' (sense) and 5'-AGTTCAGACATCTCTCTTCTCACCC-3' (antisense). PCR conditions for CXCL10 were as follows: denaturation at 94°C for 1 min, annealing at 52°C for 1 min, and extension at 72°C for 1 min. The number of PCR cycles was 30. The GAPDH housekeeping gene transcript was used as a control with the primer pair: [5'-TGAAGGTCGGAGTCAACGGATTTGGT-3' (sense) and 5'-CACCACCTGGAGTACCGGGTGTAC-3' (antisense)]. A sample of each amplified product was subjected to 1.5% agarose gel electrophoresis (Takarabio, Shiga, Japan), stained with ethidium bromide and visualized by UV illumination (Cosmobio, Tokyo, Japan). The density of each band was semi-quantified by NIH Image 1.63 software, and the relative intensities (CXCL10/GAPDH ratio) were calculated.
Immunohistochemistry
Dental pulp for immunohistochemical analysis was obtained from each tooth halved lengthwise by means of a diamond instrument, followed by a spoon excavator. Formalin-fixed, paraffin-embedded six-micrometer-thick serial tissue sections were treated with 3% H2O2 for 5 min, to eliminate intrinsic peroxidase. After the blocking procedure with 0.25% casein in phosphate-buffered saline (PBS), sections were treated with goat polyclonal anti-human CXCL10 (R&D Systems) or mouse monoclonal anti-human CXCR3 (DAKO Corporation, Carpinteria, CA, USA) antibodies overnight at 4°C. Sections were incubated with biotinylated anti-goat immunoglobulin (DAKO) or biotinylated anti-mouse immunoglobulin (DAKO) for 20 min at room temperature, and then treated with peroxidase-conjugated streptavidin (DAKO) for 10 min. The reaction was visualized by 3,3-diaminobenzidine tetrahydrochloride (DAKO). Sections were counterstained with hematoxylin and mounted with glycerol. Isotype-matched mouse antibody (DAKO) or normal goat IgG (DAKO) was used as the negative control. To confirm CXCL10-positive or CXCR3-positive cells, we performed double staining, using mouse monoclonal anti-human CD68/macrophage (DAKO) or CD45RO/T-cell (DAKO) antibody and the alkaline phosphatase-labeled streptavidin biotin method (New Fuchsin substrate system, DAKO) (Nakane, 1968).
CXCL10 Measurement
Concentrations of CXCL10 in cell culture supernatants were determined by means of a commercially available enzyme-linked immunosorbent assay (ELISA) kit (DuoSet ELISA Development system) (R&D Systems).
Statistical Analysis
Data are expressed as the mean ± standard deviation. Statistical analyses were performed by unpaired Student’s t test.
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RESULTS
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CXCL10 mRNA Expression in Human Dental Pulp Tissues
First, we examined CXCL10 expression at the mRNA level in human healthy and inflamed dental pulps. CXCL10 mRNA expression was detected in 8 of 9 inflamed samples, and in 3 of 4 healthy samples. The level of CXCL10 compared with GAPDH in the inflamed pulp group was significantly higher than that in the healthy pulp group (Fig. 1 ).
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Figure 1. Expression of CXCL10 mRNA in human dental pulp tissues. CXCL10 mRNA expression was detected by RT-PCR in 8 of 9 inflamed dental pulp samples, and in 3 of 4 healthy dental pulp samples. The level of CXCL10 compared with that of GAPDH in the inflamed pulp group was significantly higher than that in the normal pulp group.
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Localization of CXCL10- and CXCR3-positive Cells
Using immunohistochemistry, we examined whether human dental pulp tissues expressed CXCL10 and CXCR3. CXCL10 was detected in macrophages, endothelial cells, and fibroblasts in inflamed pulp (Figs. 2A–2C). CXCR3 was detected mainly on T-cells in inflamed pulp (Figs. 2D–2F). CXCL10 and CXCR3 were hardly detected in healthy pulp (data not shown).
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Figure 2. Immunolocalization of CXCL10 and its receptor, CXCR3, in inflamed dental pulp tissues. (A) CXCL10-positive cells were stained brown. (B) Section double-stained with anti-CXCL10 and anti-CD68/macrophage. Black arrows indicate CXCL10-positive macrophages (brown and pink). White arrows indicate CXCL10-positive fibroblastic cells (brown), and arrowhead indicates CXCL10-positive endothelial cells (brown). (C) Higher magnification of (B). Black arrows indicate CXCL10-expressing macrophages (brown and pink), and the white arrow indicates CXCL10-expressing fibroblastic cells (brown). (D) Arrows indicate CXCR3-positive cells (brown). (E) Arrows indicate T-cells (pink). (F) Section double-stained with anti-CXCR3 and anti-T-cells. Most T-cells were CXCR3-positive (black arrows; brown and pink). Only a few T-cells were CXCR3-negative (white arrows; pink). Bars: 50 µm.
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Induction of CXCL10 in HDPF upon Stimulation with Bacteria
To demonstrate the mechanism of CXCL10 induction in HDPF, we examined CXCL10 expression in bacteria-stimulated HDPF using RT-PCR and ELISA. HDPF stimulated with live S. mutans expressed CXCL10 mRNA at 12 hrs, and the mRNA level of CXCL10 was increased at 24 hrs (Fig. 3A). CXCL10 levels in the cell culture supernatant of HDPF were also significantly increased by live S. mutans stimulation (Fig. 3A). Similarly, the level of CXCL10 produced by live L. plantarum-stimulated HDPF was significantly higher than that of non-stimulated cells (Fig. 3B). In contrast, heat-killed bacteria did not induce CXCL10 in HDPF.
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Figure 3. Induction of CXCL10 production in cultured human dental pulp fibroblasts upon stimulation with bacteria. (A,B) Human dental pulp fibroblasts (5 x 104 cells/well) were incubated with live or heat-killed S. mutans (A), and live or heat-killed L. plantarum (B) (bacteria/fibroblasts = 0, 10, 100). After 12 or 24 hrs, cell culture supernatants were collected, and secreted CXCL10 levels were determined by ELISA. Total RNA from fibroblasts was also extracted, and CXCL10 mRNA expression was analyzed by RT-PCR. (C) Human dental pulp fibroblasts (5 x 104 cells/well) were stimulated with various concentrations of PGN for 4, 12, and 24 hrs. CXCL10 levels in the cell culture supernatants were determined by ELISA. Data are expressed as the mean + SD of triplicate cultures from 1 representative of 3 independent experiments with similar results. *P < 0.05 and **P < 0.01 compared with control (medium alone for the respective incubation time).
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We also examined the effects of LTA and PGN on CXCL10 production in HDPF. An increased level of CXCL10 protein in HDPF was observed at 12 hrs after PGN stimulation (Fig. 3C ). In contrast, the CXCL10 level in LTA-stimulated HDPF was below detection limits (data not shown).
Kinetics of Cytokine-induced CXCL10 in HDPF
We next investigated whether HDPF produced CXCL10 by stimulation with pro-inflammatory cytokines (TNF- and IL-1β ), Th1 cytokine (IFN-), and Th2 cytokine (IL-4). CXCL10 mRNA expression in HDPF was induced by the addition of = 1 ng/mL TNF-, = 0.1 ng/mL IL-1β, and = 1 ng/mL IFN- for 4 hrs, and, similarly, a significantly higher level of CXCL10 protein was produced (Figs. 4A–4C). In contrast, IL-4 did not induce the expression of CXCL10 under the conditions tested (data not shown).
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DISCUSSION
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Increased expressions of chemokines such as interleukin-8, C-C chemokine ligand (CCL) 20, and CCL2 are found in inflamed dental pulp (Huang et al., 1999; Nakanishi et al., 2005; Durand et al., 2006); however, the mechanism of activated lymphocyte infiltration in dental pulp tissues was unclear. In periapical granulomas, the presence of other chemokines (such as CCL3, CCL4, and CCL5) related to lymphocyte recruitment has been demonstrated (Marton et al., 2000; Kabashima et al., 2001). These chemokines might contribute to the formation of chemokine networks in activated T-cell infiltration in inflamed dental pulp lesions, although they have not yet been elucidated. In the present study, we first demonstrated that CXCL10 expression in inflamed dental pulp tissues was significantly increased compared with that in healthy dental pulp. In addition, many CXCR3-expressing T-cells were observed in inflamed pulp. These findings suggest that CXCL10 may act as a key chemokine in the accumulation of activated lymphocytes in pulpitis.
Fibroblasts can produce many kinds of chemokines by stimulation with bacteria and cytokines (Brouty-Boye et al., 2000). Pro-inflammatory cytokines and Toll-like receptor (TLR) ligands such as viral dsRNA are potent inducers of CXCL10 in fibroblasts (Proost et al., 2003); however, it is unknown whether dental pulp fibroblasts can produce CXCL10. Immunohistochemical analysis in this study showed that CXCL10 expression was detected on fibroblasts in inflamed pulp. Accordingly, we examined whether caries-related bacteria induced HDPF to produce CXCL10. S. mutans and L. plantarum were selected as caries-related bacteria, because they are frequently found in deep caries lesions (Hahn et al., 1991; Ozaki et al., 1994). Live, but not heat-killed, bacteria had the ability to induce CXCL10 expression in HDPF. Although it is not clear whether the bacteria affected CXCL10 induction in the fibroblasts directly or through the production of cytokines, bacterial products from live bacteria are likely to be responsible for CXCL10 expression.
Both S. mutans and L. plantarum are Gram-positive bacteria, which have bacterial cell wall components such as LTA and PGN. We next examined whether LTA and PGN play a role in inducing CXCL10 production in HDPF. Consequently, HDPF stimulated with PGN, but not LTA, were able to produce CXCL10. It is known that LTA and PGN are recognized by TLR2 (Michelsen et al., 2001). The differences in capacity between LTA and PGN to elicit CXCL10 production from HDPF might be due to nucleotide oligomerization domain (NOD) proteins, which are novel intracellular pattern-recognition molecules for PGN recognition, as well as TLR2 (Girardin et al., 2003). Indeed, it has been reported that commercially available PGN contained a specific structure recognized by NODs (Netea et al., 2005). Analysis of our preliminary data also revealed that TLR2, NOD1, and NOD2 were expressed in HDPF (data not shown). Thus, it is possible, in our experimental system, that PGN interacted with both TLR2 and NODs, but that LTA interacted only with TLR2. In addition, our present study showed that PGN, but not heat-killed bacteria, induced the up-regulation of CXCL10 in HDPF. Regarding this discrepancy, we assume that heat-killed bacteria did not contain sufficient PGN to induce CXCL10 production.
It is known that immune cells in inflamed dental pulp produce a variety of cytokines, which can modify the pathogenesis of pulpitis (Hahn et al., 2000). We next investigated whether selected cytokines, which are detected in dental pulp tissues, induced CXCL10 expression from HDPF. Our findings revealed that IFN-, but not IL-4, had the ability to induce CXCL10 production from HDPF. A recent report demonstrated that Th1 cells, in comparison with Th2 cells, preferentially induced the production of CXCL10 by skin fibroblasts (Chizzolini et al., 2006). Moreover, it has been reported that dental pulp cells produce IFN- in response to S. mutans (Hahn et al., 2000). Thus, it may be hypothesized that HDPF stimulated with S. mutans produce CXCL10 via IFN- production.
Pro-inflammatory cytokines such as TNF- and IL-1β also induced CXCL10 expression by HDPF. It has been reported that TNF- , but not IFN- , is the main inducer of CXCL10 in skin fibroblasts (Proost et al., 2003; Villagomez et al., 2004). Our results also showed that TNF- was a more powerful inducer of CXCL10 in HDPF than was IFN- . In contrast, IFN- was a more potent inducer of CXCL10 in peripheral blood mononuclear cells (Proost et al., 2003). Thus, this indicates that the inductive effect of cytokine on CXCL10 expression is dependent on the cell type.
In conclusion, we elucidated, for the first time, that an increased expression level of CXCL10 was detected in inflamed dental pulp tissues. Moreover, dental pulp fibroblasts had the capacity to produce CXCL10 in response to caries-related bacteria and pro-inflammatory cytokines. Thus, our findings suggest that the CXCL10-CXCR3 system may be involved in the pathogenesis of pulpitis.
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ACKNOWLEDGMENTS
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This research was supported by a Grant-in-Aid for Scientific Research from the Japan Society for the Promotion of Science (16591915).
Received for publication February 13, 2007.
Revision received August 4, 2007.
Accepted for publication September 12, 2007.
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Journal of Dental Research, Vol. 86, No. 12,
1217-1222 (2007)
DOI: 10.1177/154405910708601215
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ABSTRACT
INTRODUCTION