CD14 gene promoter polymorphism in di¡erent clinicalforms of tuberculosis

Eugenia Pacheco 1, Carolina Fonseca 1, Carlos Montes, Jovanny Zabaleta 2,Luis F. Garc|¤a, Mauricio A. Arias �

Grupo de Inmunolog|¤a Celular e Inmunogene¤tica, Facultad de Medicina, Universidad de Antioquia, Cra 51 D No 62-29 Lab 283 Medell|¤n, Colombia

Received 8 September 2003; received in revised form 6 November 2003; accepted 12 November 2003

First published online 14 January 2004

Abstract

Mycobacterium tuberculosis interacts with monocyte^macrophages through cell surface molecules including CD14. A soluble form ofCD14 (sCD14) exists in human serum, and higher amounts of it are found in tuberculosis. A polymorphism on CD14 gene promoter wasassociated with increased sCD14 levels in some diseases. To evaluate whether this polymorphism associates with tuberculosis, its clinicalforms, and increased sCD14, genotype/allele frequencies in tuberculosis patients were compared with the controls. Results confirmedincreased levels of sCD14 in patients with tuberculosis, and those with miliary tuberculosis had the highest levels. sCD14 decreased tonormal levels after anti-tuberculosis treatment. No association was found between the CD14 polymorphism and tuberculosis or sCD14levels. Results suggest that sCD14 may be involved in anti-tuberculosis immune response, but its increase is a consequence of infectionrather than a predisposed genetic trait. Measuring sCD14 in tuberculosis may help monitor anti-tuberculosis treatment.9 2004 Federation of European Microbiological Societies. Published by Elsevier B.V. All rights reserved.

Keywords: Tuberculosis ; Soluble CD14; Polymorphism; Polymerase chain reaction-restriction fragment length polymorphism

1. Introduction

Tuberculosis (TB) is an infectious disease of high prev-alence worldwide. The World Health Organization re-ported on estimated 8.3 million new TB cases in the worldin the year 2000, and more than 1.8 million people diedfrom TB that year [1]. The responsible pathogen, Myco-bacterium tuberculosis, has the ability to survive within thehost phagocytic cells, and the interaction between the hostand the bacteria may result in tissue damage characterisedby granuloma formation, tissue necrosis with formation ofcavities and, eventually, dissemination of the disease [2].

M. tuberculosis, similarly as an array of di¡erent Gram-

negative and Gram-positive bacteria, interacts with mono-cyte^macrophages through several cell surface moleculesincluding CD14. This is a glycosylphosphatidylinositol-linked cell surface molecule [3], which, after interactingwith either whole bacteria [4] or cell wall componentssuch as lipopolysaccharide (LPS) from Gram-negativebacteria and lipoarabinomannans (LAM) from mycobac-teria [5,6], mediates cell activation upon triggering Toll-like receptors [6]. Cell activation results in release ofpro-in£ammatory molecules such as tumor necrosis factor(TNF)-K, interleukin (IL)-6, IL-1L, nitric oxide, oxygenradicals, and complement components [7,8], which up-reg-ulate host defence mechanisms that participate in eliminat-ing bacterial infection. However, high production of thesemolecules may cause profound deleterious e¡ects, includ-ing septic shock and death [8].A soluble form of CD14 (sCD14) also exists, which

lacks the glycosylphosphatidylinositol anchor [9]. sCD14seems to be produced by both monocytes [10] and hepa-tocytes [11,12], as well as endothelial cells [13], and con-siderably high amounts (2^3 Wg ml31) are found in theserum of healthy individuals [14]. The known functionalrelevance of sCD14 is its mediation of bacteria-induced

0928-8244 / 04 / $22.00 9 2004 Federation of European Microbiological Societies. Published by Elsevier B.V. All rights reserved.doi :10.1016/S0928-8244(03)00369-9

* Corresponding author. Tel. : +57 (4) 510 6098;Fax: +57 (4) 510 6079.

E-mail address: marias@medicina.udea.edu.co (M.A. Arias).

1 The ¢rst two authors contributed equally to this study.2 Present address: Department of Pathology and Tumor Immunology

Program, Stanley S Scott Cancer Center, Louisiana State UniversityHealth Sciences Center, 533 Bolivar St, CSRB 455, New Orleans,LA 70112 USA.

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cell activation of both membrane CD14 (mCD14)-negative[15,16] and mCD14-positive cells [17]. Activation viasCD14 induces the same pro-in£ammatory e¡ects second-ary to the interaction of LPS or bacterial cell wall compo-nents with mCD14.Increased levels of serum sCD14 have been found in

non-infectious and infectious diseases such as reumathoidarthritis [18], systemic lupus erythematosus [19], polytrau-matised and severely burned patients [20], septic shock[21], periodontitis [22], HIV-infection [23], and TB withor without HIV infection [24,25]. In the case of TB, in-creased levels of serum sCD14 have been described indi¡erent geographical and racial groups without ¢ndingremarkable di¡erences [24,25]. In these reports, the e¡ectof anti-TB treatment on the levels of sCD14 showed con-tradictory results. The reason for increased levels of serumsCD14 in these diseases is still unknown. However, it isbelieved that the levels of sCD14 in individuals with in-fectious and non-infectious diseases result from the stateof activation of monocytes^macrophages [20], since it isknown that monocyte activation results in increased shed-ding of sCD14 [10]. Nevertheless, monocytes alone maynot explain the increased amounts of sCD14. In this re-gard, other cells that produce sCD14, such as hepatocytes[11,12] and endothelial cells [13], may also contribute tothe levels of sCD14 found in these diseases.Mechanisms explaining the regulation of sCD14 pro-

duction are still poorly understood. However, there is evi-dence that sCD14 levels are under genetic control [26^29].A polymorphism at position 3159 (CD14/C(-159)CT)from the transcription start site was previously described[28,30] and associated with a high risk of myocardialinfarction. At the same time, Baldini et al. [26] showedan association of the TT genotype with increased levelsof serum sCD14 in allergic individuals that correlatedwith low levels of total serum IgE and IL-4, suggestingthat sCD14 could play a role in regulating the levels ofIgE [26]. Other studies published thereafter tested theassociation of this CD14 polymorphism with Crohn’sdisease [31], ulcerative colitis [31,32], psoriasis vulgaris[33], multiple sclerosis [34] and ischemic cerebrovasculardisease [35]. These studies showed an association of theCD14 gene polymorphism with some but not all of thediseases.Studies designed to elucidate the mechanisms by which

the CD14/C(-159)CT polymorphism may a¡ect the onsetof diseases and the levels of serum sCD14 have been per-formed [11,27,29,36]. It has been reported recently that theCD14/C(-159)CT polymorphism is located at a GC boxnear the transcription start site, which serves as a bindingsite for Sp1-Sp2/Sp3 transcription factors that are involvedin the regulation of CD14 gene transcription [36]. Takingthese observations into account, our work assessedwhether elevated levels of serum sCD14 in TB patientsare in£uenced by the CD14/C(-159)CT polymorphism,and also whether this polymorphism is associated with

TB and its clinical forms. Also, we assessed whether serumsCD14 levels vary in the di¡erent forms of the disease(pulmonary^pleural^miliary) and whether they changeafter the treatment follow-up.

2. Patients and methods

2.1. Study population

Two hundred and sixty seven patients with TB, includ-ing 204 pulmonary, 33 pleural, 18 miliary, and 12 withother forms of TB, were recruited from di¡erent healthunits in the metropolitan area of Medellin, Colombia. Di-agnoses were made by use of sputum smear staining andculturing of mycobacteria. Other diagnosis criteria such asclinical and epidemiological analysis, X ray, biopsy, andtesting for levels of adenosine deaminase in pleural e¥ux[37,38] were used when direct visualisation of mycobacte-ria was not possible. One hundred and twelve tuberculin-positive healthy control individuals were recruited fromthe Facultad de Medicina at the Universidad de Antio-quia, and the institutions from where the patients wererecruited. All studied individuals were from Caucasianand Mestizo ethnic groups. The latter corresponds to themix of Caucasian and Indians [39]. Individuals, who werepositive for HIV infection, or with a history of cancer,autoimmune, metabolic or endocrine diseases, as well aspregnant women, were excluded from the study. A writteninformed consent was obtained from all subjects after ex-planation of the research study and guarantee of completeprivacy. The study has been approved by the Ethics Com-mittee from the Facultad de Medicina at the Universidadde Antioquia.

2.2. Serum sCD14 levels

Serum was obtained from all the individuals within2 weeks of starting anti-TB treatment, and from 17 pa-tients with pulmonary TB, who were followed-up at 3 and6 months after initiating anti-TB treatment. The levels ofsCD14 were measured with a commercial ELISA kit, asrecommended by the manufacturer (BioSource, Nivelles,Belgium).

2.3. Polymerase chain reaction-restriction fragment lengthpolymorphism (PCR-RFLP) analysis of CD14 genepolymorphisms

CD14 genotyping was performed in all TB patients re-gardless of the time of anti-TB treatment, and in patientswho had already ¢nished the treatment. Genomic DNAwas obtained from 10 ml of EDTA-anti-coagulated blood,and 200 ng were ampli¢ed with 1.25 U of Taq DNA poly-merase (Life Technologies, Rockville, MD, USA), as de-scribed by Baldini et al. [26]. PCR products were run on

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2% agarose gels and visualised with ethidium bromidestaining.For RFLP analysis, the obtained DNA fragment of 497

bp was digested for 16 h with 2 U AvaII (New EnglandBiolabs, Beverly, MA, USA), which is speci¢c for the se-quence CGTCC present in carriers of the CD14/-159Tallele [26], and the product was electrophoresed on a12% polyacrylamide gel. Three di¡erent banding patternswere obtained: one band of 497 bp, which corresponds toCC homozygotes, three bands of 144, 353, and 497 bp forCT heterozygotes, and two bands of 144 and 353 for TTgenotype.

2.4. Statistical analysis

All data was analysed using the statistics package Prismversion 3.0 (GraphPad Software, San Diego, CA, USA).Allelic and genotype frequencies in cases and controlswere analysed using Fisher’s exact test. Comparison ofmeans between controls and TB patients was made byStudent’s t-test. Interactions between sCD14 levels andtreatment follow-up as well as interactions betweensCD14 levels and di¡erent forms of TB, were analysedby one-way ANOVA, with Tukey’s Multiple ComparisonPost-test. Interactions among sCD14 levels, genotypes,and TB versus controls were analysed by two-way AN-OVA.

3. Results

3.1. Characteristics of studied population

Table 1 shows demographic characteristics of the pop-ulation studied. Two thirds of the controls were female,while distribution within the patients’ group was moreeven. Patients and controls were not older than 65 yearsand the mean age was similar in both control and patientgroups.

3.2. Serum sCD14 levels

Levels of serum sCD14 from 105 TB patients and 64PPD-positive healthy controls were tested by ELISA. Fig.1 shows that, in agreement with previous reports, patientswith TB exhibited higher levels of sCD14 compared tohealthy controls (8.703 T 3.568 Wg ml31 vs. 5.189T 1.191;P6 0.0001). Furthermore, Fig. 2 shows that among TB

patients, those with miliary TB had the highest sCD14levels (pulmonary TB: 8.876T 3.457; pleural TB:8.811T 2.528; miliary TB: 12.350T 5.735 Wg ml31 ;P6 0.03).To resolve whether sCD14 levels are modi¢ed by anti-

TB treatment, serum sCD14 levels from a group of 17patients with pulmonary TB were tested at 0, 3 and6 months of anti-TB treatment. Fig. 3 shows that levelsof sCD14 signi¢cantly decreased during treatment(P6 0.008).

3.3. Polymorphisms of the CD14 gene promoter and TB

Genotype and allele frequencies of 112 healthy tuber-culin-positive controls and 267 TB patients were analysedfor the CD14/-159 polymorphisms using PCR-RFLP. Thethree di¡erent genotypes (CC, CT, TT) reported previ-ously [26] were observed after digesting the ampli¢edDNA fragment of 497 bp with the restriction enzymeAvaII. Genotype frequencies in both control group andTB patients were in Hardy^Weinberg equilibrium. No as-sociation was found between allele and genotype frequen-cies and the presence of TB as a whole, or the di¡erentforms of the disease (Table 2). About half the individualswere CT heterozygous in all TB groups (range 42^59%).Thirty ¢ve percent of pulmonary TB patients were CChomozygous, whereas 23% were TT homozygous. How-ever, no di¡erences were found when compared with thecontrol group (Table 2). Similar results were found whenall TB forms were put together in only one group. Thedistribution of the alleles C and T in cases and controlswas similar, with the C allele ranging between 52 and 63%,

Table 1Characteristics of controls and TB patients

n Sex n (%) Age, meanTSD (range)

male female male female

Controls 112 38 (33.9) 74 (66.1) 34.0 T 11.0 (19^56) 37.0T 11.5 (19^58)Patients 267 149 (55.8) 118 (44.2) 40.5 T 13.9 (17^65) 35.5T 13.3 (15^64)

Fig. 1. Serum sCD14 levels in TB patients and healthy controls. Serafrom tuberculin positive healthy controls and TB patients were testedfor levels of sCD14 by use of a commercial ELISA kit (BioSource, Ni-velles, Belgium); P6 0.0001.

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and the T allele between 37 and 48%. Again, di¡erences ofallele distribution between the di¡erent groups were notsigni¢cant.An association of CD14 gene polymorphisms with the

levels of serum sCD14 in the group of TB patients and inthe control group was also tested. As shown in Fig. 4,there was no association of serum sCD14 levels with theCD14 promoter polymorphisms, neither in controls nor inpulmonary TB patients.

4. Discussion

The results presented herein con¢rm high levels of se-rum sCD14 in TB patients in a di¡erent ethnic and geo-graphical population; show that the highest levels arefound in the serum of those patients with the miliaryform of the disease; show that serum sCD14 levels de-crease to normal levels after completion of anti-TB treat-ment; and suggest a lack of association of CD14/C-(-159)CT gene polymorphisms with both the developmentof TB and its di¡erent clinical forms, and the increasedlevels of serum sCD14 in patients with pulmonary TB.These results con¢rm ¢ndings elsewhere [24,25], report-

ing on TB patients with higher levels of serum sCD14 thanhealthy controls. However, the levels found in our controlpopulation were higher than in other reports [19,23,24].These di¡erences may be associated with racial, genetic,and/or environmental factors since the individuals studiedhere were a highly mixed Colombian population, whereasin other studies individuals were either Africans [25] orCaucasians [19,23]. Ju¡ermans et al. [24] studied serumsCD14 levels in TB patients from di¡erent geographicaland ethnic origins, including European, Asian, Africanand South American, but they reported no di¡erences be-tween the ethnic groups.Our results are in agreement with the data published by

Lawn et al. [25], in that the levels of sCD14 did not de-crease after 3 months of anti-TB treatment. However, ourstudy carried out a follow-up until 6 months of anti-TBtreatment, and it was found that at this time serum sCD14levels in TB patients reached those, found in control in-dividuals. When tested for di¡erences between the threegroups (0, 3, and 6 months) using Tukey’s Multiple Com-parison test, no signi¢cant di¡erences were found between0 and 3, or between 3 and 6 months. This ¢nding suggeststhat completing anti-TB treatment is necessary to decrease

Fig. 3. Follow-up of serum sCD14 levels during anti-TB treatment. Serafrom 17 patients with pulmonary TB were tested for sCD14 levels at0, 3, and 6 months of anti-TB treatment; P6 0.008.

Table 2Allele and genotype frequencies of CD14/C(-159)CT gene promoter polymorphisms in patients with di¡erent forms of TB and healthy tuberculin-posi-tive controls

Allele na f n f n f n f n f

Controls Pulmonary TB Pleural TB Miliary TB Others TB forms

C 116 0.52 229 0.56 39 0.59 20 0.59 15 0.63T 108 0.48 181 0.44 27 0.41 14 0.41 9 0.37Total 224 1.00 410 1.00 66 1.00 34 1.00 24 1.00GenotypeCC 31 0.28 72 0.35 11 0.33 5 0.29 4 0.33CT 54 0.48 85 0.42 17 0.52 10 0.59 7 0.59TT 27 0.24 48 0.23 5 0.15 2 0.12 1 0.08Total 112 1.00 205 1.00 33 1.00 17 1.00 12 1.00

Di¡erences tested by Fisher’s Exact Test were not signi¢cant.an=number of individuals; f=gene frequency

Fig. 2. Serum sCD14 levels in di¡erent clinical forms of TB. Sera frompatients with di¡erent forms of TB and tuberculin-positive healthy con-trols were tested for levels of sCD14, P6 0.03. Dotted line is themean+2SD of the control group.

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serum sCD14 to normal levels, and that monitoringsCD14 levels during treatment may be useful to followup patients’ response to therapy.An interesting ¢nding of this study was the statistically

signi¢cant di¡erence of sCD14 levels between the miliaryform of the disease and the pulmonary form. In fact, itwas found that miliary TB patients showed the highestlevels of sCD14 within the whole group. Since miliaryTB involves a deep immune suppression, it is worth spec-ulating that sCD14 may play a role in the way patientsrespond to M. tuberculosis. In other words, sCD14 may beacting as a negative modulator of the immune system.This hypothesis is supported by previous reports [40,41],where a role of sCD14 as an immunoregulator was sug-gested by its ability to inhibit in vitro cell proliferation andcytokine production (IL-2, IFN-Q, IL-4) by human T cells,and to inhibit IL-6 and IgE while increasing IgG1 produc-tion by human tonsillar B cells. These ¢ndings were sup-ported by Baldini et al. [26], who showed a negative cor-relation between high levels of serum sCD14 and lowerlevels of serum IL-4 in a population of white non-hispanicallergic patients. This report also showed that the group ofTT homozygotes for the CD14/C(-159)CT polymorphismhad signi¢cantly higher sCD14 levels and lower levels ofIgE.Since serum from TB patients has elevated levels of

sCD14, we tested whether these high levels of sCD14and the onset of TB could be associated with the CD14/C(-159)CT gene promoter polymorphism. We found thatthe distribution of the alleles in the population studied wasin Hardy^Weinberg equilibrium, similarly as it has beenfound in other di¡erent racial and geographical popu-lations [26,28,30,31,33,35]. Also, the allele frequencies(controls C/T: 0.52/0.48; TB: 0.56/0.44) were similar tothose reported in these populations, except where an asso-ciation of the CD14/C(-159)CT polymorphism with pso-riasis vulgaris and myocardial infarction was tested [30,

33]. In these reports, Finnish and Czech individuals werestudied with two thirds of the population carrying theC allele.Even though the number of patients and controls is still

small, the data presented here do not show an associationof the CD14/C(-159)CT polymorphism with either theonset of TB or its di¡erent forms (Table 2), or the levelsof serum sCD14 (Fig. 4). This is not surprising, sincecontradictory data have been reported recently. Thesedata come from studies in di¡erent human populationsin which the CD14/C(-159)CT polymorphism is eitherassociated [42] or not [35,43,44] with high serum sCD14levels, depending on the type of study, and the ethnicgroups involved. In addition, environmental gene interac-tions might also a¡ect the outcome of the CD14 genepolymorphism in TB, as has been suggested elsewhere[45,46]. It was reported recently [47] in patients with IgAnephropathy that those with a stable disease carried theTT genotype of the CD14/C(-159)CT polymorphism,whereas those with the CC genotype had an increasedrisk of disease progression. Interestingly, in vitro stimu-lated PBMC from controls with the TT genotype pro-duced signi¢cantly higher levels of sCD14 and lower levelsof IL-6 than those with the CC genotype [47]. It is possiblethat, in the case of TB, other genetic factors apart fromthose associated with the CD14/C(-159)CT polymor-phism are a¡ecting the expression of the CD14 gene.This may be the reason why the presence of the T alleledoes not explain the increase of sCD14, as has been shownelsewhere [26,29,36,47].In summary, the functional role of sCD14 in TB is still

unknown, even though it is clear that higher levels of thismolecule are found in the serum of such patients. With anincreasing number of reports suggesting a role of sCD14as an immunoregulatory molecule, and with the ¢ndingsreported herein that miliary TB (the most aggressive formof the disease) shows the highest levels of sCD14, it issigni¢cant to research further in order to clarify the actualrole of this molecule in TB.

Acknowledgements

We thank the following Colombian health institutionsfor facilitating access to TB patients within the TB controlprogrammes: Hospital Universitario San Vicente de Pau¤l,Hospital La Mar|¤a, hospitals and health centres from Met-rosalud, Instituto de Seguros Sociales, Coomeva, Comfe-nalco, Coopsana, Salud Total, Prosalco, and Calor deHogar from Medell|¤n, and also Hospital Manuel UribeAngel from Envigado, and Hospital La Cruz from PuertoBerr|¤o. We thank the patients and healthy controls whokindly agreed to participate in the research project. Thisstudy was supported by grants 1115-05-328-96 and 1115-05-11088 from COLCIENCIAS and the Observatorio deCiencia y Tecnolog|¤a-Colombia.

Fig. 4. E¡ect of CD14 gene promoter genotype on serum sCD14 levelsin patients with pulmonary TB. Measurement of serum sCD14 levelsand genotyping of the CD14 gene promoter were performed on healthycontrols and patients with pulmonary TB by use of ELISA and PCR-RFLP, respectively. Di¡erences between sCD14 levels and genotype inPatients group were not signi¢cant.

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