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. 2005 Sep;141(3):467–474. doi: 10.1111/j.1365-2249.2005.02852.x

CXCL12 and CXCR4 expression by human gingival fibroblasts in periodontal disease

Y Hosokawa *, I Hosokawa *, K Ozaki *, H Nakae *, K Murakami †, Y Miyake †, T Matsuo *
PMCID: PMC1809465  PMID: 16045736

Abstract

CXCL12 is a CXC chemokine that is related to lymphocyte infiltration and angiogenesis in inflammatory sites such as arthritis. However, the expression and roles of CXCL12 in periodontal disease are uncertain. The aim of this study was to assess the expression of CXCL12 and its receptor, CXCR4, in periodontal tissue and to investigate the properties of CXCL12 and CXCR4 expression by human gingival fibroblasts (HGF). RT-PCR analysis revealed that CXCL12 and CXCR4 mRNA were expressed in both normal gingival tissues and periodontal diseased tissues. Immunohistochemistry disclosed that CXCL12 was expressed and CXCR4 positive cells were found in both normal and periodontal diseased gingival tissues. Our in vitro experiments elucidated that HGF constitutively produced CXCL12, and the levels were enhanced by stimulation with tumour necrosis factor-α (TNF-α), interferon-γ (IFN-γ), transforming growth factor-β (TGF–β), regulated upon activation normal T cell expressed and secreted (RANTES) and macrophage inflammatory protein 3α (MIP-3α). On the other hand, heat killed Porphyromonas gingivalis (P. gingivalis) and P. gingivalis LPS reduced the CXCL12 production by HGF. Flow cytometry analysis clarified that CXCR4 was highly expressed on HGF, and CXCR4 expression was abrogated by TNF-α, IFN-γ and P. gingivalis LPS. Moreover, CXCL12 induced vascular endothelial growth factor (VEGF) production by HGF. Our results demonstrated that CXCL12 might be related to CXCR4+ cells infiltration and angiogenesis both in normal periodontal tissues and periodontal diseased tissue. P. gingivalis, a known periodontal pathogen, inhibits the production of CXCL12 and the expression of CXCR4 by HGF. This fact means that P. gingivalis may inhibit CXCR4+ cells infiltration and neovascularization in periodontal tissue and escape from the immune response.

Keywords: CXCL12, CXCR4, fibroblast, periodontal disease, Porphyromonas gingivalis

Introduction

Periodontal disease is characterized as chronic inflammation associated with pathogenic bacteria in subgingival plaque, including P. gingivalis[1,2], resulting in soft tissue destruction and periodontal bone resorption. The host-immune response to these bacteria has been suggested to be associated with the alteration or even progression of this disease [3,4]. A wide variety of cytokines, chemokines and their receptors are synthesized by gingival fibroblasts, epithelial cells, endothelial cells and inflammatory cells [5,6].

Approximately 40 chemokines and 16 chemokine receptors are known at present [7,8], with several studies demonstrating the production of chemokines in gingival tissue [9–11]. Constitutive expression of monocyte chemotactic protein-1 (MCP-1) and interleukin-8 (IL-8), along with the lesser expression of growth-related gene product γ (GRO-γ), macrophage inflammatory protein-1α (MIP-1α) and macrophage inflammatory protein-1 β (MIP-1β) were measured by RT-PCR in healthy gingivae [9]. Up-regulated production of MIP-1α and MIP-3α in diseased tissue were observed to correlate with the degree of inflammation [10,11]. Migration of various types of lymphocytes into effector sites appears to be regulated by specific chemokine receptors expressed on the cells [7,8].

Chemokines induce cell migration and activation by binding to specific G-protein coupled cell surface receptors [7,8]. Some chemokine receptors are also expressed by nonhematopoietic cells, including neurones [12], astrocytes [13], epithelial cells [14], and endothelial cells [15]. This suggests that the chemokine system has many functions other than leucocyte chemotaxis.

Recently, much attention has been paid to one particular member of the chemokine receptor family, named CXCR4. Current investigations show that CXCR4 is functionally expressed on several tissues and cell types [16], but has not been observed in periodontal tissue. It has been reported that CXCR4 has the unique ligand CXCL12, and CXCL12 costimulates CD4+ T cells to induce cytokine production, activation markers and proliferation [17]. In rheumatoid arthritis, it has been reported that synoviocytes overexpress CXCL12 [18]. CXCL12 is reportedly involved in angiogenesis [19], bone resorption and MMP production [20–22] in rheumatoid arthritis synovium. However, CXCL12 expression has not been described in periodontal tissue.

Fibroblasts were previously considered important connective tissue cells that construct a supporting framework crucial for tissue integrity and repair [18]. Recently, fibroblasts have been suggested to be important sentinel cells in the immune system [19]. Fibroblasts actively define the structure of tissue microenvironments and regulate inflammatory responses by the production of cytokines such as IL-1β, IL-8 [18,19]. However, CXCL12 production and chemokine receptor expression by HGF are still poorly understood.

In the present study, we aimed to elucidate the expression and role of CXCL12/CXCR4 in periodontal disease. We examined CXCL12/CXCR4 expression in periodontal tissue and further evaluated the effects of cytokines, chemokines and bacterial components on CXCL12 and CXCR4 expression by HGF. Moreover, we examined VEGF production by HGF stimulated with CXCL12 to reveal the role of CXCL12 on HGF.

Materials and methods

Gingival tissue biopsies and cell culture

Tissue biopsies were sampled from the inflamed gingivae of patients at surgery who were diagnosed with chronic periodontitis, or from the gingivae of clinically healthy subjects. All gingival biopsy sites in the chronic periodontitis group exhibited radiographic evidence of bone destruction, as well as having clinical probing depths greater than 4 mm, with sulcular bleeding on probing, otherwise the patients were systemically healthy. Samples of gingival tissues were obtained from 17 chronic periodontitis patients (4 males and 13 females, aged 49–82 years old) and 5 healthy control subjects (5 females, aged 23–40 years old). We used 2 clinically healthy gingival samples and 8 chronic periodontitis samples for RT-PCR, and 2 clinically healthy gingival samples and 9 chronic periodontitis for immunohistochemical staining. We used HGF isolated from 3 clinically healthy gingivae during routine distal wedge surgical procedure. Gingival specimens were cut into small pieces and transferred to culture dishes. HGF that grew from the gingivae were primarily cultured on 100 mm2 uncoated plastic dishes in Dulbecco's modified Eagle medium (DMEM; Sigma, St. Louis, MO, USA) supplemented with 10% fetal bovine serum (FBS; Gibco, Grand Island, NY, USA) and antibiotics (penicillin G; 100 units/ml, streptomycin; 100 mg/ml) at 37 °C in humidified air with 5% CO2 Confluent cells were transferred and cultured for use in the present study. After three to four subcultures by trypsinization, cultures contained homogeneous, slim, spindle-shaped cells growing in characteristic swirls. The cells were used for experiments after five passages. Informed consent was obtained from all subjects participating in this study. The study was performed with the approval and compliance of the Tokushima University Ethical Committee.

Bacteria culture

P. gingivalis w83 was grown in GAM broth (Nissui, Tokyo, Japan) in a 5% CO2 atmosphere using the Anaeropack system (Mitsubishi Gas Chemical, Tokyo, Japan). An overnight culture of P. gingivalis was harvested by centrifugation (2000 g for 15 min), then washed twice with phosphate-buffered saline (PBS). Cells were killed by heating to 60°C for 30 min. P. gingivalis LPS was purified by hot phenol-water extraction [23].

RNA extraction and reverse transcriptional-PCR (RT-PCR) analysis

Total RNA was prepared from gingival biopsies using the Rneasy total RNA isolation Kit (Qiagen, Hilden, Germany). Single-strand cDNA for a PCR template was synthesized from 48 ng of total RNA using a primer, oligo(dT)12−18 (Invitrogen, Carlsbad, CA, USA) and superscript‡V reverse transcriptase (Invitrogen) under the conditions indicated by the manufacture. Specific primers were designed from cDNA sequence for CXCL12, CXCR4 and glyceraldehydes-3-phosphate dehydrogenase (GAPDH). Each cDNA was amplified by PCR using Hot star Taq DNA polymerase (Qiagen). The sequences of the primers were as follows: CXCL12-F (5′-CTG GGC AAA GCC TAG TGA-3′), CXCL12-R (5′-GTC CTG AGA GTC CTT TTG CG-3′), CXCR4-F (5′-GGA CCT GTG GCC AAG TTC TTA GTT-3′), CXCR4-R (5′-ACT GTA GGT GCT GAA ATC AAC CCA-3′), GAPDH-F (5′-TGA AGG TCG GAG TCA ACG GAT TTG GT-3′), and GAPDH-R (5′-CAT GTG GGC CAT GAG GTC CAC CAC-3′). The conditions for PCR were 1× (95 °C, 15 min), 35× (94 °C, 1 min, 60 °C, 1 min, 72 °C, 1 min) and 1× (72 °C, 10min). The products were analysed on a 1·5% agarose gel containing ethidium bromide. The expected sizes of the PCR products for CXCL12, CXCR4 and GAPDH were 286 bp, 273 bp and 985 bp, respectively.

Immunohistochemistry

Gingival biopsies were immediately embedded in the O.C.T. compound (Miles Laboratories Inc., Elkhart, IN, USA) and quenched and stored in liquid nitrogen. The specimens were cut at 6 µm sections using a cryostat (SFS, Bright instrumental Company, Huntingdon, England) and collected on poly L-lysine-coated slides. CXCL12, CXCR4 and CD3 were analysed with specific antibodies; the anti-human CXCL12 mAb (clone 79014·111, DAKO, Kyoto, Japan, 5 µg/ml), the anti-human CXCR4 mAb (clone 44716·111, Sigma, 5 µg/ml) or the anti-human CD3 mAb (clone UCHT1, Ancell, Bayport, MN, USA, 5 µg/ml), and we used an isotype matched control mAb (DAKO) as the negative control. The sections were reacted with specific antibodies overnight at 4°C. After washing with PBS, the sections were incubated with biotinylated anti-mouse and rabbit immunoglobulins (Universal Ab; DAKO) for 20 min at room temperature and washed with PBS to remove any unreacted antibodies. The sections were then treated with peroxidase-conjugated streptavidin (DAKO) for 10 min, and washed and reacted with DAB (3,3-diamino-benzidine tetrahydrochrolide; DAKO) in the presence of 3% H2O2 to develop colour. The sections were counterstained with haematoxylin and mounted with glycerol.

Cytokine production by HGF

HGF was stimulated with IL-1β (Peprotech, Rocky Hill, NJ, USA), TNF-α (Peprotech), IFN-γ (Peprotech), IL-4 (Peprotech), TGF-β1 (Peprotech), RANTES (Peprotech), MIP-3α (Peprotech), interferon-inducible protein 10 (IP-10) (Peprotech), fractalkine (Peprotech), Escherichia coli (E. coli) 0111; B4 LPS (Sigma), P. gingivalis w83 LPS, oligodeoxynucleotides containing unmethylated CpG motifs (CpG DNA; Hokkaido System Science, Sapporo, Japan) or CXCL12 (Peprotech) for 24 h. Supernatants from HGF were collected and the CXCL12, IL-8 and VEGF concentrations of the culture supernatants were measured in triplicate by ELISA. Duoset (R & D systems, Minneapolis, MI) was used for CXCL12 and IL-8 determinations and human VEGF ELISA development Kit (Peprotech) for VEGF. Detection ranges for the CXCL12, IL-8 and VEGF ELISAs were 20–4000, 20–2000, and 32–4000 pg/ml, respectively. All assays were performed according to the manufacturer's instructions and cytokine levels were determined using a standard curve prepared for each assay.

Flow cytometric analyses

Cultured HGF were detached using Trypsin-EDTA (Gibco) and washed twice with PBS. Cells were incubated with anti-human CCR1 (clone 53504, R & D systems, 5 µg/ml), the anti-human CCR5 mAb (clone 45523, R & D systems, 5 µg/ml), the anti-human CCR6 mAb (clone 53103, R & D systems, 5 µg/ml), the anti-human CXCR3 mAb (clone 49801, R & D systems, 5 µg/ml), the anti-human CXCR4 mAb (clone 44716·111, Sigma), or an isotype control antibody (DAKO) on ice for 30 min. After washing three times with PBS-1% bovine serum albumen (BSA; Sigma), the cells were incubated with the FITC-conjugated rabbit anti-mouse F(ab′)2 fragment (DAKO) for 30 min on ice. After washing three times with PBS-1% BSA, cells were immediately analysed by flow cytometry (Epics XL-MCL; Coulter, Hialeah, FL, USA). The percentage of positive cells was calculated after defining a cut off value according to the isotype control.

Statistical analysis

Statistical significance was analysed by the Student's t-test. P-values < 0·05 were considered significant.

Results

CXCL12 and CXCR4 expression in periodontal tissue

We first examined CXCL12 and CXCR4 mRNA expression by whole gingival tissue. Both CXCL12 mRNA and CXCR4 mRNA were detected in extracts from 2 samples of clinically normal gingiva. CXCL12 and CXCR4 mRNA was detected in six of eight samples and seven of eight samples derived from periodontal diseased tissues, respectively (Fig. 1). We carried out immunohistochemical staining to investigate the expression of CXCL12 and CXCR4 in periodontal tissues (Fig. 2). Small numbers of CD3 positive cells were infiltrated in normal gingival tissues (Fig. 2a) and CXCR4 positive cells (Fig. 2c) were present in normal gingival tissues together with CXCL12 positive fibroblasts, epithelial cells and mononuclear cells. In periodontal diseased tissue, anti-CXCL12 mAb stained endothelial cells as well as mononuclear cells and fibroblasts (Fig. 2f). Diseased tissues also contained many CXCR4-positive and CD3 positive cells (Figs 2b–d).

Fig. 1.

Fig. 1

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RT-PCR analysis of CXCL12 and CXCR4 mRNA expressions in periodontal diseased tissue. Total RNA was prepared from 2 clinically healthy gingival samples (pocket depth 2 mm) and 8 periodontal diseased gingival samples (pocket depth 4–10 mm). Expressions of CXCL12, CXCR4 and GAPDH mRNAs in periodontal tissues were analysed by RT-PCR as described in Methods.

Fig. 2.

Fig. 2

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CXCR4 and CXCL12 immunostainings in periodontal diseased tissue. Immunohistochemical analysis of human periodontal tissues. (a) Normal gingiva stained with anti-CD3 MoAb. (b) inflamed gingiva stained with anti-CD3 MoAb. (c) normal gingiva stained with anti-CXCR4 MoAb. (d) inflamed gingiva stained with anti-CXCR4 MoAb. (e) normal gingiva stained with CXCL12 MoAb. (f) inflamed gingiva stained with CXCL12 MoAb. Original magnification for each photograph was ×200.

Production of CXCL12 by HGF

Because fibroblasts expressed CXCL12 in periodontal tissues, we examined the effects of proinflammatory cytokines, chemokines and bacteria-related products on CXCL12 production by HGF. Figure 3 shows that nonstimulated HGF produced CXCL12 constitutively, and that CXCL12 production was enhanced by TNF-α, IFN-γ and TGF-β1 in a dose dependent manner (Fig. 3a). However, IL-1β and IL-4 did not modulate CXCL12 production by HGF. Furthermore, RANTES and MIP-3α cause a 4–5-fold enhancement of CXCL12 production by HGF, whereas Fractalkine only induced moderate increase (Fig. 3b). On the other hand, IP-10 did not affect the production of CXCL12. Although high concentrations of E. coli LPS and CpG DNA enhanced the production of CXCL12, P. gingivalis LPS reduced the production of CXCL12 by HGF (Fig. 3c).

Fig. 3.

Fig. 3

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CXCL12 production by HGF stimulated with cytokines, chemokines and PAMPs. HGF were stimulated with cytokines (IL-1β, TNF-α, IFN-γ, IL-4, TGF-β1), chemokines (RANTES, MIP-3α, IP-10, fractalkine) or PAMPs (E. coli LPS, P. gingivalis LPS, CpG DNA) for 24 h. The amount of CXCL12 in the culture medium was measured by ELISA. Data are representative of three different HGF from three different donors. The results were calculated as mean and SD of one representative experiment performed in triplicates. Error bars show the SD of the values. *P < 0·05 significantly different from the medium.

P. gingivalis inhibit CXCL12 production by HGF

P. gingivalis LPS reduced the production of CXCL12 by HGF, we postulated that intact P. gingivalis might also reduce CXCL12 production. We therefore stimulated HGF by heat killed P. gingivalis w83, and examined CXCL12 production. Figure 4 indicates that heat killed P. gingivalis w83 enhanced IL-8 production in a dose dependent manner. On the other hand, CXCL12 production by HGF decreased when the number of P. gingivalis w83 increased.

Fig. 4.

Fig. 4

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(a) CXCL12 and (b) IL-8 productions by HGF stimulated with heat killed P. gingivalis w83. HGF were stimulated with heat killed P. gingivalis w83 (OD0·0001–0·01) for 24 h. The amounts of CXCL12 and IL-8 in the culture medium were measured by ELISA. Data are representative of three different HGF from three different donors. The results were calculated as mean and SD of one representative experiment performed in triplicates. Error bars show the SD of the values. *P < 0·05, **P < 0·01 significantly different from the medium.

HGF expressed high levels of CXCR4

As HGF express CXCL12, we hypothesized that they might also express CXCR4 and other chemokine receptors. Therefore, the expression of chemokine receptors was investigated by flow cytometry. HGF expressed low levels of CCR1, CCR5, CCR6 and CXCR3 but high levels of CXCR4 (Fig. 5).

Fig. 5.

Fig. 5

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Chemokine receptors expressions on HGF.HGF were incubated with each chemokine receptor mouse antibody (CCR1, CCR5, CCR6, CXCR3, CXCR4). Then cells were stained with FITC-labelled goat anti-mouse IgG and analysed with flow cytometry to determine the expression of each chemokine receptor. Clear zones and grey zones show cell staining by the isotype matched mouse IgG and chemokine receptor antibody, respectively. Data are representative of three different HGF from three different donors.

TNF-α, IFN-γ and P. gingivalis LPS reduced CXCR4 expression by HGF

The finding of CXCR4 expression by nonstimulated HGF led us to examine whether proinflammatory cytokine and LPS can regulate this expression. IL-1β did not affect CXCR4 expression and E. coli LPS slightly increased the expression of CXCR4 by HGF. On the other hand, TNF-α, IFN-γ and P. gingivalis LPS decreased CXCR4 expression by HGF (Fig. 6).

Fig. 6.

Fig. 6

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CXCR4 expression on HGF stimulated with proinflammatory cytokines or LPS. HGF were stimulated with IL-1β (10 ng/ml), TNF-α (10 ng/ml), IFN-γ (10 ng/ml), E.coli LPS (1 mg/ml) or P. gingivalis LPS (1 µg/ml) for 24 h. Then, cells were recovered and incubated with the anti-CXCR4 mAb. After that, cells were stained with the FITC-labelled goat anti-mouse IgG and analysed with flow cytometry to determine the expression of CXCR4. Black lines show stimulated HGF stained by anti-CXCR4 mAb. Dark grey lines show nonstimulated HGF stained by anti-CXCR4 mAb. Light grey lines show staining by the isotype matched control mAb. Data are representative of three different HGF from three different donors.

CXCL12 induced VEGF production by HGF

It has been reported that CXCL12 is related to angiogenesis [24]. Consequently, we examined the effects of CXCL12 on VEGF production by HGF. CXCL12 induced VEGF production by HGF whereas RANTES (1 ng/ml), IP-10 (1 ng/ml) and MIP-3α (1 ng/ml) did not (Fig. 7).

Fig. 7.

Fig. 7

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VEGF production by HGF treated with CXCL12.HGF were stimulated with CXCL12 (0·01–1 ng/ml), RANTES (1 ng/ml), MIP-3α (1 ng/ml) and IP-10 (1 ng/ml) for 24 h. The amounts of VEGF in the culture medium were measured by ELISA. Data are representative of three different HGF from three different donors. The results were calculated as mean and SD of one representative experiment performed in triplicates. Error bars show the SD of the values. *P < 0·05, significantly different from the medium alone.

Discussion

Our data demonstrated that CXCL12 was expressed in clinically normal periodontal tissues. Experiments in vitro showed nonstimulated HGF produced CXCL12 constitutively. It has been reported that healthy tissues basally express CXCL12 mRNA [16]. Our results agree with these reports. These results might explain that CXCL12 production by nonstimulated HGF is related to the physiological condition in normal periodontal tissues. Furthermore, we showed that CXCL12 induced VEGF production by HGF. It has been suggested that leucocytes in normal tissues are important for defense against foreign bodies such as microorganisms [25,26], and it is known that vessels are necessary for remodeling tissues. In this study, CD3 positive cells and CXCR4 positive cells were detected in normal periodontal tissues. Put together, these facts might suggest that CXCL12 controls leucocytes migration in normal periodontal tissue and, that is related to protection against periodontal disease associated bacteria in normal gingival tissue and remodeling periodontal tissues to induce the production of VEGF from HGF.

Immunohistochemical analysis revealed CXCL12 was detected in the region that CXCR4+ lymphocytes infiltrated in periodontal diseased tissue. Nanki et al. [27] reported that CXCL12 played a central role in CD4+ lymphocyte accumulation in arthritis synovium. It has been reported that CXCR4 is expressed on CD3+ memory T cells [28]. Our immunohistochemical results demonstrated that CD3+ lymphocytes and CXCR4+ cells infiltrated in the same area of periodontal diseased tissue. These results mean that CXCL12 may control CD3+CXCR4+ cells migration not only in normal periodontal tissues but also periodontal diseased tissue. Further investigation might be necessary to elucidate the roles of CXCL12 on CXCR4+ cells infiltration in periodontal disease.

In this study, heat killed P. gingivalis enhanced IL-8 production but reduced CXCL12 production by HGF. Moreover, P. gingivalis LPS down-regulated the expression of CXCR4 on HGF. It is known that P. gingivalis is one of the pathogenic bacteria involved in periodontal disease [29]. We suggested that CXCL12 expression by HGF might be involved in the physiological condition from our results. Therefore, reduction of CXCL12 and CXCR4 by HGF might disturb the homeostasis of normal periodontal tissue, including the inhibition of new vessel formation in periodontal tissue. These results suggest that the inhibitory effect of P. gingivalis on CXCL12 production and CXCR4 expression by HGF may contribute to the disease process. Furthermore, our results suggest that CXCL12 are involved in CXCR4+ cells migration in periodontal tissues. Figure 4 might explain that P. gingivalis escape from the immune response in periodontal tissue to inhibit CXCL12 production by HGF.

Chemokine receptor expression on HGF has been unknown. However, our results demonstrated that CCR1, CCR5, CCR6, CXCR3 and CXCR4 were expressed on nonstimulated HGF, and CXCR4 expression was much stronger than any other chemokine receptor examined in this experiment. On the other hand, CXCR4 expression was reduced by TNF-α or IFN-γ stimulation. However, the function of CXCR4 on HGF is not certain. Further investigation about the roles of CXCR4 on HGF might be necessary in normal conditions and inflammatory conditions.

In conclusion, our results mean that CXCL12 might be related to CXCR4+ cells infiltration and angiogenesis in both normal periodontal tissue and periodontal diseased tissue. HGF may be a major source of CXCL12 in periodontal tissue, and CXCL12 and CXCR4 expressions by HGF might be complexly regulated by many factors in periodontal tissue. Moreover, P. gingivalis is an important factor of the CXCL12/CXCR4 expression by HGF in periodontal tissue, and the inhibition of CXCL12 production and CXCR4 expression might be related to the progression of periodontal disease.

Acknowledgments

This study was supported by a grant-in-aid from the Ministry of Education, Science, and Culture of Japan.

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