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Single-nucleotide Polymorphism in the CD14 Promoter and Periodontal Disease Expression in a Japanese Population
K. Yamazaki*,1,
K. Ueki-Maruyama1,
T. Oda1,
K. Tabeta1,
Y. Shimada1,
H. Tai1,
T. Nakajima1,
H. Yoshie1,
D. Herawati2 and
G.J. Seymour3
1 Division of Periodontology, Department of Oral Biological Science, Niigata University Graduate School of Medical and Dental Sciences, 5274, Gakkocho-Dori 2-ban-cho, Niigata 951-8514, Japan;
3 Oral Biology and Pathology, Department of Dentistry, The University of Queensland, Brisbane, Australia;
Correspondence: * corresponding author, kaz{at}dent.niigata-u.ac.jp
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ABSTRACT
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It has been reported that there is a relationship between a single-nucleotide polymorphism (SNP) in the promoter region of the CD14 gene at position -159 (C T) and infectious diseases. The aim of the present study was to test the hypthesis that expression of this SNP correlates with periodontal disease in a Japanese population. The CD14 genotype was determined in 163 subjects with periodontitis and in 104 age- and gender-matched control subjects without periodontitis. The genotype distribution and allele frequency within the periodontitis patients were not significantly different from those of control subjects. There was, however, a significant difference in the genotype distribution between young patients (< 35 yrs) and older patients ( 35 yrs). These findings suggest that CD14 -159C/T polymorphism is not related to the development of periodontitis in a Japanese population, but that, within the periodontitis subjects, expression of the SNP may be related to early disease activity.
Key Words: CD14 • polymorphism • chronic periodontitis • Japanese
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INTRODUCTION
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Studies in twins have shown that genetic factors can account for a large part of the variation seen in periodontal disease expression in humans (Michalowicz et al., 1991; Michalowicz, 1994). In recent years, several studies have sought to identify the basis of this genetic association, and in particular, several gene polymorphisms, especially those of the IL-1 gene complex, have been investigated (Kornman et al., 1997; Diehl et al., 1999; Cullinan et al., 2001). Aggressive periodontitis (AgP) is a form of chronic inflammatory periodontal disease, characterized by localized or generalized rapid loss of connective tissue attachment and alveolar bone early in life (Tonetti and Mombelli, 1999). Genetic factors seem to have a strong influence on susceptibility to AgP, as evidenced by population and family studies (Schenkein and Van Dyke, 1994; Hart, 1996). However, to date, studies on pro-inflammatory cytokine gene polymorphisms have produced conflicting results (Kinane et al., 1999; Parkhill et al., 2000; Endo et al., 2001; Hodge et al., 2001; Shapira et al., 2001; Craandijk et al., 2002).
CD14, a glycoprotein localized on the cell surfaces of myeloid cells, functions as a pattern recognition receptor for various bacterial products, such as lipopolysaccharide (LPS) (Ulevitch and Tobias, 1995). It has been reported to be expressed on neutrophils, monocytes/macrophages, and fibroblasts, all of which are present in periodontitis lesions and produce pro-inflammatory cytokines such as IL-1 and TNF- in response to stimulation with LPS derived from periodontopathic bacteria (Yamazaki et al., 1992; Shapira et al., 1994; Agarwal et al., 1995; Watanabe et al., 1996). Although CD14 lacks transmembrane and cytoplasmic domains and does not elicit intracellular signaling, this molecule binds to LPS and acts by transferring LPS to the Toll-like receptor 4 (TLR4)/MD-2 complex (da Silva Correia et al., 2001). In a transfection experiment, co-expression of CD14 and TLR4 dramatically enhanced the cellular response to LPS, indicating that the level of CD14 expression influences the subsequent production of pro-inflammatory cytokines (Akashi et al., 2000).
It has also been reported that there is a single nucleotide polymorphism in the promoter region of the CD14 gene at position -159 (CT) (Hubacek et al., 1999). The transcriptional activity of the T allele was found, by the luciferase reporter assay, to be increased in monocytic Mono Mac 6 cells (LeVan et al., 2001). Homozygous carriers of the T allele have a significant increase in serum levels of soluble CD14 (Baldini et al., 1999). In this context, therefore, it is reasonable to speculate that the T allele may be associated with increased levels of pro-inflammatory cytokines and hence lead to increased progression of periodontal disease.
Therefore, the aim of the present study was to determine whether the SNP at position -159 in the promoter region of the CD14 gene is associated with periodontal disease status in a Japanese population.
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MATERIALS & METHODS
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Subjects
The study consisted of 163 subjects with periodontitis referred to the Periodontal Clinic of Niigata University Dental Hospital, and 104 age- and gender-matched periodontally healthy subjects. None of the subjects had a history or current manifestation of systemic disease. The study design was approved by the Ethics Committee at Niigata University Graduate School of Medical and Dental Sciences, and written informed consent was obtained from all patients and control subjects before inclusion in the study. Both current and previous smokers were excluded from the study population. A subject was considered as a non-smoker if he or she had never smoked or had stopped smoking more than 5 years prior to the date of examination and had a pack-year history of fewer than 10 (Engebretson et al., 1999).
Clinical Assessment and Diagnosis
We assessed periodontal tissue destruction by measuring the clinical attachment level and probing pocket depth at 6 sites for each tooth. The periodontitis subjects were divided on the basis of disease expression in terms of radiographic and clinical criteria (Kornman et al., 1997) and on the basis of age (younger than 35 yrs and older than 35 yrs).
CD14 C(-159)T Genotype Determination
Genomic DNA was extracted from peripheral blood by the use of a DNA extraction kit (Wako Pure Chemical Industries, Inc., Osaka, Japan). A DNA fragment containing a reported polymorphism within the CD14 promoter region was amplified by means of polymerase chain-reaction (PCR) with the use of a specific primer set (5'-TTGGTGCCAACAGATGAGGTTCAC-3' and 5'-TTCTTTCCTACACAGCGGCACCC-3').
The PCR product was digested with each of 20 units of HaeIII and 10 units of AvaII restriction enzymes (both from Promega Co., Madison, WI, USA) in appropriate buffer at 37°C overnight. The DNA fragments were separated by electrophoresis on 2% agarose gel and visualized with ethidium-bromide staining (Hubacek et al., 1999).
Reporter Assays
CD14 5'-flanking region that contains the proximal promoter and polymorphic region was amplified by PCR of genomic DNA from individuals homozygous for either C or T at position -159, with, as primers, 5'-AACTGCAGAATGGCAGCAAAAGACGTAAAATTC-3' and 5'-CGGAATTCCGCTGAGCAGGGAGGAATTACATC-3'. The PCR products were digested with Xho-I/HindIII and cloned into pGL3-Basic (Promega). Generated constructs (-159C/luc and -159T/luc) were confirmed by sequence analysis and prepared for transfection with the use of Nucleobond AX (Macherey-Nagel GmbH & Co. KG, Duren, Germany).
THP-1 cells were incubated in a six-well culture plate (Coster, Cambridge, MA, USA) at a concentration of 1 x 106 cell/well in 2 mL of RPMI 1640 supplemented with 10% fetal calf serum, 100 U/mL penicillin, and 100 µg/mL streptomycin. The cells were transiently transfected with -159C/luc, -159T/luc, or pGL3-Basic (1 µg) and a control Renilla luciferase reported plasmid (pRL-TK, 25 ng; Promega) with FuGENE6 Transfection Reagent (Roche, Indianapolis, IN, USA). Cells were then incubated at room temperature for 15 min before the addition of growth medium with 10% human serum. Following 24-hour incubation, luciferase activity was determined with use of the Dual-Luciferase Reporter Assay system (Promega) on a TD 20/20 Luminometer (Promega). Results were normalized for Renilla activity and were expressed as relative luciferase activity.
CD14 Genotypes and the Expression of CD14 on Monocytes
Four TT homozygotes, 4 CT heterozygotes, and 4 CC homozygotes were randomly selected from control subjects after being genotyped. Peripheral blood mononuclear cells (PBMC) were separated by Ficoll-Paque density gradient centrifugation. The cells were washed and re-suspended at a concentration of 1 x 106 cells/50 µL in PBS containing 1% FCS and 0.1% NaN3. The cells were incubated with saturating amounts of either Pc5-conjugated anti-CD14 (RMO52; Immunotech., Marseilles, France) or isotype control. The cells were analyzed by means of an FACscan flow cytometer (Becton Dickinson, San Jose, CA, USA) equipped with Consort 30 software (Hewlett-Packard, Cupertino, CA, USA). Data analysis was performed with CELLQuest software (Becton Dickinson). Monocytes were gated according to their forward- and side-scatter characteristics.
CD14 Genotypes and the Production of TNF- by Monocytes Stimulated with P. gingivalis LPS
PBMCs were re-suspended in RPMI 1640 supplemented with 10% human AB serum (C-six Diagnostics, Inc., Mequon, WI, USA) and inoculated onto a 96-well plate at a concentration of 5 x 106/mL, then allowed to adhere for 2 hrs at 37°C. The adherent cells were stimulated with 1 µg/mL P. gingivalis LPS for 24 hrs in RPMI 1640 supplemented with 10% human serum. The supernatants were collected, and the TNF- activity was determined by means of an ELISA kit (Endogen Inc., Woburn, MA, USA).
Statistical Analysis
We calculated genotype frequencies on patients and control groups by direct counting. The differences in the frequencies of the CD14 genotypes and alleles were analyzed by the  2 test with Yates’ correction. The difference in the levels of luciferase activity, surface expression of CD14, and TNF- production between each allele and genotype were analyzed by unpaired t test. A probability value < 0.05 was considered statistically significant.
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RESULTS
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Genotype Frequency of CD14 -159 Polymorphism in AgP Patients
The clinical profile of the patients is shown in Table 1 . The genotype distribution of total periodontitis patients showed that 24.5% were CC, 43.6% were CT, and 31.9% were TT, and this was not significantly different from the distribution in control subjects (Table 2; 18.3% were CC, 47.1% were CT, and 34.6% were TT). Similarly, allele frequency of the CD14 gene promoter at position -159 did not differ significantly between the two groups. Further, there was no obvious skewing in either genotype distribution, or allele frequency, with disease severity. However, young subjects were statistically different from older subjects and from older subjects with advanced disease in terms of genotype distribution (Table 2; young vs. older, 2 = 6.34, p = 0.042; young vs. older advanced, 2 = 7.08; p = 0.029).
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Table 2. Allele and Genotype Frequencies of Polymorphism in the CD14 Gene Promoter in Periodontitis Patients and Controls
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Transcriptional Activity of CD14/-159C and CD14/-159T
The wild-type CD14 promoter (-159C/luc) had strong basal transcriptional activity. Further, the SNP at position -159 from C to T resulted in an 80% increase in constitutive gene expression (Fig. 1). This difference in transcriptional activity was significant (p < 0.05).
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Figure 1. Transcriptional activity of CD14/-159C and CD14/-159T. CD14/-159T has enhanced transcriptional activity in reporter assays. THP-1 cells were transiently transfected with -159C/luc, -159T/luc, or pGL3-Basic (1 µg). Luciferase activity was normalized for transfection efficiency with the use of a control plasmid pRL-TK (25 ng) and protein concentration. Results are fold increase in relative luciferase activity (RLA) of the CD14 reporter constructs compared with the empty control vector pGL3-Basic. The data are presented as mean ± SEM of 3 replicates. *Significant at p = 0.015 (unpaired t test).
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CD14 Expression on Monocyte, TNF- Production, and Genotypes
The expression of CD14 on monocytes tended to correlate with genotype, where mean fluorescence intensity was the highest on monocytes with the TT genotype, the lowest in those with the CC genotype, and intermediate in those with the CT heterozygous genotype (Fig. 2A). However, the difference between genotypes was not statistically significant.
The stimulatory effect of P. gingivalis LPS was the most evident in TT homozygous genotype followed by CT heterozygous and CC homozygous, in that order. TT homozygous was significantly higher than CC homozygous (p = 0.0036) and CT heterozygous (p = 0.0028) (Fig. 2B ).
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DISCUSSION
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The results of the present study were unable to show any difference in CD14 genotype distribution between subjects and controls, or between subjects with various levels of disease severity. In contrast, there was a statistically significant difference in genotype distribution between young (under 35 yrs of age) and older (over 35 yrs of age) subjects and between young subjects and older subjects with advanced disease. Previously so-called Early-onset Periodontitis (EOP) was classified on the basis of subjects being under 35 yrs of age. Whether these young subjects had Aggressive Periodontitis (AgP) could not be determined. Clearly, none of them had classic Localized or Generalized Juvenile Periodontitis according to the accepted criteria, and while a diagnosis of AgP could not be ruled out, it is highly likely that they had Chronic Periodontitis manifesting at an earlier age. In this context, the results of the present study show that the SNP at position -159 in the promoter region of the CD14 gene is not associated with periodontal disease status in this Japanese population. At the same time, however, within the diseased population, it may be associated with the early expression of the disease. This would suggest that, like other polymorphisms, the SNP at position -159 in the promoter region of the CD14 gene is not essential for periodontal disease, but when present in a susceptible population, it may contribute to early disease expression. In this context, while other genetic and environmental factors defining susceptibility to periodontitis need to be determined, this SNP of the CD14 gene may represent a non-essential risk factor in the Japanese population.
The biological mechanisms of this association are nevertheless obscure, since the differences involve an increased proportion of CC homozygotes and decreased proportion of CT heterozygotes.
The polymorphic site, C or T at position -159, within the Sp1 transcription factor binding site has been shown to have a major influence on CD14 expression (Baldini et al., 1999). The present study has confirmed previous findings that a common SNP in the proximal CD14 promoter resulted in increased transcriptional activity in Mono Mac 6 cells. It is generally believed that the function of Sp protein-dependent promoters is regulated by the relative ratio between activating and repressing members of the Sp family. In this context, the -159CT polymorphism increases transcription by lowering the affinity of the CD14 regulatory region for Sp3, a factor known to inhibit the activity of several promoters.
Since LPS stimulation up-regulates CD14 expression on monocytes (Marchant et al., 1992; Landmann et al., 1996), it was reasonable to hypothesize that repeated stimulation may lead to substantial differences in the levels of CD14 expression, resulting in much higher pro-inflammatory cytokine production in monocytes with -159T than in those with -159C in vivo. While the present study showed that monocytes with TT genotype demonstrated slightly higher mean fluorescence intensity for CD14, the difference between the TT genotype and the CC genotype was much smaller than that expected from the transcriptional activity. A possible explanation for this discrepancy could be that the polymorphism at -159 may not be responsible for altered CD14 expression, but may be in linkage disequilibrium with another mutation that influences CD14 gene transcription (Lichy et al., 2002). However, this remains to be verified.
The production of TNF- by PBMC with each CD14 genotype showed little difference between CC and CT genotypes. However, monocytes with TT genotype showed elevated TNF- production in response to LPS stimulation, in spite of the fact that surface expression of the CD14 was similar. The reason for this finding is not known, although it is known that P. gingivalis LPS also binds to the TLR2 receptor and may be less dependent on CD14 binding (Hirschfeld et al., 2001). In this context, other molecules, such as TLRs, may be more important than CD14, or there may be other genetic variants affecting cytokine production within the CD14 gene.
In summary, we found that the CD14 -159 polymorphism did not affect the disease phenotype or severity of periodontitis in this Japanese population, but when present in susceptible subjects, it may be associated with early disease expression.
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ACKNOWLEDGMENTS
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This work was supported by grants from the Ministry of Education, Science, Sports and Culture of Japan (13470462, 14657553) and by a Grant for Promotion of Niigata University Research Projects (KY and TN).
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FOOTNOTES
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2 present address, Department of Periodontology, Faculty of Dentistry, Gajah Mada University, Yogyakarta, Indonesia; 
Received for publication October 20, 2002.
Revision received March 12, 2003.
Accepted for publication May 28, 2003.
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Journal of Dental Research, Vol. 82, No. 8,
612-616 (2003)
DOI: 10.1177/154405910308200808
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ABSTRACT
INTRODUCTION