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Regulation of CXCL9/10/11 in Oral Keratinocytes and Fibroblasts
K. Ohta*,
H. Shigeishi,
M. Taki,
H. Nishi,
K. Higashikawa,
M. Takechi and
N. Kamata
Department of Oral and Maxillofacial Surgery, Division of Cervico-Gnathostomatology, Programs for Applied Biomedicine, Graduate School of Biomedical Sciences, Hiroshima University, 1-2-3 Kasumi, Minami-Ku, Hiroshima 734-8553, Japan;
Correspondence: * corresponding author, otkouji{at}hiroshima-u.ac.jp
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ABSTRACT
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Th1 and Th2 cytokines such as interferon- (IFN- ) , tumor necrosis factor-  (TNF- ), and IL-4 are expressed in T-cell-mediated inflammation in the oral cavity. We tested the hypothesis that those cytokines may act on CXCR3-agonistic chemokines, T-cell recruiting factors, and on neighboring cells, including oral keratinocytes and fibroblasts. Human immortalized oral keratinocytes (RT7) and fibroblasts (GT1) after 24-hour stimulation with IFN- showed increased mRNA levels of CXCL9 (600- and 700-fold), CXCL10 (10,000- and 150-fold), and CXCL11 (5000- and 300-fold), respectively. In contrast, TNF- caused an increase in CXCL9 (300-fold), CXCL10 (2000-fold), and CXCL11 (2000-fold) mRNA levels in GT1, but not RT7 cells, at 24 hrs. IL-4 reinforced the promotion of CXCL9, CXCL10, and CXCL11 expression by IFN- in RT7 cells, whereas IL-4 inhibited the increased levels by IFN- and TNF- in GT1 cells. Thus, IFN- , TNF- , and IL-4 appear cooperatively to regulate CXCR3-agonistic chemokines in oral keratinocytes and fibroblasts in T-cell-mediated oral inflammation sites.
Key Words: Chemokine • oral keratinocytes • oral fibroblasts • Th1 cytokine • Th2 cytokine
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INTRODUCTION
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Thelper 1 (Th1) cytokines, interferon- (IFN- ), and tumor necrosis factor- (TNF- ), which Th1 cells produce, have been detected in the lesions of periodontal disease characterized by dense T-cell infiltration (Taubman and Kawai, 2001). They also determine immunological activity in oral mucosal inflammatory disease, such as lichen planus, which is characterized by a band-like accumulation of T-cells in the connective tissue adjacent to the basement membrane (Khan et al., 2003). Thus, IFN- and TNF- are associated with oral chronic inflammation mediated by T-cells (Taubman and Kawai, 2001). The cytokines may modulate functioning of neighboring cells, including oral keratinocytes and fibroblasts, in T-cell-mediated oral inflammation.
Chemokines induce leukocyte migration into inflammation sites and regulate leukocyte trafficking through lymphoid tissues (Yoshie et al., 2001). Four chemokine subfamilies are composed of the cysteine residue positions CXC, CC, C, and CX3C (Yoshie et al., 2001). The chemotactic activity of various chemokines shows relatively rigid patterns of target cell selectivity (Table 1 ). Previous reports have suggested inducible expression of chemokines in oral keratinocytes and fibroblasts. Th1 cytokines, such as IFN- or TNF- , induce CCL2, CCL5, and CXCL8 in oral keratinocytes, while CCL20, CXCL8, CXCL12, and CXCL16 are induced in gingival fibroblasts (Takashiba et al., 1992; Li et al., 1996; Hosokawa et al., 2005a,b, 2007). However, it remains unknown how several chemokines are regulated by IFN- or TNF- in oral keratinocytes or fibroblasts.
CXCR3-agonistic chemokines such as CXCL9, CXCL10, and CXCL11 play an important role in inflammation through their T-cell chemotactic and adhesion-promoting activities. CXCL10 is highly expressed in aggressive periodontitis, and coincides with IFN- expression (Garlet et al., 2003). CXCL9 is highly expressed in skin lichen planus compared with other chemokines, such as CCL2, CCL20, and CXCL8 (Spandau et al., 1998). IFN- or TNF- enhances CXCL10 expression in skin keratinocytes and fibroblasts (Boorsma et al., 1998; Villagomez et al., 2004). These findings imply that oral keratinocytes and fibroblasts produce CXCL9, CXCL10, and CXCL11 when stimulated with IFN- or TNF- and are implicated in the development process of T-cell-mediated oral inflammation.
T helper 2 (Th2) cytokines such as IL-4, IL-10, and IL-13, released from Th2 cells, suppress the destructive actions of inflammatory responses mediated by Th1 cytokines (Essner et al., 1989). It has been reported that both Th1 and Th2 cells infiltrate inflamed oral tissues and produce Th1 cytokines, IFN- and TNF- , and the Th2 cytokine, IL-4, respectively (Taubman and Kawai, 2001). Therefore, IL-4 may modulate the expression of chemokines that are involved in the recruitment of T-cells by IFN- or TNF- at inflammation sites.
In this study, we hypothesized that the regulation of chemokine expression such as CXCL9, CXCL10, and CXCL11 by IFN- , TNF- , and IL-4 in oral keratinocytes and fibroblasts participates in the development of T-cell-mediated oral inflammation. To prove this hypothesis, we first examined which chemokines IFN- and TNF- influenced in immortalized human oral keratinocytes (RT7) and gingival fibroblasts (GT1). Subsequently, we investigated the effect of IL-4 on any chemokine expression strongly affected by IFN- and TNF- in the 2 cell types.
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MATERIALS & METHODS
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Cell Lines
RT7, an immortalized human oral keratinocyte cell line, was established by transfection of hTERT and E7, as described previously (Fujimoto et al., 2002), and GT1, a human oral fibroblast cell line, was established by transfection of hTERT, as described previously (Kamata et al., 2004).
RT7 was cultured with Keratinocytes-SFM (Gibco BRL, Gaithersburg, MD, USA), including 25 µ g/mL bovine pituitary extract, 0.05 ng/mL epidermal growth factor, 100 U/mL penicillin, and 100 µ g/mL streptomycin. GT1 was cultured with Dulbecco’s modified Eagle’s medium (Sigma Chemical Co., St. Louis, MO, USA) containing 10% fetal calf serum, 100 U/mL penicillin, and 100 µ g/mL streptomycin. We also prepared a primary culture of human gingival keratinocytes and fibroblasts (Kamata et al., 2004; Uchida et al., 2005). Informed consent for the acquisition was obtained under a protocol approved by the Ethical Committee of Hiroshima University.
RNA Preparation
RT7 (5 x 104 cells/well) and GT1 (5 x 104 cells/well) cells were seeded into six-well cell culture plates in each medium. After a three-day incubation, the cells reached approximately 80% confluence. Subsequently, they were exposed to recombinant human IFN- (10 ng/mL), TNF- (10 ng/mL), IL-4, or their combination (10 ng/mL) (R & D Systems, Minneapolis, MN, USA) for various periods. RNA from each culture was extracted by means of an RNAeasy Mini Kit (QIAGEN, Hilden, Germany).
Real-time PCR
Total RNA (1 µ g) was subjected to a reverse-transcriptase reaction by means of a First Strand cDNA Synthesis Kit (Amersham Biosciences, Uppsala, Sweden). The synthesized cDNAs were used for quantitative PCR analysis with oligonucleotide primers (Table 1 ). Quantitative PCR analysis was performed with an ABI PRISM 7700 Sequence Detection System (Perkin-Elmer, Foster City, CA, USA) and SYBR-Green Master Mix (Applied Biosystems, Warrington, UK) for 40 cycles at 95°C for 15 sec and 60°C for 60 sec. The quantification of chemokine mRNA levels, shown as relative to the internal control, β-actin, are the mean ± standard deviation from 3 independent experiments.
Determination of Chemokine Production
RT7 (1 x 104 cells /well) and GT1 (1 x 104 cells /well) cells were seeded into 48-well cell culture plates in each medium. After a two-day culture, the cells were exposed to 10 ng/mL recombinant human IFN- , TNF- , IL-4, or their combination for 48 hrs. The collected media were centrifuged, and the supernatant fluids were stored at –80°C before the assays. The protein levels of each chemokine in the medium were determined by means of an ELISA kit (R&D Systems), according to the protocol recommended by the manufacturer.
Statistical Analysis
Data were analyzed by one-way analysis of variance (ANOVA) according to the Bonferroni or Dunn method, and the results are presented as the mean ± standard deviation.
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RESULTS
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Time-course Effect of IFN- or TNF- on Various Chemokine mRNA Expressions in RT7 and GT1 Cells
We examined the time-course effect of IFN- or TNF- on 10 chemokine mRNA expressions in RT7 and GT1 cells. Ten chemokines were selected based on their ability to recruit different target leukocytes. The optimal additional cytokine concentration (the concentration required for maximal mRNA expression of the 10 chemokines) was found, by RT-PCR, to be 10 ng/mL (data not shown). IFN- dramatically increased the mRNA levels of CXCL9, CXCL10, and CXCL11 in a time-dependent manner up to 24 hrs in RT7 cells (Fig. 1). IFN- slightly stimulated CCL5 mRNA expression in a time-dependent manner (Fig. 1). The 12-hour exposure to IFN- culminated in the maximum increase in the mRNA levels of CCL2, CCL20, CXCL12, and CX3CL1 in RT7 (Fig. 1). IFN- did not induce CXCL8 or CXCL5 mRNA expression at any exposure time (Fig. 1). When RT7 cells were stimulated by TNF- , CCL5, CCL20, CXCL5, CXCL8, CXCL10, and CXCL11, mRNA levels increased in the first 2 hrs, and the increases were maintained over the next 12 hrs (Fig. 1). CCL2, CXCL12, and CX3CL1 mRNA expression induced by TNF- reached peak levels at 2 hrs, and decreased to almost basal levels at 6 hrs (Fig. 1). TNF- did not increase CXCL9 mRNA expression (Fig. 1).
The mRNA levels of CXCL9, CXCL10, and CXCL11 in GT1 cells were dramatically increased by IFN- , and the effects reached a plateau at 2 hrs (Fig. 2). IFN- stimulated CCL5 mRNA expression at only 24 hrs (Fig. 2). CCL2 mRNA expression induced by IFN- reached peak levels at 2 hrs, and then declined to basal levels at 24 hrs (Fig. 2). IFN- did not increase CCL20, CXCL5, CXCL8, CXCL12, or CX3CL1 mRNA expression (Fig. 2). TNF- increased the mRNA levels of CCL2, CCL5, CCL20, CXCL5, CXCL8, CXCL9, CXCL10, and CXCL11 in a time-dependent manner up to 24 hrs (Fig. 2). TNF- did not significantly induce CXCL12 or CX3CL1 mRNA expression (Fig. 2).
We summarized the effects of IFN- and TNF- on the 10 chemokine mRNA expressions by comparing no exposure with a 24-hour exposure. IFN- caused a 600-fold increase in CXCL9 mRNA levels, a 10,000-fold increase in CXCL10 mRNA levels, and a 5000-fold increase in CXCL11 mRNA levels in RT7 cells, and a 700-fold increase in CXCL9 mRNA levels, a 150-fold increase in CXCL10 mRNA levels, and a 300-fold increase in CXCL11 mRNA levels in GT1 cells. The addition of IFN- to cultures of RT7 and GT1 cells resulted in a much greater increase in CXCL9, CXCL10, and CXCL11 than did that of the other chemokines. TNF- caused a 100-fold increase in CCL2 mRNA levels, a 700-fold increase in CCL5 mRNA levels, a 100-fold increase in CCL20 mRNA levels, a 70-fold increase in CXCL5 mRNA levels, a 100-fold increase in CXCL8 mRNA levels, a 300-fold increase in CXCL9 mRNA levels, a 2000-fold increase in CXCL10 mRNA levels, and a 2000-fold increase in CXCL11 mRNA levels in GT1 cells. In a subsequent experiment, CXCL9, CXCL10, and CXCL11 were the main focus.
CXCL9, CXCL10, and CXCL11 Expressions Induced by Combinations of IFN- , TNF- , and IL-4
A combination of IFN- and TNF- enhanced CXCL9, CXCL10, and CXCL11 mRNA expression in both cell lines in comparison with stimulation with either alone (Table 2). IL-4 enhanced the increase in CXCL9, CXCL10, and CXCL11 mRNA levels by IFN- or TNF- in RT7 cells, with exceptional CXCL9 mRNA levels observed in the presence TNF- . In contrast, IL-4 abolished the increase in CXCL9, CXCL10, and CXCL11 mRNA levels by IFN- or TNF- in GT1 cells (Table 2). The effects of the various combinations on the mRNA expression of CXCL9, CXCL10, and CXCL11 were similar to those on the protein expression (Appendix Table 1). In addition, the primary oral keratinocytes and fibroblasts showed the same response as RT7 and GT1 cells when exposed to IFN- , TNF- , IL-4, or their combinations (Appendix Table 2).
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Table 2. CXCL9, CXCL10, and CXCL11 mRNA Expressions in RT7 and GT1 Cells Stimulated with IFN- , TNF- , IL-4, or Their Combinationsa
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DISCUSSION
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The present study showed that the increase in the mRNA levels of CXCL9, CXCL10, and CXCL11 stimulated with IFN- in RT7 and GT1 cells was much higher than that of other chemokines. These T-cell-specific chemokines share an approximately 40% amino acid sequence identity, and each binds to the chemokine receptor CXCR3, which is mainly expressed by memory/activated T-cells associated with Th1-type reactions (Qin et al., 1998). Previous studies have shown that IFN- increases CXCL10 mRNA expression in skin keratinocytes, skin fibroblasts, and dental pulp fibroblasts in a time-dependent manner (Boorsma et al., 1998; Villagomez et al., 2004; Adachi et al., 2007). CXCL10 and CXCR3 are highly expressed in aggressive and chronic periodontitis associated with IFN- (Garlet et al., 2003). In oral lichen planus, infiltrating T-cells predominantly express CXCR3 or CCR5, and oral keratinocytes overexpress CXCL9, CXCL10, and CXCL11 in T-cell-mediated infiltration lesions adjacent to the epithelial basement membrane (Iijima et al., 2003). Thus, IFN--induced CXCR3-agonistic chemokines in oral keratinocytes and fibroblasts seem to mobilize a large number of T-cells toward the inflammatory sites, leading to the development of T-cell-mediated oral inflammation.
TNF- does not increase CXCL10 mRNA levels in skin keratinocytes (Boorsma et al., 1998). However, TNF- up-regulates CXCL10 expression in skin fibroblasts and dental pulp fibroblasts (Villagomez et al., 2004; Adachi et al., 2007). In this study, TNF- did not markedly increase CXCL10 or CXCL11, and did not induce CXCL9 in RT7 cells. However, TNF- did cause an increase to high levels of these chemokines in GT1 cells. These findings suggest that TNF- , but not IFN- , is the main inducer of CXCL9, CXCL10, and CXCL11 in oral fibroblasts. Further, TNF- showed appreciable enhancement of mRNA levels of CCL2, CCL5, CCL20, CXCL5, and CXCL8 in GT1 cells. These chemokines released from gingival fibroblasts may contribute to the regulation of trafficking of different leukocyte subsets in the inflammatory status.
IL-4 secreted by Th2-type CD4+ T-cells is known to down-regulate cellular responses to Th1 cytokines in several cell types. IL-4 inhibits CXCL10 production by IFN- in human neutrophils, mouse macrophages, and human intestinal epithelium cells (Deng et al., 1994; Gasperini et al., 1999; Dwinell et al., 2001). IL-4 reinforced the increased effect of IFN- on the expression of CXCL9, CXCL10, and CXCL11 in RT7 cells. IL-4 also enhanced TNF- –induced CXCL10 in RT7 cells. IL-4 may exacerbate inflammation by enhancing the induction of CXCR3-agonistic chemokines by IFN- in oral keratinocytes. In contrast, the effects of IL-4 on the increase in CXCL9, CXCL10, and CXCL11 by IFN- or TNF- in gingival fibrobrasts have not yet been examined. In this study, IL-4 abolished the increases in these chemokine levels due to IFN- or TNF- in gingival fibroblasts. From these findings, the effects of IL-4 on the expressions of CXCR3-agonistic chemokines induced by IFN- or TNF- differ among cell types.
IFN--induced mRNA levels of CXCL9, CXCL10, and CXCL11 in RT7 and GT1 cells were much higher than those of other chemokines, as well as of TNF--induced chemokines, in GT1 cells. CXCL10 levels in gingival crevicular fluid were reported to differ significantly between persons with chronic periodontitis and healthy gingiva (Sakai et al., 2006). Higher expressions of CXCL9, CXCL10, and CXCL11 mRNA have also been observed in oral lichen planus in comparison with normal tissue (Ichimura et al., 2006). These findings suggest that these CXCR3-agonistic chemokines may act as key chemokines in T-cell-mediated oral inflammatory disease. Previous studies have attempted to reveal the Th1/Th2 profile in periodontal disease (Taubman and Kawai, 2001). Th1 cells are involved in periodontal destruction, and Th2 cells abrogate periodontal disease symptoms (Taubman et al., 1992), whereas Th2 cells have been associated with progressing regions in periodontal disease (Seymour et al., 1993). Different patterns of Th1/Th2 imbalance have been detected in oral lichen planus (Rhodus et al., 2007). However, it is not certain what regulates the Th1/Th2 balance in these diseases. Differential CXCR3-agonistic chemokine expression by IFN- , TNF- , and IL-4 among cell types may control the Th1/Th2 balance in the epithelium and connective tissue in these diseases. These cytokines cooperatively regulate CXCL9, CXCL10, and CXCL11 in oral keratinocytes and fibroblasts, and may promote and shape T-cell-mediated oral inflammation sites.
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ACKNOWLEDGMENTS
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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. 17791460).
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FOOTNOTES
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A supplemental appendix to this article is published electronically only at http://jdr.iadrjournals.org/cgi/content/full/87/12/1160/DC1.
Received for publication December 27, 2007.
Revision received July 29, 2008.
Accepted for publication September 2, 2008.
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Journal of Dental Research, Vol. 87, No. 12,
1160-1165 (2008)
DOI: 10.1177/154405910808701211
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ABSTRACT
INTRODUCTION
) for the indicated periods. The chemokine mRNA levels, which are shown as relative to the internal control, β-actin, are shown as the mean ± standard deviation from 3 independent experiments. *Significantly different from non-treated RT7 (p < 0.05).