A CD14 promoter polymorphism is associated with CD14 ... - Nature

Genes and Immunity (2004) 5, 426–430 & 2004 Nature Publishing Group All rights reserved 1466-4879/04 $30.00 www.nature.com/gene

BRIEF COMMUNICATION

A CD14 promoter polymorphism is associated with CD14 expression and Chlamydia-stimulated TNFa production HL Eng1, CH Wang2, CH Chen3, MH Chou4, CT Cheng2 and TM Lin2 Department of Pathology, Chang Gung University and Memorial Hospital, Kaohsiung Medical Center, Kaohsiung, Taiwan, Republic of China; 2Department of Medical Technology, College of Medicine, National Cheng Kung University, Tainan, Taiwan, Republic of China; 3 Department of Neurology, College of Medicine, National Cheng Kung University, Tainan, Taiwan, Republic of China; 4Institute of Basic Medical Sciences, College of Medicine, National Cheng Kung University, Tainan, Taiwan, Republic of China 1

CD14, a pattern recognition receptor on monocyte and macrophage, plays a central role in innate immunity through recognition of bacterial lipopolysaccharide and initiation of inflammatory response. Recently, CD14/260C4T promoter gene polymorphism has been found to be related to a risk of inflammatory diseases. Our results showed that the C allele frequency among Chinese in Taiwan was lower than those in Western countries. The membrane CD14 expression was significantly higher in TT as compared with CT and CC genotypes (P ¼ 0.034, 0.044, respectively). There was a higher level of soluble CD14 in TT and CT genotypes than in CC genotypes. In addition, TNFa production in whole blood was significantly higher in TT genotype than in CC genotype after stimulation by Chlamydiae. In conclusion, the single base pair polymorphism of CD14 promoter gene is associated with CD14 expression and Chlamydia-stimulated TNFa production, and may thus play some role in the chlamydia-induced inflammatory response. Genes and Immunity (2004) 5, 426–430. doi:10.1038/sj.gene.6364100 Published online 27 May 2004 Keywords: CD14 polymorphism; TNFa; Chlamydia; LPS

Introduction As a pattern recognition receptor on the membrane of monocyte and macrophage, CD14, a membrane glycoprotein, plays a central role in innate immunity through recognition of bacterial lipopolysaccharide (LPS), and initiating an antimicrobial response.1 Membrane CD14 (mCD14) is a 54-kDa glycoprotein that was initially known as a myeloid differentiation marker. Soluble CD14 (sCD14) is released in two isoforms (49 and 55 kDa) from monocytes, both are lacking the glycosylphosphatidylinositol (GPI) anchoring.2,3 At the molecular level, CD14 acts by transferring LPS and other bacterial ligands from circulating LPS-binding protein to Toll-like receptor 4/MD-2 signaling complex.4 Engagement of this complex resulted in activation of innate host defense mechanisms such as the release of inflammatory cytokines, and in upregulation of co-stimulatory molecules, Correspondence: Dr TM Lin, Department of Medical Technology, College of Medicine, National Cheng Kung University, Tainan, Taiwan 701, Republic of China. E-mail: [email protected] This study was presented in part as a poster (P036) at the 22nd World Congress of Pathology and Laboratory Medicine, August 2003, Busan, Korea. Informed consent was obtained from participants, and the study was approved by the National Cheng Kung University Hospital Research Ethics Committee. Received 26 January 2004; revised 24 March 2004; accepted 30 March 2004; published online 27 May 2004

thus providing cues that are essential to directing adaptive immune response.5,6 A 260C4T (also known as 159C4T) polymorphism in the CD14 gene promoter results in decreased affinity of DNA/protein interaction at a GC box that contains a binding site for Sp proteins and modulates the activity of the promoter.7 Two groups have studied the promoter gene polymorphism in patients with myocardial infarction, and found that individuals homozygous for the T nucleotide had an increased risk for myocardial infarction.8,9 Thus, the two allelic variants of CD14 promoter may be important for infectious and inflammatory diseases, and this CD14 polymorphism could be a genetic factor responsible for interindividual differences in the susceptibility to infection. In our previous study, a significant association of TT genotype and Chlamydia pneumoniae (C pneumoniae) infection was found.10 Chlamydiae are obligate intracellular Gram negative bacteria. Infection with Chlamydiae is usually followed by a chronic inflammatory response that releases various cytokines. Recently, there has been increasing evidence that chronic or recurrent chlamydial infection of vessel walls might contribute to atherogenesis and possibly to plaque destabilization, as well as to acute coronary syndromes.11 Several investigators have reported that whole chlamydial elementary bodies (EB) and LPS preparations are capable of stimulating mononuclear phagocytes.5,12 In addition, macrophage activation and

subsequent release of tumor necrosis factor alpha (TNFa), a pleiotropic cytokine, has been reported as an important part of the cell-mediated and nonspecific immune response following chlamydial infection.13 Variation in the production of TNFa after various stimuli in healthy subjects might explain predispositions to the development of certain complications or phenotypes of infectious and immuno-inflammatory diseases. It was also reported that differences in the capacity for TNFa production might be related to genetic background.14 This study is aimed at investigating the relationship between the expression of monocyte mCD14 and plasma levels of sCD14, and the CD14 genotype in healthy subjects; to assess the intersubject variations in TNFa production after LPS and Chlamydial stimulation in whole blood cell culture, and to evaluate whether individuals of variant CD14/260 gene polymorphism are significantly different in basal level and induced variability of TNF-a production upon LPS stimulation and chlamydial infection.

a

Results and discussion

sCD14 (ng/ml)

CD14 polymorphism and TNFa production HL Eng et al

CD14/260 genotypes were identified for 165 healthy subjects (age 19–45 years; mean7s.d., 26.875.3 years; 84 females and 81 males). The frequencies of TT, CT, and CC were 24.2, 55.8, and 20.0% respectively. The T allele was 52.7% and C allele was 47.9%. Allele frequency and genotype distribution was very similar to the expected Hardy–Weinberg equilibrium. The distribution of CC genotype in Taiwan is significantly low as compared with those in Western countries.9,15,16 The mean fluorescence intensity (MFI) of mCD14 on PBMC and plasma levels of sCD14 were measured in 84 healthy individuals (age 20–45 years; mean7s.d., 23.874.3 years; 44 females and 40 males). As shown in Figure 1, mCD14 in TT genotypes (mean: 1818) are significantly higher than those in CT (mean: 1517; P ¼ 0.044) and CC (mean: 1603; P ¼ 0.034) genotypes. The plasma levels of sCD14 in CC (mean: 466 ng/ml) genotype are lower than TT (mean: 622 ng/ml; P ¼ 0.105) and CT (mean: 644 ng/ml; P ¼ 0.046) genotypes. Subjects with T allele at 260 site have significantly higher sCD14 levels than subjects with non-T allele (635 vs 466 ng/ml; P ¼ 0.035). In contrast, the mCD14 of subjects with non-C allele was significantly higher than C allele (1818 vs 1507; P ¼ 0.021). Our findings confirm that CD14 protein expression is under genetic control. Our data are consistent with previous reports, where Hubacek et al8 demonstrated an increased expression of CD14 on the surface of monocytes in carriers of the TT genotype, Baldini et al17 also found the polymorphism to associate with circulating sCD14 levels. However, these findings are in contrast with the results obtained by two other groups, who did not find an association between CD14 expression and CD14 promoter gene polymorphism.18,19 Ito et al18 found no association of CD14 promoter gene polymorphism with either the concentration of sCD14 or the density of mCD14 on monocytes in patients with cerebrovascular disease (CVD). It could be speculated that the monocytes were activated in CVD, but the sample size was small (n ¼ 30) for mCD14 determination. Heesen et al19 analyzed 95 healthy blood donors and found no statistically significant difference between the

427 p = 0.034 p = 0.044

mCD14 (MFI)

3000

2000

1000

0 TT

CT

b

CC p = 0.046

1500

1000

500

0 TT

CT

CC

Figure 1 Membrane CD14 expression on monocytes and plasma levels of sCD14 in different CD14 260C4T genetypes. (a) Peripheral blood mononuclear cells (PBMC) were separated by Ficoll-Paque and used for mCD14 analysis and DNA extraction for CD14 promoter genotyping. The density of mCD14 on the monocyte surface was stained with phycoerythrin-labeled antiCD14 monoclonal antibodies (Mfp9, Becton Dickinson, San Jose, CA, USA) and measured by flow cytometry. (b) Plasma levels of sCD14 were measured using a commercially available enzymelinked immunosorbent assay kit (R & D systems, Minneapolis, USA). A total of 26 TT homozygotes, 36 CT heterozygotes, and 22 CC homozygotes were randomly selected from healthy subjects after genotyping. The numbers of TT, CT, and CC were 26, 36, and 22, respectively. The line represents the mean of the genotypes. The mCD14 intensity and sCD14 level were compared with unpaired ttest.

mCD14 density of individuals with various genotypes. A possible explanation for the different finding between our study and that of Heesen et al19 is that the polymorphism at site 260 per se may not be responsible for altering CD14 expression. It may be in the linkage dysequilibrium with another mutation that influences CD14 expression in different ethic groups. To further investigate the influence of the polymorphism on proinflammatory cytokines induction, we analyzed TNFa production in two CD14 (260) genotypes (12 CC and 14 TT), randomly selected from healthy subjects after genotyping, by ex vivo LPS and Chlamydia stimulations of whole blood. In both CC and TT groups, LPS induced TNFa production in a dose-dependent manner (Figure 2). Particularly, after 24 h incubation, the mean TNFa production by 100 ng/ml LPS stimulation Genes and Immunity


428 4500

a TT

4000

8000 2*10^5 IFU

*

CC 2*10^6 IFU 1*10^7 IFU

3000

6000

TNFα production (pg/ml)


3500

2500 2000 1500 1000 500

4000

2000

0 0

0.01

0.1

1

10

100

1000

LPS concentration (ng/ml)

was higher in TT genotype than in CC genotype (2086.6 vs 1691.2 pg/ml). However, it is not statistically significant (P ¼ 0.363). The mean amounts of TNFa production in a 24-h whole blood cell culture after being stimulated with C pneumoniae (TW183) and C trachomatis (UW5) were also in a dose-dependent manner (Figure 3). The mean production of TNFa in the TT group is significantly higher than in the CC group, when they were stimulated by C. pneumoniae, TW183 with doses of (3683.5 vs 5526.0 pg/ml, P ¼ 0.014) 1 107 IFU (Figure 3a). Similar results were observed when they were stimulated by C. trachomatis, UW-5 with doses of 2 106 and 1 107 IFU (P ¼ 0.041 and 0.017, respectively) (Figure 3b). Genetically determined increased expression of CD14 could be associated with a high response to LPS. Temp et al20 reported a higher TNFa mRNA level in the TT subjects than in the CC after incubation with LPS or Escherichia coli. In our present study, after LPS stimulation the TT also had higher TNFa production than the CC genotypes, but the difference was not statistically significant. However, a statistically significant difference was observed only by chlamydial infection either by C pneumoniae or C trachomatis. Although chlamydial LPS had been reported to be less potent than E coli LPS in eliciting TNFa production,21 Chlamydiae might still have components other than LPS to stimulate monocytes mediated CD14 to produce TNFa. The outer surface of Chlamydiae contains two major antigens, LPS and the major outer membrane protein (MOMP). During the course of natural and experimental infection, both Genes and Immunity

0

CC

TT

b 6000

*

2*10^5 IFU

5000

2*10^6 IFU 1*10^7 IFU


Figure 2 Influence of CD14/260 gene polymorphism (CC and TT genotypes) on LPS dose–response curves of TNFa production in healthy subjects. A total of 14 TT homozygotes and 12 CC homozygotes were randomly selected from healthy subjects after genotyping. Heparin-anticoagulated venous blood was diluted 1 : 12.5 (v/v) with serum-free RPMI 1640 (GIBCO BRL, Karlsruhe, Germany) and stimulated with various concentration (range 0.01– 1000 ng/ml) of LPS (Escherichia coli O2:B22, Sigma Chemical, St Louis, MO). TNFa levels of the culture supernatants were determined by ELISA kit (Duoset, R&D system, Minneapolis, USA). Data represent the mean with 25th and 75th percentiles of 12 volunteers in the CC group and 14 volunteers in the TT group.

4000

*

3000

2000

1000

0

CC

TT

Figure 3 Influence of CD14/260 gene polymorphism (CC and TT genotypes) on TNFa production by stimulation of C pneumoniae and C trachomatis in healthy subjects. Heparin-anticoagulated venous blood was diluted 1 : 12.5 (v/v) with serum-free RPMI 1640 (GIBCO BRL, Karlsruhe, Germany) and stimulated with various inclusion forming units (IFU) (2 105, 2 106, and 2 107 IFU) of (a) C pneumoniae (TW183) and (b) C trachomatis (UW-5) for 24 h at 371C. TNF-a was determined in cell-free supernatants after 24 h of incubation by ELISA. Data are depicted as a box and whiskers blot that gives the range of values, with the box being subdivided into the 25 and 75% quantiles by the median; 50% of all values that lie within the box. Data are means7s.e. represented as the mean with 25th and 75th percentiles of the 12 volunteers in the CC group and 14 volunteer in the TT group. The stimulated TNFa production was statistically analyzed by Mann–Whitney test. *Po0.05 when compared with the CC and TT groups.

antigens interact with the immune system to induce antibodies directed against MOMP22 and LPS23 in the sera of infected patients. Recently, a second protein has


been associated with pathogenic chlamydia, it is the heat shock protein, Hsp-60, which produces inflammatory changes in experimental models of trachoma.24 Netea et al25 demonstrated that chlamydial endotoxin and acellular components of C pneumoniae are potent stimulus for cytokine production, and this mechanism may have an important role in the inflammatory aspects of atherogenesis. LPS and Hsp-60 are both biologically active in vitro and can induce secretion of inflammatory cytokines, and they are implicated in the pathogenesis of other chronic inflammatory chlamydial diseases (ie, trachoma, pelvic inflammatory disease).26 Hsp-60 was reported to activate human PBMC and monocytederivated macrophages through CD14 signaling and p38 mitogen-activated protein kinase, and sharing this pathway with LPS.27 However, the role of these protein antigens, MOMP and Hsp-60, relative to LPS, in the inflammatory response to infection has not been well characterized. With the infection of C pneumoniae and with the increased production of their TNFa, it may lead to chronic inflammatory reaction. Therefore, the TT subjects may have an increased inflammatory reaction to chlamydial infection, a process that may promote atherogenesis and plaque rupture. In conclusion, the single base pair polymorphism of CD14 promoter gene is associated with CD14 expression and Chlamydia-stimulated TNFa production, and may thus play some role in the chlamydia-induced inflammatory response. In addition, Toll-like receptors (TLR) have been shown to be important components of CD14 signaling complex and TLR2 and TLR4 have both been shown to contribute to the LPS and Hsp-60 responses of chlamydial infection.28 Further study on the roles of TLR2 and TLR4 gene polymorphisms contributes to the TNFa production of LPS stimulation and chlamydial infection is necessary.

Acknowledgements We express our thanks to Ms Yuan-Jen Huang and Ms Chin-Fang Tsai for their technical assistance. This study was supported by grants NSC89-2314B-182A-056 from the National Science Council, CMRPG8055 from Chang Gung University and Memorial Hospital, and MOE program for Promoting Academic Excellent of University under the Grant Number 91-B-FA09-2-4 from Ministry of Education, Taiwan, Republic of China.

429 5 6 7

8

9

10

11

12 13 14

15

16

17

18 19

References 1 Wright SD, Ramos RA, Tobias PS, Ulevitch RJ, Mathison JC. CD14, a receptor for complexes of lipopolysaccharide (LPS) and LPS binding protein. Science 1990; 249: 1431–1433. 2 Bazil V, Baudys M, Hilgert I et al. Structural relationship between the soluble and membrane-bound forms of human monocyte surface glycoprotein CD14. Mol Immunol 1989; 26: 657–662. 3 Durieux JJ, Vita N, Popescu O et al. The two soluble forms of the lipopolysaccharide receptor, CD14: characterization and release by normal human momocyte. Eur J Immunol 1994; 24: 2006–2012. 4 da Silva Correia J, Soldau K, Christen U, Tobias PS, Ulevitch RJ. Lipopolysaccharide is in close proximity to each of the

20

21

22

proteins in its membrane receptor complex: transfer from CD14 to TLR4 and MD-2. J Biol Chem 2001; 276: 21129–21135. Wright SD. CD14 and innate recognition of bacteria. J Immunol 1995; 155: 6–8. Ulevitch RJ, Tobias PS. Recognition of Gram-negative bacteria and endotoxin by the innate immune system. Curr Opin Immunol 1999; 11: 19–22. LeVan TD, Bloom JW, Bailey TJ et al. A common single nucleotide polymorphism in the CD14 promoter decreases the affinity of Sp protein binding and enhance transcriptional activity. J Immunol 2001; 167: 5838–5844. Hubacek JA, Rothe G, Pit’ha J et al. C(260)-T polymorphism in the promoter of the CD14 monocyte receptor gene as a risk factor for myocardial infarction. Circulation 1999; 99: 3218–3220. Unkelbach K, Gardemann A, Kostrzewa M et al. A new promoter polymorphism in the gene of lipopolysaccharide receptor CD14 is associated with expired myocardial infarction in patients with low atherosclerotic risk profile. Arterioscler Thromb Vasc Biol 1999; 19: 932–938. Eng HL, Chen CH, Kuo CC, Wu JS, Wang CH, Lin TM. Association of CD14 gene promotor polymorphism and Chlamydia pneumoniae infection. J Infect Dis 2003; 188: 90–97. Bauriedel G, Welsch U, Likungu JA, Welz A, Luderitz B. Chlamydia pneumoniae in coronary plaques: increased detection with acute coronary syndrome. Dtsch Med Wochensch 1999; 124: 375–380. Rothermel CD, Schachter J, Lavrich P, Lipsitz EC, Francus T. Chlamydia trachomatis-induced production of interleukin-1 by human monocytes. Infect Immun 1989; 57: 2705–2711. Williams DM, Magee DM, Bonewald L et al. A role in vivo for tumor necrosis factor alpha in host defense against Chlamydia trachomatis. Infect Immun 1990; 58: 1572–1576. Louis E, Franchimont D, Piron A et al. Tumor necrosis factor (TNF) gene polymorphism influences TNF-a production in lipopolysaccharide (LPS)-stimulated whole blood culture in health human. Clin Exp Immunol 1998; 113: 401–406. Zee RY, Lindpaintner K, Struk B, Hennekens CH, Ridker PM. A prospective evaluation of CD14(260) T gene polymorphism and the risk of myocardial infarction. Atherosclerosis 2001; 154: 699–702. Karhukorpi J, Yan Y, Niemela S et al. Effect of CD14 promoter polymorphism and H pylori infection and its clinical outcomes on clinical CD14. Clin Exp Immunol 2002; 128: 326–332. Baldini M, Lohman IC, Halonen M et al. A polymorphism in the 50 flanking region of the CD14 gene is associated with circulating soluble CD14 levels and with total serum immunoglobulin E. Am J Respir Cell Mol Biol 1999; 20: 976–983. Ito D, Murata M, Tanahashi N et al. Polymorphism in the promoter of lipopolysaccharide receptor CD14 and ischemic cerebrovascular disease. Stroke 2000; 31: 2661–2664. Heesen M, Blomeke B, Schluter B, Heussen N, Rossaint R, Kunz D. Lack of association between the 260C-T promoter polymorphism of the endotoxin receptor CD14 gene and the CD14 density of unstimulated human monocytes and soluble CD14 plasma levels. Intensive Care Med 2001; 27: 1770–1775. Temple SE, Cheong KY, Almeida CM, Price P, Waterer GW. Polymorphisms in lymphotoxin alpha and CD14 influence TNFa production induced by Gram-positive and Gramnegative bacteria. Gene Immun 2003; 4: 283–288. Van der Linden MW, Huizinga TW, Stoeken DJ, Sturk A, Westendorp RG. Determination of tumor necrosis factor-alpha and interleukin-10 production in a whole blood stimulation system: assessment of laboratory error and individual variation. J Immunol Methods 1998; 218: 63–71. Jones J, Schachter MJ, Stephens RS. Evaluation of the humoral immune response in trachoma to Chlamydia trachomatis major Genes and Immunity


430 outer membrane proteins by sequence-defined immunoassay. J Infect Dis 1992; 166: 915–919. 23 Brade L, Brunnemann H, Ernst M et al. Occurrence of antibodies against chlamydial lipopolysaccharide in human sera measured by ELISA using an artificial glycoconjugated antigen, FEMS. Immunol Med Microbiol 1994; 8: 27–42. 24 Morrison RP, Su H, Lyng K, Yuan Y. The Chlamydia trachomatis hyp operon is homologous to the groE stress response operon of Escherichia coli. Infect Immun 1990; 58: 2701–2705. 25 Netea MG, Selzman CH, Kullberg BJ et al. Acellar components of Chlamydia pneumoniae stimulate cytokine production in human blood mononuclear cells. Eur J Immunol 2000; 30: 541–549.

Genes and Immunity

26 LaVerda D, Kalayoglu MV, Byme GI. Chlamydial heat shock proteins and disease pathology: new paradigms for old problems? Infect Dis Obstet Gynecol 1999; 7: 64–71. 27 Kol A, Lichtman AH, Finberg RW, Libby P, Kurt-Jones EA. Heat shock protein (HSP)60 activates the innate immune response:CD14 is an essential receptor for HSP60 activation of mononuclear cells. J Immun 2000; 164: 13–17. 28 Bulut Y, Faure E, Thomas L et al. Chlamydial heat shock protein 60 activates macrophages and endothelial cells through Toll-like receptor 4 and MD2 in MyD88-dependent pathway. J Immunol 2002; 168: 1435–1440.