CD14 gene promoter polymorphism in different clinical forms of

FEMS Immunology and Medical Microbiology 40 (2004) 207^213
www.fems-microbiology.org
CD14 gene promoter polymorphism in di¡erent clinical
forms 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 of
CD14 (sCD14) exists in human serum, and higher amounts of it are found in tuberculosis. A polymorphism on CD14 gene promoter was
associated with increased sCD14 levels in some diseases. To evaluate whether this polymorphism associates with tuberculosis, its clinical
forms, and increased sCD14, genotype/allele frequencies in tuberculosis patients were compared with the controls. Results confirmed
increased levels of sCD14 in patients with tuberculosis, and those with miliary tuberculosis had the highest levels. sCD14 decreased to
normal levels after anti-tuberculosis treatment. No association was found between the CD14 polymorphism and tuberculosis or sCD14
levels. Results suggest that sCD14 may be involved in anti-tuberculosis immune response, but its increase is a consequence of infection
rather 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 prevalence worldwide. The World Health Organization reported on estimated 8.3 million new TB cases in the world
in the year 2000, and more than 1.8 million people died
from TB that year [1]. The responsible pathogen, Mycobacterium tuberculosis, has the ability to survive within the
host phagocytic cells, and the interaction between the host
and the bacteria may result in tissue damage characterised
by granuloma formation, tissue necrosis with formation of
cavities and, eventually, dissemination of the disease [2].
M. tuberculosis, similarly as an array of di¡erent Gram-
* 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.
Present address: Department of Pathology and Tumor Immunology
Program, Stanley S Scott Cancer Center, Louisiana State University
Health Sciences Center, 533 Bolivar St, CSRB 455, New Orleans,
LA 70112 USA.
2
negative and Gram-positive bacteria, interacts with monocyte^macrophages through several cell surface molecules
including CD14. This is a glycosylphosphatidylinositollinked cell surface molecule [3], which, after interacting
with either whole bacteria [4] or cell wall components
such as lipopolysaccharide (LPS) from Gram-negative
bacteria and lipoarabinomannans (LAM) from mycobacteria [5,6], mediates cell activation upon triggering Tolllike receptors [6]. Cell activation results in release of
pro-in£ammatory molecules such as tumor necrosis factor
(TNF)-K, interleukin (IL)-6, IL-1L, nitric oxide, oxygen
radicals, and complement components [7,8], which up-regulate host defence mechanisms that participate in eliminating bacterial infection. However, high production of these
molecules may cause profound deleterious e¡ects, including septic shock and death [8].
A soluble form of CD14 (sCD14) also exists, which
lacks the glycosylphosphatidylinositol anchor [9]. sCD14
seems to be produced by both monocytes [10] and hepatocytes [11,12], as well as endothelial cells [13], and considerably high amounts (2^3 Wg ml31 ) are found in the
serum of healthy individuals [14]. The known functional
relevance 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
FEMSIM 1661 10-3-04
208
E. Pacheco et al. / FEMS Immunology and Medical Microbiology 40 (2004) 207^213
cell activation of both membrane CD14 (mCD14)-negative
[15,16] and mCD14-positive cells [17]. Activation via
sCD14 induces the same pro-in£ammatory e¡ects secondary to the interaction of LPS or bacterial cell wall components with mCD14.
Increased levels of serum sCD14 have been found in
non-infectious and infectious diseases such as reumathoid
arthritis [18], systemic lupus erythematosus [19], polytraumatised and severely burned patients [20], septic shock
[21], periodontitis [22], HIV-infection [23], and TB with
or without HIV infection [24,25]. In the case of TB, increased levels of serum sCD14 have been described in
di¡erent geographical and racial groups without ¢nding
remarkable di¡erences [24,25]. In these reports, the e¡ect
of anti-TB treatment on the levels of sCD14 showed contradictory results. The reason for increased levels of serum
sCD14 in these diseases is still unknown. However, it is
believed that the levels of sCD14 in individuals with infectious and non-infectious diseases result from the state
of activation of monocytes^macrophages [20], since it is
known that monocyte activation results in increased shedding of sCD14 [10]. Nevertheless, monocytes alone may
not explain the increased amounts of sCD14. In this regard, other cells that produce sCD14, such as hepatocytes
[11,12] and endothelial cells [13], may also contribute to
the levels of sCD14 found in these diseases.
Mechanisms explaining the regulation of sCD14 production are still poorly understood. However, there is evidence 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 myocardial
infarction. At the same time, Baldini et al. [26] showed
an association of the TT genotype with increased levels
of serum sCD14 in allergic individuals that correlated
with low levels of total serum IgE and IL-4, suggesting
that sCD14 could play a role in regulating the levels of
IgE [26]. Other studies published thereafter tested the
association of this CD14 polymorphism with Crohn’s
disease [31], ulcerative colitis [31,32], psoriasis vulgaris
[33], multiple sclerosis [34] and ischemic cerebrovascular
disease [35]. These studies showed an association of the
CD14 gene polymorphism with some but not all of the
diseases.
Studies designed to elucidate the mechanisms by which
the CD14/C(-159)CT polymorphism may a¡ect the onset
of diseases and the levels of serum sCD14 have been performed [11,27,29,36]. It has been reported recently that the
CD14/C(-159)CT polymorphism is located at a GC box
near the transcription start site, which serves as a binding
site for Sp1-Sp2/Sp3 transcription factors that are involved
in the regulation of CD14 gene transcription [36]. Taking
these observations into account, our work assessed
whether elevated levels of serum sCD14 in TB patients
are 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 serum
sCD14 levels vary in the di¡erent forms of the disease
(pulmonary^pleural^miliary) and whether they change
after the treatment follow-up.
2. Patients and methods
2.1. Study population
Two hundred and sixty seven patients with TB, including 204 pulmonary, 33 pleural, 18 miliary, and 12 with
other forms of TB, were recruited from di¡erent health
units in the metropolitan area of Medellin, Colombia. Diagnoses were made by use of sputum smear staining and
culturing of mycobacteria. Other diagnosis criteria such as
clinical and epidemiological analysis, X ray, biopsy, and
testing for levels of adenosine deaminase in pleural e¥ux
[37,38] were used when direct visualisation of mycobacteria was not possible. One hundred and twelve tuberculinpositive healthy control individuals were recruited from
the Facultad de Medicina at the Universidad de Antioquia, and the institutions from where the patients were
recruited. All studied individuals were from Caucasian
and Mestizo ethnic groups. The latter corresponds to the
mix of Caucasian and Indians [39]. Individuals, who were
positive for HIV infection, or with a history of cancer,
autoimmune, metabolic or endocrine diseases, as well as
pregnant women, were excluded from the study. A written
informed consent was obtained from all subjects after explanation of the research study and guarantee of complete
privacy. The study has been approved by the Ethics Committee from the Facultad de Medicina at the Universidad
de Antioquia.
2.2. Serum sCD14 levels
Serum was obtained from all the individuals within
2 weeks of starting anti-TB treatment, and from 17 patients with pulmonary TB, who were followed-up at 3 and
6 months after initiating anti-TB treatment. The levels of
sCD14 were measured with a commercial ELISA kit, as
recommended by the manufacturer (BioSource, Nivelles,
Belgium).
2.3. Polymerase chain reaction-restriction fragment length
polymorphism (PCR-RFLP) analysis of CD14 gene
polymorphisms
CD14 genotyping was performed in all TB patients regardless of the time of anti-TB treatment, and in patients
who had already ¢nished the treatment. Genomic DNA
was obtained from 10 ml of EDTA-anti-coagulated blood,
and 200 ng were ampli¢ed with 1.25 U of Taq DNA polymerase (Life Technologies, Rockville, MD, USA), as described by Baldini et al. [26]. PCR products were run on
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E. Pacheco et al. / FEMS Immunology and Medical Microbiology 40 (2004) 207^213
2% agarose gels and visualised with ethidium bromide
staining.
For RFLP analysis, the obtained DNA fragment of 497
bp was digested for 16 h with 2 U AvaII (New England
Biolabs, Beverly, MA, USA), which is speci¢c for the sequence CGTCC present in carriers of the CD14/-159T
allele [26], and the product was electrophoresed on a
12% polyacrylamide gel. Three di¡erent banding patterns
were obtained: one band of 497 bp, which corresponds to
CC homozygotes, three bands of 144, 353, and 497 bp for
CT heterozygotes, and two bands of 144 and 353 for TT
genotype.
2.4. Statistical analysis
All data was analysed using the statistics package Prism
version 3.0 (GraphPad Software, San Diego, CA, USA).
Allelic and genotype frequencies in cases and controls
were analysed using Fisher’s exact test. Comparison of
means between controls and TB patients was made by
Student’s t-test. Interactions between sCD14 levels and
treatment follow-up as well as interactions between
sCD14 levels and di¡erent forms of TB, were analysed
by one-way ANOVA, with Tukey’s Multiple Comparison
Post-test. Interactions among sCD14 levels, genotypes,
and TB versus controls were analysed by two-way ANOVA.
209
Fig. 1. Serum sCD14 levels in TB patients and healthy controls. Sera
from tuberculin positive healthy controls and TB patients were tested
for levels of sCD14 by use of a commercial ELISA kit (BioSource, Nivelles, Belgium) ; P 6 0.0001.
patients, those with miliary TB had the highest sCD14
levels (pulmonary TB: 8.876 T 3.457; pleural TB :
8.811 T 2.528; miliary TB: 12.350 T 5.735 Wg ml31 ;
P 6 0.03).
To resolve whether sCD14 levels are modi¢ed by antiTB treatment, serum sCD14 levels from a group of 17
patients with pulmonary TB were tested at 0, 3 and
6 months of anti-TB treatment. Fig. 3 shows that levels
of sCD14 signi¢cantly decreased during treatment
(P 6 0.008).
3.3. Polymorphisms of the CD14 gene promoter and TB
3. Results
3.1. Characteristics of studied population
Table 1 shows demographic characteristics of the population studied. Two thirds of the controls were female,
while distribution within the patients’ group was more
even. Patients and controls were not older than 65 years
and the mean age was similar in both control and patient
groups.
3.2. Serum sCD14 levels
Levels of serum sCD14 from 105 TB patients and 64
PPD-positive healthy controls were tested by ELISA. Fig.
1 shows that, in agreement with previous reports, patients
with TB exhibited higher levels of sCD14 compared to
healthy controls (8.703 T 3.568 Wg ml31 vs. 5.189 T 1.191;
P 6 0.0001). Furthermore, Fig. 2 shows that among TB
Genotype and allele frequencies of 112 healthy tuberculin-positive controls and 267 TB patients were analysed
for the CD14/-159 polymorphisms using PCR-RFLP. The
three di¡erent genotypes (CC, CT, TT) reported previously [26] were observed after digesting the ampli¢ed
DNA fragment of 497 bp with the restriction enzyme
AvaII. Genotype frequencies in both control group and
TB patients were in Hardy^Weinberg equilibrium. No association was found between allele and genotype frequencies and the presence of TB as a whole, or the di¡erent
forms of the disease (Table 2). About half the individuals
were CT heterozygous in all TB groups (range 42^59%).
Thirty ¢ve percent of pulmonary TB patients were CC
homozygous, whereas 23% were TT homozygous. However, no di¡erences were found when compared with the
control group (Table 2). Similar results were found when
all TB forms were put together in only one group. The
distribution of the alleles C and T in cases and controls
was similar, with the C allele ranging between 52 and 63%,
Table 1
Characteristics of controls and TB patients
n
Controls
Patients
112
267
Sex n (%)
Age, mean T SD (range)
male
female
male
female
38 (33.9)
149 (55.8)
74 (66.1)
118 (44.2)
34.0 T 11.0 (19^56)
40.5 T 13.9 (17^65)
37.0 T 11.5 (19^58)
35.5 T 13.3 (15^64)
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E. Pacheco et al. / FEMS Immunology and Medical Microbiology 40 (2004) 207^213
Fig. 2. Serum sCD14 levels in di¡erent clinical forms of TB. Sera from
patients with di¡erent forms of TB and tuberculin-positive healthy controls were tested for levels of sCD14, P 6 0.03. Dotted line is the
mean+2SD of the control group.
Fig. 3. Follow-up of serum sCD14 levels during anti-TB treatment. Sera
from 17 patients with pulmonary TB were tested for sCD14 levels at
0, 3, and 6 months of anti-TB treatment; P 6 0.008.
and the T allele between 37 and 48%. Again, di¡erences of
allele distribution between the di¡erent groups were not
signi¢cant.
An association of CD14 gene polymorphisms with the
levels of serum sCD14 in the group of TB patients and in
the control group was also tested. As shown in Fig. 4,
there was no association of serum sCD14 levels with the
CD14 promoter polymorphisms, neither in controls nor in
pulmonary TB patients.
ing on TB patients with higher levels of serum sCD14 than
healthy controls. However, the levels found in our control
population 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 studied
here were a highly mixed Colombian population, whereas
in other studies individuals were either Africans [25] or
Caucasians [19,23]. Ju¡ermans et al. [24] studied serum
sCD14 levels in TB patients from di¡erent geographical
and ethnic origins, including European, Asian, African
and South American, but they reported no di¡erences between 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 decrease after 3 months of anti-TB treatment. However, our
study carried out a follow-up until 6 months of anti-TB
treatment, and it was found that at this time serum sCD14
levels in TB patients reached those, found in control individuals. When tested for di¡erences between the three
groups (0, 3, and 6 months) using Tukey’s Multiple Comparison test, no signi¢cant di¡erences were found between
0 and 3, or between 3 and 6 months. This ¢nding suggests
that completing anti-TB treatment is necessary to decrease
4. Discussion
The results presented herein con¢rm high levels of serum sCD14 in TB patients in a di¡erent ethnic and geographical population; show that the highest levels are
found in the serum of those patients with the miliary
form of the disease ; show that serum sCD14 levels decrease to normal levels after completion of anti-TB treatment; and suggest a lack of association of CD14/C(-159)CT gene polymorphisms with both the development
of TB and its di¡erent clinical forms, and the increased
levels of serum sCD14 in patients with pulmonary TB.
These results con¢rm ¢ndings elsewhere [24,25], report-
Table 2
Allele and genotype frequencies of CD14/C(-159)CT gene promoter polymorphisms in patients with di¡erent forms of TB and healthy tuberculin-positive controls
Allele
na
f
Controls
C
T
Total
Genotype
CC
CT
TT
Total
n
f
Pulmonary TB
n
f
Pleural TB
n
f
Miliary TB
n
f
Others TB forms
116
108
224
0.52
0.48
1.00
229
181
410
0.56
0.44
1.00
39
27
66
0.59
0.41
1.00
20
14
34
0.59
0.41
1.00
15
9
24
0.63
0.37
1.00
31
54
27
112
0.28
0.48
0.24
1.00
72
85
48
205
0.35
0.42
0.23
1.00
11
17
5
33
0.33
0.52
0.15
1.00
5
10
2
17
0.29
0.59
0.12
1.00
4
7
1
12
0.33
0.59
0.08
1.00
Di¡erences tested by Fisher’s Exact Test were not signi¢cant.
n = number of individuals; f = gene frequency
a
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E. Pacheco et al. / FEMS Immunology and Medical Microbiology 40 (2004) 207^213
Fig. 4. E¡ect of CD14 gene promoter genotype on serum sCD14 levels
in patients with pulmonary TB. Measurement of serum sCD14 levels
and genotyping of the CD14 gene promoter were performed on healthy
controls and patients with pulmonary TB by use of ELISA and PCRRFLP, respectively. Di¡erences between sCD14 levels and genotype in
Patients group were not signi¢cant.
serum sCD14 to normal levels, and that monitoring
sCD14 levels during treatment may be useful to follow
up patients’ response to therapy.
An interesting ¢nding of this study was the statistically
signi¢cant di¡erence of sCD14 levels between the miliary
form of the disease and the pulmonary form. In fact, it
was found that miliary TB patients showed the highest
levels of sCD14 within the whole group. Since miliary
TB involves a deep immune suppression, it is worth speculating that sCD14 may play a role in the way patients
respond to M. tuberculosis. In other words, sCD14 may be
acting 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 suggested by its ability to inhibit in vitro cell proliferation and
cytokine production (IL-2, IFN-Q, IL-4) by human T cells,
and to inhibit IL-6 and IgE while increasing IgG1 production by human tonsillar B cells. These ¢ndings were supported by Baldini et al. [26], who showed a negative correlation between high levels of serum sCD14 and lower
levels of serum IL-4 in a population of white non-hispanic
allergic patients. This report also showed that the group of
TT homozygotes for the CD14/C(-159)CT polymorphism
had signi¢cantly higher sCD14 levels and lower levels of
IgE.
Since serum from TB patients has elevated levels of
sCD14, we tested whether these high levels of sCD14
and the onset of TB could be associated with the CD14/
C(-159)CT gene promoter polymorphism. We found that
the distribution of the alleles in the population studied was
in Hardy^Weinberg equilibrium, similarly as it has been
found in other di¡erent racial and geographical populations [26,28,30,31,33,35]. Also, the allele frequencies
(controls C/T : 0.52/0.48; TB: 0.56/0.44) were similar to
those reported in these populations, except where an association of the CD14/C(-159)CT polymorphism with psoriasis vulgaris and myocardial infarction was tested [30,
211
33]. In these reports, Finnish and Czech individuals were
studied with two thirds of the population carrying the
C allele.
Even though the number of patients and controls is still
small, the data presented here do not show an association
of the CD14/C(-159)CT polymorphism with either the
onset of TB or its di¡erent forms (Table 2), or the levels
of serum sCD14 (Fig. 4). This is not surprising, since
contradictory data have been reported recently. These
data come from studies in di¡erent human populations
in which the CD14/C(-159)CT polymorphism is either
associated [42] or not [35,43,44] with high serum sCD14
levels, depending on the type of study, and the ethnic
groups involved. In addition, environmental gene interactions might also a¡ect the outcome of the CD14 gene
polymorphism in TB, as has been suggested elsewhere
[45,46]. It was reported recently [47] in patients with IgA
nephropathy that those with a stable disease carried the
TT genotype of the CD14/C(-159)CT polymorphism,
whereas those with the CC genotype had an increased
risk of disease progression. Interestingly, in vitro stimulated PBMC from controls with the TT genotype produced signi¢cantly higher levels of sCD14 and lower levels
of IL-6 than those with the CC genotype [47]. It is possible
that, in the case of TB, other genetic factors apart from
those associated with the CD14/C(-159)CT polymorphism are a¡ecting the expression of the CD14 gene.
This may be the reason why the presence of the T allele
does not explain the increase of sCD14, as has been shown
elsewhere [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 this
molecule are found in the serum of such patients. With an
increasing number of reports suggesting a role of sCD14
as an immunoregulatory molecule, and with the ¢ndings
reported herein that miliary TB (the most aggressive form
of the disease) shows the highest levels of sCD14, it is
signi¢cant to research further in order to clarify the actual
role of this molecule in TB.
Acknowledgements
We thank the following Colombian health institutions
for facilitating access to TB patients within the TB control
programmes : Hospital Universitario San Vicente de Pau¤l,
Hospital La Mar|¤a, hospitals and health centres from Metrosalud, Instituto de Seguros Sociales, Coomeva, Comfenalco, Coopsana, Salud Total, Prosalco, and Calor de
Hogar from Medell|¤n, and also Hospital Manuel Uribe
Angel from Envigado, and Hospital La Cruz from Puerto
Berr|¤o. We thank the patients and healthy controls who
kindly agreed to participate in the research project. This
study was supported by grants 1115-05-328-96 and 111505-11088 from COLCIENCIAS and the Observatorio de
Ciencia y Tecnolog|¤a-Colombia.
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FEMSIM 1661 10-3-04

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