SEARCH

SEARCH BY CITATION

Keywords:

  • Chemokine;
  • oral keratinocytes;
  • oral fibroblasts;
  • Toll-like receptor

ABSTRACT

  1. Top of page
  2. ABSTRACT
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. ACKNOWLEDGMENTS
  7. DISCLOSURE
  8. REFERENCES

Oral keratinocytes and fibroblasts may be the first line of host defense against oral microorganisms. Here, the contention that oral keratinocytes and fibroblasts recognize microbial components via Toll-like receptors (TLRs) and participate in development of oral inflammation was examined. It was found that immortalized oral keratinocytes (RT7), fibroblasts (GT1) and primary cells express mRNA of TLRs 1–10. Interleukin-8 (IL-8) production by RT7 cells was induced by treatment with TLRs 1–9 with the exception of TLR7 agonist, whereas GT1 cells were induced to produce IL-8 by all TLR agonists tested except for TLR7 and TLR9. GT1 cells showed increased CXCL10 production following treatment with agonists for TLR1/2, TLR3, TLR4, and TLR5, whereas only those for TLR3 and TLR5 increased CXCL10 production in RT7 cells. Moreover, TLR agonists differentially regulated tumor necrosis factor-alpha-induced IL-8 and CXCL10 production by the tested cell types. These findings suggest that recognition of pathogenic microorganisms in oral keratinocytes and fibroblasts by TLRs may have important roles in orchestrating host immune responses via production of various chemokines.

List of Abbreviations
ASMC

airway smooth muscle cells

CpG

cytosine-phosphorothioate-guanine

E. coli

Escherichia coli

hTERT

human telomerase reverse transcriptase

IFN-γ

interferon-gamma

IL

interleukin

IRF

interferon regulatory factor

LPS

lipopolysaccharide

LTA

lipoteichoic acid

MALP

mycoplasma lipopeptide

MAPK

mitogen-activated protein kinase

MyD

myeloid differentiation primary response gene

NF-κB

nuclear factor-kappa

ODN

oligodeoxynucleotide

PAMPS

pathogen-associated molecules patterns

poly I:C

polyinosinic-polycytidylic acid

TLR

Toll-like receptor

TNF-α

tumor necrosis factor-alpha

The innate immune response is the first line of host defense. Recognition of different classes of microorganisms involves signaling through specific receptors, one major group of which is TLRs [1]. TLRs recognize various PAMPS conserved in microorganisms, including triacylated lipoproteins (TLR1/2 agonist), diacylated lipoproteins (TLR2/6 agonist), double-stranded RNA (TLR3 agonist), LPS (TLR4 agonist), flagellin (TLR5 agonist), single-stranded RNA (TLR7, TLR8 agonist) [2] and CpG motifs in DNA (TLR9 agonist) [3]. Following pathogen detection, these TLRs mediate activation of innate and adaptive immune responses through modulation of gene expression by immune cells [1]. Recently, the connective tissue and epithelial cell layers of both healthy and infected gingival tissues were shown to express various TLRs [4], implying that oral keratinocytes and fibroblasts play an essential role in orchestrating immune responses against pathogens via TLRs.

Interleukin-8, a potent neutrophil chemoattractant and activator, has been associated with the pathogenesis of various forms of periodontitis, accumulation and degranulation of neutrophils with subsequent destruction of normal tissue being a common feature of these diseases [5]. On the other hand, CXCL10, which is highly expressed in aggressive periodontitis and lichen planus, plays an important role in T-cell-mediated oral inflammation through its T-cell chemotactic and adhesion-promoting activities [6, 7]. TLR agonists cause secretion of proinflammatory chemokines and double-stranded RNA (TLR3) was found to dramatically increase IL-8 and CXCL10 in airway epithelial cells [8, 9]. Also, stimulation with LTA (TLR2) and LPS (TLR4) increases IL-8 production by intestinal myofibroblasts [10]. On the other hand, TNF-α is a major inflammatory cytokine in inflammatory diseases related to periodontitis [11]. Oral keratinocytes and fibroblasts produce IL-8 and CXCL10 in response to TNF-α production of these chemokines by oral keratinocytes is thought to lead to oral inflammation [12, 13]. However, it remains unknown whether direct recognition of PAMPS, rather than immune cells, regulates IL-8 and CXCL10 or augments TNF-α-mediated inflammatory responses by oral keratinocytes and fibroblasts in oral inflammatory diseases.

We postulated that oral keratinocytes, as well as oral fibroblasts, recognize PAMPS from bacterial pathogens via TLRs and cooperatively participate in oral inflammatory diseases. In the present study, we examined IL-8 and CXCL10 production by immortalized human oral keratinocytes (RT7) and gingival fibroblasts (GT1) in response to various TLR agonists. Subsequently, we investigated the effects of TLR agonists combined with TNF-α on production of IL-8 and CXCL10.

MATERIALS AND METHODS

  1. Top of page
  2. ABSTRACT
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. ACKNOWLEDGMENTS
  7. DISCLOSURE
  8. REFERENCES

Cell lines

RT7, an immortalized human oral keratinocyte cell line, was established by transfection of hTERT and E7, as previously described [14], whereas GT1, a human oral fibroblast cell line, was established by transfection of hTERT, as previously described [15]. RT7 was cultured in keratinocyte-serum-free medium (Gibco BRL, Gaithersburg, MD, USA), which includes 25 μg/mL bovine pituitary extract, 0.05 ng/mL epidermal growth factor, 100 U/mL penicillin and 100 μg/mL streptomycin. GT1 was cultured in Dulbecco's modified Eagle's medium (Sigma Chemical, St. Louis, MO, USA) containing 10% FCS, 100 U/mL penicillin and 100 μg/mL streptomycin. Primary cultures of human gingival keratinocytes and fibroblasts obtained from a healthy volunteer were also prepared [14]. Informed consent for acquisition was obtained according to a protocol approved by the Ethical Committee of Hiroshima University.

Toll-like receptor agonists

The TLR agonists tested in this study were purchased from Imgenex (San Diego, CA, USA). Those included Pam3CSK4, a synthetic bacterial lipopeptide (TLR1/2 agonist), poly I:C, a synthetic viruses double stranded RNA (TLR3 agonist), Escherichia coli LPS, a synthetic cell wall component of gram-negative bacteria (TLR4 agonist), flagellin from Salmonella typhimurium, a synthetic bacterial flagellin (TLR5 agonist), MALP-2, a synthetic Mycoplasma lipopeptide (TLR2/6 agonist), imiquimod (R-837), a synthetic molecule of the imidazoquinoline family (TLR7 agonist) and synthetic ODN containing CpG motifs, a synthetic bacterial DNA (TLR9 agonist).

RNA extraction and reverse transcription polymerase chain reaction

Total RNA was prepared from the cell lines using an RNeasy total RNA isolation kit (Qiagen, Hilden, Germany). Single-stranded cDNA for a PCR template was synthesized from a First Strand cDNA Synthesis kit (Amersham Biosciences, Uppsala, Sweden). Target cDNA was amplified by PCR using an RT-PCR High Plus system (Toyobo, Osaka, Japan), using specific primers (Table 1). The PCR conditions were one cycle at 95°C for 15 min, 40 cycles at 95°C for 2 min, one cycle at 55°C for 30 s, one cycle at 72°C for 1 min and one cycle at 72°C for 7 min. The products were analyzed on 2% agarose gels. β-actin was included as an internal control.

Table 1. Specific primers of main target mRNA expression in the present study
Target of mRNA expressionPrimer
TLR15′-CACCAAGTTGTCAGCGATGT-3′
 5′-CCACATCCAGGAAGGTCAGT-3′
TLR25′-GCCAAAGTCTTGATTGATTGG-3′
 5′-TTGAAGTTCTCCAGCTCCTG-3′
TLR35′-AAATTGGGCAAGAACTCACAGG-3′
 5′-GTGTTTCCAGAGCCGTGCTAA-3′
TLR45′-GGTGGAAGTTGAACGAATGG-3′
 5′-CTGTCCTCCCACTCCAGGATA-3′
TLR55′-CCTTACAGCGAACCTCATCC-3′
 5′-AAGAGGGAAACCCCAGAGAA-3′
TLR65′-TTGACAGTTTTGAGACTTTCCC-3′
 5′-TGGACCTCTGGTGAGTCCTG-3′
TLR75′-TCCAGTGTCTAAAGAACCTGG-3′
 5′-TGGTAAATATACCACACATCCC-3′
TLR85′-TAATAGGCTGAAGCACATCCC-3′
 5′-TCCCAGTAAAACAAATGGTGAG-3′
TLR95′-GTGCCCCACTTCTCCATG-3′
 5′-GGCACAGTCATGATGTTGTTG-3′
TLR105′-GGATGCTAGGTCAATGCACA-3′
 5′-ATAGCAGCTCGAAGGTTTGC-3′
IL-85′-ATGACTTCCAAGCTGGCCGTGGC-3′
 5′-TCTCAGCCCTCTTCAAAAACTTCTC-3′
CXCL105′-GCAGCTGATTTGGTGACCATCATTGG-3′
 5'-TGCAAGCCAATTTTGTCCACGTGTTG-3'
β-actin5′-TCACCCACACTGTGCCCATCTACGA-3′
 5′-CAGCGGAACCGCTCATTGCCAATGG-3′

Real-time polymerase chain reaction

RT7 (5 × 104 cells/well) and GT1 (5 × 104 cells/well) were seeded into six-well cell culture plates in each medium. After 3 days of incubation, the cells reached approximately 80% confluence, then were exposed to Pam3CSK4 1 μg/mL, poly I:C 1 μg/mL, E. coli LPS 10 μg/mL, flagellin 100 ng/mL, MALP-2 50 ng/mL, and imiquimod 10 μg/mL for 12 hr. RNA from each culture was extracted and synthesized with cDNA, as noted above. The synthesized cDNAs were used for quantitative PCR analysis with oligonucleotide primers (Table 1). Quantitative PCR analysis was performed using an ABI PRISM 7700 Sequence Detection System (Perkin-Elmer, Foster City, CA, USA) and SYBR-Green Master Mix (Applied Biosystems) for 40 cycles at 95°C for 15 s and 60°C for 60 s. Values for quantification of amounts of chemokine mRNA are shown as relative to the internal control, β-actin and as the mean ± standard deviation from three independent experiments.

Quantification of IL-8 and CXCL10 protein

RT7 and GT1were seeded into 96-well cell culture plates in the appropriate medium. To determine the concentration of TLR agonists corresponding to maximal effectiveness in regard to their activating capacity, IL-8 and CXCL10 protein production following stimulation with TLR agonists were assessed at various doses (Pam3CSK4 100 ng/mL to 5 μg/mL, poly I:C 100 ng/mL to 5 μg/mL, LPS 1–20 μg/mL, flagellin 10–500 ng/mL, MALP-2 1–50 ng/mL, imiquimod 0.1–50 μg/mL). Based on those results, cells were exposed to Pam3CSK4 1 μg/mL, poly I:C 1 μg/mL, E. coli LPS 10 μg/mL, flagellin 100 ng/mL, MALP-2 50 ng/mL, imiquimod 10 μg/mL, CpG-ODN (1 and 10 μg/mL) negative control GpC (negative ODN) (1 and 10 μg/mL) instead of CpG, and TNF-α 10 ng/mL or combinations thereof for 48 hr. The collected media were centrifuged and the supernatant fluids stored at −80°C prior to performing assays. The protein concentrations of each chemokine in the medium were determined using an ELISA kit (R&D Systems, Minneapolis, MI, USA), according to the protocol recommended by the manufacturer.

Statistical analysis

Data were analyzed by Student's t-test or ANOVA using the Bonferroni or Dunn method and the results presented as the mean ± standard deviation.

RESULTS

  1. Top of page
  2. ABSTRACT
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. ACKNOWLEDGMENTS
  7. DISCLOSURE
  8. REFERENCES

mRNA expression of Toll-like receptors in RT7 and GT1

We first examined whether oral keratinocytes and fibroblasts express TLR 1-10. As shown in Figure 1, primary oral keratinocytes and RT7 growing in culture expressed the mRNA of TLR 1-10, whereas primary gingival fibroblasts and GT1 also constitutively expressed the mRNA of TLRs 1–10 (Fig. 1).

image

Figure 1. mRNA expression of TLRs 1–10 in RT7, GT1 and primary cells. Total RNA was isolated from each cell line at confluence, after which RT-PCR was performed for TLRs 1–10 and β-actin (control).

Download figure to PowerPoint

Effects of Toll-like receptor agonists on interleukin-8 expression in RT7 and GT1

Because functional TLRs lead biological responses, we examined the effects of various TLR agonists on IL-8 mRNA expression in RT7 and GT1. As demonstrated in Figure 2, Pam3CSK4 (TLR1/2), poly I:C (TLR3), E. coli LPS (TLR4), flagellin (TLR5) and MALP-2 (TLR2/6) increased IL-8 mRNA expression in both cell lines; imiquimod (TLR7) was the only exception (Fig. 2). Compatible with the level of mRNA expression, we detected significant IL-8 production by these cells in culture medium after treatment with each agonist at various concentrations for 48 hr (Fig. 3). On the other hand, CpG-ODN (TLR9) increased IL-8 production in RT7, but not in GT1 (Fig. 4). Imiquimod (TLR7) did not have an effect on IL-8 production within the range of 0.1–50 μg/mL (data not shown).

image

Figure 2. Effects of various TLR agonists on expression of IL-8 and CXCL10 mRNA in RT7 and GT1. IL-8 or CXCL10 mRNA expression in (a, c) RT7 and (b, d) GT1 stimulated with TLR agonists for TLRs 1–7. Cells were cultured as described in the Materials and Methods section and exposed to Pam3CSK4 1 μg/mL, poly I:C 1 μg/mL, E. coli LPS 10 μg/mL, flagellin 100 ng/mL, MALP-2 50 ng/mL, and imiquimod 10 μg/mL for 24 hr. Levels of chemokine mRNA expression are shown as relative to the internal control, β-actin and values are presented as the mean ± standard deviation of three independent experiments. *Significantly different from non-treated cells (Student's t-test: P < 0.05).

Download figure to PowerPoint

image

Figure 3. Effects of various TLR agonists on IL-8 protein in RT7 and GT1. IL-8 protein concentrations in (a) RT7 and (b) GT1 stimulated with different concentration of TLR agonists for TLRs 1-2/6. Cells were cultured as described in the Materials and Methods section and exposed to various concentrations of TLR agonists for TLRs 1-2/6 for 48 hr. Data are shown as the mean ± standard deviation of three independent experiments. *Significant increase as compared with non-treated cells (Dunnett's test: P < 0.05).

Download figure to PowerPoint

image

Figure 4. Effects of various TLR agonists on TNF-α − induced IL-8 protein in RT7 and GT1. IL-8 protein expressions in (a) RT7 and (b) GT1 stimulated with TLR agonists for TLR1-7 or TNF-α combinations. IL-8 protein expression in (c) RT7 and (d) GT1 stimulated with TLR agonists for TLR 9 or TNF-α combinations. Cells were cultured as described in the Materials and Methods section, then exposed to Pam3CSK4 1 μg/mL, poly I:C 1 μg/mL, E. coli LPS 10 μg/mL, flagellin 100 ng/mL, MALP-2 50 ng/mL, imiquimod 10 μg/mL, CpG-ODN (1 and 10 μg/mL) negative control GpC (negative oligo) (1 and 10 μg/mL) instead of CpG and TNF-α 10 ng/mL for 48 hr, after which the concentrations of IL-8 in the culture supernatants were measured by ELISA. Data are shown as the mean ± standard deviation of three independent experiments. *Significant increase compared with non-treated cells (Student's t-test: P < 0.05). #Significant increase compared with TNF-α or TLR agonist alone (Student's t-test: P < 0.05).

Download figure to PowerPoint

Effects of Toll-like receptor agonists on CXCL10 production by RT7 and GT1

Next, we examined whether TLR agonists affect CXCL10 production by RT7 and GT. Poly I:C (TLR3) and flagellin (TLR5) increased CXCL10 mRNA and production in RT7 (Figs. 2 and 5). In contrast, CXCL10 mRNA was induced by Pam3CSK4 (TLR1/2), poly I:C (TLR3), E. coli LPS (TLR4) and flagellin (TLR5) in GT1 (Fig. 2). These TLR agonists increased CXCL10 protein in a dose-dependent manner, the results being compatible with those regarding mRNA expression (Fig. 5). In particular, poly I:C (TLR3) dramatically increased CXCL10 in both cells (Fig. 5). In contrast, CXCL10 was not induced by imiquimod (TLR7) within the range of 0.1–50 μg/mL (data not shown).

image

Figure 5. Effects of various TLR agonists on CXCL10 protein in RT7 and GT1. CXCL10 protein in (a) RT7 and (b) GT1 stimulated with different concentration of TLR agonists for TLRs 1-2/6. Cells were cultured as described in the Materials and Methods section and exposed to various concentrations of TLR agonists for TLR 1-2/6 for 48 hr. Data are shown as the mean ± standard deviation of three independent experiments. *Significant increase as compared with non-treated cells (Dunnett's test: P < 0.05).

Download figure to PowerPoint

Pathogen-associated molecules patterns modify tumor necrosis factor-alpha induced interleukin-8 and CXCL10 production by RT7 and GT1

We examined the effects of a combination of TLR agonists and TNF-α on IL-8 and CXCL10 expression. Pam3CSK4, poly I:C, LPS, flagellin, and MALP-2 enhanced TNF-α-induced IL-8 production by both RT7 and GT1 (Fig. 4), whereas addition of Pam3CSK4 and poly I:C enhanced TNF-α-induced CXCL10 production by both cell lines (Fig. 6). On the other hands, CpG-ODN enhanced TNF-α-induced IL-8 by RT7 but not GT1 (Fig. 4), whereas the addition of LPS and CpG-ODN abolished TNF-α-induced CXCL10 in GT1, but not RT7 (Fig. 6).

image

Figure 6. Effects of various TLR agonists on TNF-α-induced CXCL10 proteins in RT7 and GT1. CXCL10 protein expression in (a) RT7 and (b, c) GT1 stimulated with TLR agonists for TLRs 1–7 or TNF-α combinations. CXCL10 protein expression in (d) RT7 and (e) GT1 stimulated with TLR agonists for TLR 9 or TNF-α combinations. Cells were cultured as described in the Materials and Methods section, then exposed to Pam3CSK4 1 μg/mL, poly I:C 1 μg/mL, E. coli LPS 10 μg/mL, flagellin 100 ng/mL, MALP-2 50 ng/mL, imiquimod 10 μg/mL, CpG-ODN (1 and 10 μg/mL), negative control GpC (negative oligo) (1 and 10 μg/mL) instead of CpG and TNF-α 10 ng/mL for 48 hr, after which the concentrations of CXCL10 in the culture supernatants were measured by ELISA. Data are shown as the mean ± standard deviation of three independent experiments. *Significant increase compared with non-treated cells (Student's t-test: P < 0.05). #Significant increase compared with TNF-α or TLR agonist alone (Student's t-test: P < 0.05). $Significant decrease compared with TNF-α alone (Student's t-test: P < 0.05).

Download figure to PowerPoint

DISCUSSION

  1. Top of page
  2. ABSTRACT
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. ACKNOWLEDGMENTS
  7. DISCLOSURE
  8. REFERENCES

Toll-like receptors have been found in many types of cells and are known to play a central role in pathogen recognition by the innate immune system [16]. Some investigators have reported that mRNAs of various TLRs are expressed in oral keratinocytes and fibroblasts [17, 18]. The present results demonstrate that both oral keratinocytes and gingival fibroblasts constitutively express mRNA of TLRs 1–10 (Fig. 1). An immunohistochemical study has shown that TLRs 1–9 (i.e. excluding TLR10) are present in epithelial and fibroblastic cells in gingival tissue [4]. Thus, TLR10 mRNA may not have the ability to produce protein in oral keratinocytes and fibroblasts.

It has also been reported that various TLR agonists (e.g., TLR1/2, 4, 9 for gingival epithelial cells, TLR3 and 5 for gingival fibroblasts) induce IL-8 in primary cultures [19, 20]. In agreement with these findings, we found that various TLR agonists induce IL-8 production by RT7 and GT1 (Figs. 3 and 5). The marked IL-8 production by RT7 in response to MALP-2 (TLR2/6 agonist) is noteworthy (Fig. 3) because it implies that oral keratinocytes are specifically involved in host defense responses against mycoplasma through TLR2/6, which has not been previously reported.

CXCL10 binds to the chemokine receptor CXCR3, which is mainly expressed by memory/activated T cells associated with Th1-type responses [21]. In a more recent study, expressions of CXCL10 and CXCR3 were more prevalent and stronger in gingival tissues from patients with aggressive periodontitis than in those from healthy subjects [22]. Poly I:C (TLR3 agonist) has been shown to increase CXCL10 in cultures of human dendritic cells, macrophages, endothelial cells and synovial fibroblasts isolated from rheumatoid arthritis joint tissue [23]. The present findings demonstrate that poly I:C directly induces production of CXCL10 in oral keratinocytes and fibroblasts (Fig. 5), suggesting that oral keratinocytes and fibroblasts infected by viruses may attract T cells toward the site of infection via CXCL10 secretion, leading to the development of T-cell-mediated oral inflammation.

A previous study found that TNF-α induces increases in expression of various chemokines, such as IL-8 and CXCL10, in oral keratinocytes and fibroblasts [12]. Also, the amounts of TNF-α in tissue biopsy specimens correlate strongly with the severity of periodontitis [24]. TLR agonists are known to enhance TNF-α-mediated inflammatory responses in ASMCs; a recent study reported that Pam3CSK4 increases TNF-α-induced IL-8 production via TLR1/2 [25]. Poly I:C (TLR3 agonist) treatment also results in marked enhancement of TNF-α-mediated CXCL10 production in ASMCs [26]. In the present study, we found that almost all TLR agonists, except for imiquimod (TLR7 agonist), enhanced TNF-α-dependent production of IL-8 by RT7 and GT1, whereas marked enhancement of TNF-α-dependent CXCL10 was induced only by poly I:C and Pam3CSK4 in these cells (Figs. 4 and 6). Therefore, TNF-α-dependent acute and chronic inflammation may be affected by gingival tissues through direct recognition of microorganisms by TLRs.

Some TLR agonists act not only as immune stimulatory agents but also induce strong immune suppression in inflammatory conditions [27]. For example, LPS (TLR4 agonist) abolishes IFN-γ-induced CXCL10 in peripheral blood mononuclear cells [23], whereas other investigations have demonstrated suppressive effects of CpG ODNs (TLR9 agonist) on skin and intestinal inflammation in mouse models [28, 29]. In our study, addition of LPS and CpG-ODN suppressed TNF-α dependent CXCL10 production by GT1, but not by RT7 (Figs. 4 and 6). These results indicate that microorganisms with specific PAMPS, such as CpG-ODN, and LPS, may take advantage of the anti-inflammatory properties of oral fibroblasts to escape from TNF-α dependent inflammatory protection.

Most TLR agonists activate NF-κB and MAPK via a MyD88-dependent pathway for induction of inflammatory cytokines [30]. Although the same TLR agonists, such as poly I:C, flagellin and Pam3CSK4 in GT1, tended to dramatically increase both IL-8 and CXCL10, the extent of induction of IL-8 and CXCL10 by the same TLR agonist differed in RT7 (Figs. 4 and 6). This differential induction of IL-8 and CXCL10 may be related to activation of diverse signaling pathways in RT7 by the TLR agonists. On the other hand, TNF-α activates transcription factors such as NF-κB, which are involved in transcription of IL-8 through MAPK in human airway epithelial cells [31]. Nearly all of the tested TLR agonists enhanced TNF-α-induced IL-8 production regardless of cell type (Figs. 4 and 6), which might be explained by TNF-α and TLR agonists (e.g., NF-kB and MAPK) sharing some signaling pathways for inducing IL-8. However, some TLR agonists differently modulate induction of TNF-α-induced CXCL10 production in different cell types. For example, in our study both flagellin and poly I:C alone increased CXCL10 production, whereas flagellin failed to enhance TNF-α-induced CXCL10 production (Figs. 4 and 6). Moreover, LPS and CpG-ODN decreased TNF-α-induced CXCL10 in GT1. Since specific TLRs (e.g., TLR3, TLR4, and TLR9) are able to activate the IRF signaling pathway [32], CXCL10 induction may be regulated through complex modification of or interference with the signal transduction pathway between TNF-α and TLR agonists in different cell types. Thus, differential modulation between keratinocytes and fibroblasts of IL-8 and CXCL10 by specific PAMPS and TNF-α may shape various leukocyte migration patterns in epithelium and connective tissue in oral inflammation disease.

In conclusion, our study demonstrates that: (i) oral keratinocytes and fibroblasts express mRNA of TLRs 1–10 mRNA; (ii) IL-8 and CXCL10 production by oral keratinocytes and fibroblasts is induced by treatment with various TLR agonists; and (iii) TLR agonists differentially regulate TNF-α-induced IL-8 and CXCL10 production by the tested cell types. Therefore, direct recognition of microorganism pathogens by those cell types may have an important role in promoting hyper/hypo reactions associated with oral inflammatory diseases.

ACKNOWLEDGMENTS

  1. Top of page
  2. ABSTRACT
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. ACKNOWLEDGMENTS
  7. DISCLOSURE
  8. REFERENCES

This work was supported by a Grant-in-Aid for scientific research from the Japan Society for Young Scientists (B) from the Ministry of Education, Culture, Sports, Science and Technology (No. 21109871).

DISCLOSURE

  1. Top of page
  2. ABSTRACT
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. ACKNOWLEDGMENTS
  7. DISCLOSURE
  8. REFERENCES

All authors declare no conflict of interest related to this work.

REFERENCES

  1. Top of page
  2. ABSTRACT
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. ACKNOWLEDGMENTS
  7. DISCLOSURE
  8. REFERENCES
  • 1
    Takeda K., Akira S. (2001) Roles of Toll-like receptors in innate immune responses. Genes Cells 6: 73342.
  • 2
    Kawai T., Akira S. (2007) Signaling to NF-kappaB by Toll-like receptors. Trends Mol Med 13: 4609.
  • 3
    Akira S., Hemmi H. (2003) Recognition of pathogen-associated molecular patterns by TLR family. Immunol Lett 85: 8595.
  • 4
    Beklen A., Hukkanen M., Richardson R., Konttinen Y.T. (2008) Immunohistochemical localization of Toll-like receptors 1- 10 in periodontitis. Oral Microbiol Immunol 23: 42531.
  • 5
    Takashiba S., Takigawa M., Takahashi K., Myokai F., Nishimura F., Chihara T., Kurihara H., Nomura Y., Murayama Y. (1992) Interleukin-8 is a major neutrophil chemotactic factor derived from cultured human gingival fibroblasts stimulated with interleukin-1 beta or tumor necrosis factor alpha. Infect Immun 60: 52538.
  • 6
    Garlet GP., Martins W, Jr., Ferreira B.R., Milanezi CM., Silva J.S. (2003) Patterns of chemokines and chemokine receptors expression in different forms of human periodontal disease. J Periodontal Res 38: 2107.
  • 7
    Iijima W., Ohtani H., Nakayama T., Sugawara Y., Sato E., Nagura H., Yoshie O., Sasano T. (2003) Infiltrating CD8+ T-cells in oral lichen planus predominantly express CCR5 and CXCR3 and carry respective chemokine ligands RANTES/CCL5 and IP-10/CXCL10 in their cytolytic granules: a potential self-recruiting mechanism. Am J Pathol 163: 2618.
  • 8
    Sha Q., Truong-Tran A.Q., Plitt J.R., Beck L.A., Schleimer R.P. (2004) Activation of airway epithelial cells by Toll-like receptor agonists. Am J Respir Cell Mol Biol 31: 35864.
  • 9
    Spurrell J.C., Wiehler S., Zaheer R.S., Sanders S.P., Proud D. (2005) Human airway epithelial cells produce IP-10 (CXCL10) in vitro and in vivo upon rhinovirus infection. Am J Physiol Lung Cell Mol Physiol 289: 8595.
  • 10
    Otte J.M., Rosenberg I.M., Podolsky D.K. (2003) Intestinal myofibroblasts in innate immune responses of the intestine. Gastroenterology 124: 186678.
  • 11
    Taubman M.A., Kawai T. (2001) Involvement of T-lymphocytes in periodontal disease and in direct and indirect induction of bone resorption. Crit Rev Oral Biol Med 12: 12535.
  • 12
    Ohta K., Shigeishi H., Taki M., Nishi H., Higashikawa K., Takechi M., Kamata N. (2008) Different regulation of CXCL9, CXCL10, and CXCL11 expression induced by IFN-γ, TNF-α and IL-4 in human oral keratinocytes and fibroblasts. J Dent Res 87: 11615.
  • 13
    Li J., Ireland G.W., Farthing P.M., Thornhill M.H. (1996) Epidermal and oral keratinocytes are induced to produce RANTES and IL-8 by cytokine stimulation. J Invest Dermatol 106: 6616.
  • 14
    Fujimoto R., Kamata N., Yokoyama K., Taki M., Tomonari M., Tsutsumi S., Yamanouchi H., Tomonari M., Nagayama M. (2002) Establishment of immortalized human oral keratinocytes by gene transfer of a telomerase component. J Jpn Oral Muco Membr 8: 18.
  • 15
    Kamata N., Fujimoto R., Tomonari M., Taki M., Nagayama M., Yasumoto S. (2004) Immortalization of human dental papilla, dental pulp, periodontal ligament cells and gingival fibroblasts by telomerase reverse transcriptase. J Oral Pathol Med 33: 41723.
  • 16
    Akira S., Uematsu S., Takeuchi O. (2006) Pathogen recognition and innate immunity. Cell 124: 783801.
  • 17
    Kinane D.F., Shiba H., Stathopoulou P.G., Zhao H., Lappin D.F., Singh A., Eskan M.A., Beckers S., Waigel S., Alpert B., Knudsen T.B. (2006) Gingival epithelial cells heterozygous for Toll-like receptor 4 polymorphisms Asp299Gly and Thr399ile are hypo-responsive to Porphyromonas gingivalis. Genes Immun 7: 190200.
  • 18
    Mahanonda R., Sa-Ard-Iam N., Montreekachon P., Pimkhaokham A., Yongvanichit K., Fukuda M.M., Pichyangkul S. (2007) IL-8 and IDO expression by human gingival fibroblasts via TLRs. J Immunol 178: 11517.
  • 19
    Onishi S., Honma K., Liang S., Stathopoulou P., Kinane D., Hajishengallis G., Sharma A. (2007) Toll-like receptor 2-mediated interleukin-8 expression in gingival epithelial cells by the Tannerella forsythia leucine-rich repeat protein BspA. Infect Immun 76: 198205.
  • 20
    Kim Y., Jo A.R., Jang D.H., Cho Y.J., Chun J., Min B.M., Choi Y. (2011) Toll-like receptor 9 mediates oral bacteria-induced IL-8 expression in gingival epithelial cells. Immunol Cell Biol 90: 65563, doi: 10.1038/icb.2011.85
  • 21
    Qin S., Rottman J.B., Myers P., Kassam N., Weinblatt M., Loetscher M., Koch A.E., Moser B., Mackay CR. (1998) The chemokine receptors CXCR3 and CCR5 mark subsets of T cells associated with certain inflammatory reactions. J Clin Invest 101: 74654.
  • 22
    Garlet G.P., Martins W.Jr., Ferreira B.R., Milanezi C.M., Silva J.S. (2003) Patterns of chemokines and chemokine receptors expression in different forms of human periodontal disease. J Periodontal Res 38: 2107.
  • 23
    Lundberg A.M., Drexler S.K., Monaco C., Williams L.M., Sacre S.M., Feldmann M., Foxwell B.M. (2007) Key differences in TLR3/poly I:C signaling and cytokine induction by human primary cells: a phenomenon absent from murine cell systems. Blood 110: 324552.
  • 24
    Górska R., Gregorek H., Kowalski J., Laskus-Perendyk A., Syczewska M., Madaliński K. (2003) Relationship between clinical parameters and cytokine profiles in inflamed gingival tissue and serum samples from patients with chronic periodontitis. J Clin Periodontol 30: 104652.
  • 25
    Manetsch M., Seidel P., Heintz U., Che W., Hughes J.M., Ge Q., Sukkar M.B., Ammit A.J. (2012) TLR2 ligand engagement upregulates airway smooth muscle TNFα-induced cytokine production. Am J Physiol Lung Cell Mol Physiol 302: 83845.
  • 26
    Morris G.E., Parker L.C., Ward J.R., Jones E.C., Whyte M.K., Brightling C.E., Bradding P., Dower S.K., Sabroe I. (2006) Cooperative molecular and cellular networks regulate Toll-like receptor-dependent inflammatory responses. FASEB 20: 21535.
  • 27
    Dorn A., Ludwig R.J., Bock A., Thaci D., Hardt K., Bereiter-Hahn J Kaufmann R., Bernd A., Kippenberg. (2007) Oligonucleotides suppress IL-8 in skin keratinocytes in vitro and offer anti-inflammatory properties in vivo. J Invest Dermatol 127: 84654.
  • 28
    Proost P., Vynckier A.K., Mahieu F., Put W., Grillet B., Struyf S., Wuyts A., Opdenakker G., Van Damme J. (2003) Microbial Toll-like receptor ligands differentially regulate CXCL10/IP-10 expression in fibroblasts and mononuclear leukocytes in synergy with IFN-gamma and provide a mechanism for enhanced synovial chemokine levels in septic arthritis. Eur J Immunol 33: 314653.
  • 29
    Hofmann C., Dunger N., Grunwald N., Hämmerling G.J., Hoffmann P., Schölmerich J., Falk W., Obermeier F. (2010) T cell-dependent protective effects of CpG motifs of bacterial DNA in experimental colitis are mediated by CD11c+ dendritic cells. Gut 59: 134754.
  • 30
    Takeda K., Akira S. (2004) TLR signaling pathways. Semin Immunol 16: 39.
  • 31
    Li J., Kartha S., Iasvovskaia S., Tan A., Bhat R.K., Manaligod J.M., Page K., Brasier A.R., Hershenson M.B. (2002) Regulation of human airway epithelial cell IL-8 expression by MAP kinases. Am J Physiol Lung Cell Mol Physiol 283: 6909.
  • 32
    Honda K., Taniguchi T. (2006) IRFs: master regulators of signalling by Toll-like receptors and cytosolic pattern-recognition receptors. Nat Rev Immunol 6: 64458.