original papers - Advances in Clinical and Experimental Medicine

original papers
Adv Clin Exp Med 2011, 20, 4, 413–421
ISSN 1230-025X
© Copyright by Wroclaw Medical University
Tomasz Zaborowski1, Kamila Wojas-Krawczyk1, Paweł Krawczyk1,
Olga Jankowska1, Jan Siwiec1, Tomasz Kucharczyk1, 2, Maciej Grzybek3,
Janusz Milanowski1, 4
The Effect of CD14 and TLR4 Gene Polymorphisms
on the Occurrence of Atopic and Non-Atopic Asthma
Wpływ polimorfizmu genu CD14 i TLR4 na występowanie atopowej
i nieatopowej astmy oskrzelowej
Department of Pneumonology, Oncology and Allergology, Medical University, Lublin, Poland
Postgraduate School of Molecular Medicine, Warsaw Medical University, Warsaw, Poland
3
Faculty of Biology and Animal Breeding, University of Life Science, Lublin, Poland
4
Institute of Agriculture Medicine, Lublin, Poland
1
2
Abstract
Background. A significant increase in the incidence of asthma which has been observed in recent years leads many
researchers to the opinion that it is not caused only by hereditary factors but also by exposure to external factors.
The fact that environmental changes cause asthma should not exclude other causes though, especially in atopic,
genetically-predisposed individuals. Evidence of this phenomenon is the protective effect of lipopolysaccharide
on the incidence of atopic asthma in carriers of certain polymorphic variations of LPS receptor genes: TLR4 and
CD14.
Objectives. The aim of this study was to determine the genetic predisposition to the occurrence of asthma, depending on the variability of the endotoxin receptor gene.
Material and Methods. The study included a group of 106 individuals diagnosed with asthma. The control group
numbered 159 healthy individuals. Using the RFLP-PCR technique, polymorphism –159C < T of the CD14 gene
and 1187A < G of the TLR4 gene were identified. In addition, the number of eosinophiles in peripheral blood and
the total concentration of IgE in serum were determined in the group of individuals with asthma.
Results. The TT genotype in position 159 of CD14 and the AA genotype in position 1187 of TLR4 are present significantly less frequently in atopic asthma and are associated with a lower concentration of IgE in blood serum in
individuals with asthma. CC homozygotes in the CD14 gene and AG heterozygotes in the TLR4 gene correspond
to a higher risk of occurrence of atopic asthma, while the TT genotype of the CD14 gene and AA of the TLR4 gene
are connected with a higher risk of non-atopic asthma.
Conclusion. Atopic and non-atopic asthma differ in etiology at the molecular level. Exposure to endotoxin in
patients with the characteristic polymorphisms of genes for LPS receptors may have a protective effect on the
development of atopic asthma (Adv Clin Exp Med 2011, 20, 4, 413–421).
Key words: asthma, polymorphism, TLR4 gene, CD14 gene, lipopolysaccharide.
Streszczenie
Wprowadzenie. Znaczące zwiększenie zachorowań na astmę oskrzelową obserwowane w ostatnich dekadach sprawia, że większość badaczy przyczyny tego zjawiska dopatruje się bardziej po stronie zmieniającego się narażenia na
czynniki zewnętrzne, niż w predyspozycjach dziedzicznych. Nie można jednak wykluczyć, że zmiany środowiska
wywołują astmę oskrzelową, zwłaszcza atopową u osób predysponowanych genetycznie. Dowodem na to ma być
protekcyjny wpływ lipopolisacharydu na częstość występowania astmy atopowej u nosicieli pewnych polimorficznych odmian genów receptorów LPS: TLR4 i CD14.
Cel pracy. Określenie genetycznej predyspozycji do wystąpienia astmy oskrzelowej w zależności od zmienności
genów receptorów dla endotoksyny.
Materiał i metody. Badaniem objęto grupę 106 chorych z rozpoznaną astmą oskrzelową. Grupę kontrolną stanowiło 159 osób zdrowych. Z użyciem metody PCR RFLP oznaczono polimorfizm –159C < T genu CD14 oraz poli-
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T. Zaborowski et al.
morfizm 1187A < G genu TLR4. W grupie osób chorych na astmę oskrzelową oznaczono ponadto liczbę eozynofili
we krwi obwodowej oraz stężenie IgE całkowitego w surowicy krwi.
Wyniki. Genotyp TT w pozycji 159 genu CD14 oraz genotyp AA w pozycji 1187 genu TLR4 wiąże się z istotnie
rzadszym występowaniem atopowej astmy oskrzelowej i mniejszym stężeniem IgE całkowitego w surowicy krwi
osób chorych na astmę oskrzelową. Homozygoty CC w genie CD14 oraz heterozygoty AG w genie TLR4 charakteryzują się większym ryzykiem wystąpienia astmy atopowej. Genotyp TT genu CD14 i AA genu TLR4 sprzyja
natomiast powstawaniu astmy nieatopowej.
Wnioski. Atopowa i nieatopowa astma oskrzelowa różnią się etiologią na poziomie molekularnym. Ekspozycja na
endotoksynę u chorych z odpowiednimi polimorfizmami genów receptorów dla LPS może wywierać efekt protekcyjny na rozwój atopowej astmy oskrzelowej (Adv Clin Exp Med 2011, 20, 4, 413–421).
Słowa kluczowe: astma, polimorfizm, TLR4, CD14, lipopolisacharyd.
The number of people with asthma in Poland
and in the world is growing steadily. It is estimated
that by the year 2015 the number of patients worldwide will grow to 400 million. A rapid increase in
the growth rate observed in recent decades leads
many researchers to the opinion that it is not only
caused by hereditary factors but also by exposure
to external factors [32].
One of the most widely discussed hypotheses
explaining the increase of incidence of asthma and
other allergic diseases is the ‘hygienic hypothesis’,
by David P. Strachan, who in 1989 observed a significantly less frequent occurrence of hay fever and
contact dermatitis in children from large families [31]. The author explained these findings by
greater exposure to infectious agents in children
from large families, which significantly influences
the development of the immune system [31]. The
‘hygienic hypothesis’ was developed by further research which showed that increased exposure to
infection causes stimulation of the immune system
towards development of the Th1 lymphocyte and
a lack of infectious agents in the environment results in the survival of the fetal Th2 lymphocyte
phenotype conducive to the development of asthma [20, 27].
One of the most important environmental
factors affecting Th1/Th2 balance is lipopolysaccharide (LPS, endotoxin), a component of the
cell wall of gram-negative bacteria. Some of the
recent studies have shown a protective influence
of lipopolysaccharide (LPS) on the incidence of
allergic diseases in children and adults [4, 5, 9,
11, 16, 25, 26], whereas other studies have not
confirmed this relationship [6, 10]. The reason
for the discrepancies of the results could be the
different time and duration of exposure to endotoxin, its concentration in various environmental
subjects and the existence of different polymorphic forms of genes encoding receptors for endotoxin in patients.
CD14 and TLR4 are the most important receptors responsible for transmitting signals by LPS.
After entering the body, endotoxin binds to an
LPB protein [34, 37]. The LBP transports lipopoly-
saccharide and forwards it to the mCD14 (membrane CD14), a receptor present on the surface
of monocytes, the majority of macrophages, and
granulocytes or sCD14 circulating in peripheral
blood. sCD14 enables the binding of LPS to cells
that do not possess an mCD14 receptor – endothelial, epithelial and dendritic cells [8, 35]. mCD14
and sCD14 in the final stage allow LPS to interact
with the target receptor, which is TLR4.
Gene encoding of the CD14 receptor is located
on the long arm of chromosome 5 in the close vicinity of the 5q31-q33 loci, in which genes are responsible for the production of IgE and increased
risk of asthma and overactive bronchitis [19, 22,
37, 38]. CD14 receptor activity is connected with
the polymorphism of promoter region of the
CD14 gene (–159T < C). The presence of the C allele at position –159 of the gene correlates with
increased production of IgE and the prevalence of
positive skin tests. However, TT homozygotes are
recorded with a higher concentration of sCD14 in
serum [2].
The TLR4 gene is located on chromosome 8.
The two most common polymorphic forms are
1187A < G substitution (D299G, rs4986790) and
1487C < T substitution (T399I, rs4986791). Increased response to LPS in terms of FEV 1 decline
in the provocative test of endotoxin and a higher
incidence of overactive bronchitis after inhaling
LPS occurs in carriers of A and C alleles in the
TLR4 gene. The occurrence of A allele in position
1187 is also associated with a higher affinity TLR4
receptor to LPS [1, 23]. On the basis of the results
of these tests it can be supposed that the prevalence of these polymorphisms may be related to
the occurrence of asthma.
The aim of this study was to determine the
risk of asthma development, depending on the
prevalence of polymorphisms –159C < T of the
CD14 gene and 1187A < G of the TLR4 gene. It
was also to demonstrate the relationship between
polymorphisms of given genes and the percentage
and number of peripheral blood eosinophiles and
concentration of IgE in the serum of patients with
bronchial asthma.
CD14 and TLR4 Polymorphisms in Asthma
Material and Methods
Study Group
The study included a group of 106 patients
(including 32 men and 74 women, median age =
49 years) with diagnosed bronchial asthma treated
in the Department of Pneumonology, Oncology
and Allergology, Medical University of Lublin in
the years 2007–2008. Based on the results of skin
tests and clinical symptoms, the patients were
divided into groups: with atopic asthma (55 persons, median age = 45 years) and with non-atopic
asthma (51 patients, median age = 52 years). The
results of skin tests were as follows: 33 individuals
were mites positive; 45 individuals were tree pollen, grasses or weeds positive; 23 individuals were
pet fur positive, and 7 individuals were mould
positive. The control group included 159 healthy
unrelated individuals with the excluded presence
of any allergic diseases in the past (96 men and
63 women, median age = 41 years).
In all of the patients, peripheral blood samples
were collected into EDTA tubes to prepare genetic
tests. In addition, the number of eosinophiles in
peripheral blood and the level of total IgE in serum was determined in the group of patients with
asthma. The studies have obtained the acceptance
of the local Bioethics Committee operating at the
Medical University in Lublin.
DNA Isolation
DNA was isolated using the Blood Mini Kit
(A & A Biotechnology, Poland) according to the
manufacturer’s instructions. In brief, after removal of erythrocytes, leukocytes were lysed in a buffer
and proteinase K. The buffer contained chaotropic
salts and nonionic detergents (LT buffer). In addition, proteinase K was also used in the lysis process. After rinsing the column with proteins, purified DNA was eluted with a Tris buffer (10 mM
TRIS.HCl pH 8.5). DNA concentration was calculated using BioPhotometer Plus through the use of
microliter cells Hellma TrayCell (Eppendorf AG,
Germany). DNA was stored in the Tris buffer at
–20°C until the examination of CD14 and TLR4
polymorphisms.
PCR-RFLP of –159C < T
Polymorphism of the CD14 Gene
The CD14 gene fragment containing the polymorphic site was amplified in 50 µl of a reaction
mixture containing the following: 20 pmol of
each of the primers: forward 5-’GTGCCAACA-
415
GATGAGGTTCAC-3’ and reverse 5’-GCCTCTGACAGTTTATGTAATC-3’, Taq DNA polymerase buffer (1.5mM MgCl2), 0.25 U Taq DNA
polymerase and a 0.2mM dNTP nucleotide mixture. The mixture was supplemented with nuclease-free water. The amplification was carried out
in a T-Personal thermocycler (Biometer, USA) under the following conditions: initial denaturation
at 94°C for 1 min followed by 35 cycles of 30 sec at
94°C, 30 sec at 60°C and 45 sec at 72°C with a final
elongation step of 10 min at 72°C.
The PCR products were digested with AvaII
Fast Digest (Fermentas, Germany) according to
instruction. The restricted products were analyzed
by electrophoresis in 2% agarose gel (constant amperage 8 mA/cm2) containing ethidium bromide.
The place of cutting for the restriction enzyme
appeared in the amplified DNA fragment if the
T allele was found in –159 position of the CD14
gene. In the case of CC homozygotes, an undigested fragment of 497bp length occurred. In the
case of CT heterozygotes, fragments with a length
of 497bp, 353bp and 144bp were seen. For TT homozygotes, fragments with a length of 353bp and
144bp were seen (Fig. 1) [3].
PCR-RFLP of 1187A < G
Polymorphism of the TLR4 Gene
A TLR4 gene fragment containing the polymorphic site was amplified in 50 µl of a reaction
mixture containing all the reagents described
above and 20 pmol of each specified primer: forward 5-’GATTAGCATACTTAGACTACTACCTCCATG-3’ and reverse 5’-GATCAACTTCTGAAAAAGCATTCCCAC-3’. PCR reaction was
done under the following conditions: initial denaturation (94°C for 4 min), 35 proper cycles, that
included denaturation (94°C for 30 sec), hybridization with primers (55°C for 10 min), elongation
(72°C for 30 sec) and final elongation (72°C for 10
min) [18].
The PCR products were incubated with the
NcoI restriction enzyme (Fermentas, Germany)
according to manufacturer’s instructions. The restricted products were detected by electrophoresis on 2% agarose gel as described previously. The
cutting place for the restriction enzyme appeared
in amplified DNA fragments if the allele G of the
TLR4 gene occurred in 1187 position. In the case of
AA homozygotes, an undigested fragment of 249bp
length occurred. While in the case of AG heterozygotes, fragments with 249bp, 223bp and 26bp were
seen. For GG homozygotes, fragments of 223bp and
26bp occurred (there were no such homozygotes in
the investigated material) (Fig. 2).
416
T. Zaborowski et al.
497 bp
353 bp
144 bp
10
9
8
7
6
5
4
3
2
1
M
M – DNA marker ladder; lines 2, 5, 6 and 8 – genotype CC; lines 1, 7 and 9 – genotype CT;
lines 3, 4 and 10 – genotype TT.
M – drabinka DNA; linie 2, 5, 6 i 8 – genotyp CC; linie 1, 7 i 9 – genotyp CT; linie 3, 4 i 10 – genotyp TT.
Fig. 1. Representative example of polymorphism –159C < T CD14 gene
Ryc. 1. Przykładowa analiza polimorfizmu –159C < T genu CD14
223 bp
249 bp
14 13
12 11 10
9
8
7
6
5
4
3
2
1
M
M – DNA marker ladder; lines 2 and 12 – genotype AG; the other lines – genotype AA.
M – drabinka DNA; linie 2 i 12 – genotyp AG; pozostałe linie – genotyp AA.
Fig. 2. Representative example of polymorphism 1187A < G TLR4 gene
Ryc. 2. Przykładowa analiza polimorfizmu 1187A < G genu TLR4
Statistical Analysis
Statistical analysis was carried using the
U Mann-Whitney test to examine the differences
in analyzed parameters between the two independent groups. The Kruskal-Wallis test was used to
compare performance in three or more independent groups. Statistical analysis was performed using Statistica v. 8.0 (StatSoft, Poland). The accuracy Fisher test was used to establish the distribution
of genotypes according to the Hardy-Weinberg
equilibrium. Differences in the frequency of different alleles in patients with asthma and the control
group were assessed by a Pearson χ2 test. Hazard
ratio (HR) and 95% confidence interval calculated
for the likelihood of asthma, depending on the frequency of different genotypes, was calculated by
using logistic regression (http://ihg.gsf.de). An assumption of 5% error of inference and the related
level of significance p < 0.05 indicate the existence
of statistically significant differences or dependencies.
Results
The genotype distribution of the tested genes
remained in compliance with the Hardy-Weinberg
equilibrium. There was no connection between the
presence of polymorphic forms of the investigated
417
CD14 and TLR4 Polymorphisms in Asthma
genes and the risk of bronchial asthma. The incidence of different genotypes in a group of individuals with asthma and healthy subjects was similar
(Table 1).
AG genotype of the TLR4 gene (p < 0.005) and
CC genotype of the CD14 gene (p < 0.00001) occurred significantly more often in patients with atopic
asthma than in subjects with non-atopic asthma,
while the TT genotype of CD14 gene was observed
significantly less frequently (p < 0.01). There were no
statistically significant differences in the incidence of
CT genotype in these groups (Table 2).
The risk of atopic asthma was significantly
higher in carriers of AG genotype of the TLR4
gene than in those with AA genotype (HR = 2.33;
95% CI = 1.033–5.261; p < 0.05). By contrast, individuals with AG genotype in the TLR4 gene had
a significantly lower risk of non-atopic asthma
than those with AA genotype (HR = 0.081; 95%
CI = 0.005–1.365; p < 0.05).
AG genotype in the TLR4 gene and CC genotype in the CD14 gene were observed significantly more frequently in the group of patients
with atopic asthma than in healthy individuals.
TT genotype of the TLR4 gene was observed with
a significantly lower frequency (p < 0.05, Table 3)
in atopic asthma patients. By contrast, in patients
with non-atopic asthma AA genotype of the TLR4
gene was significantly more frequent than in
healthy individuals (p < 0.05). AG heterozygotes
were not found in the examined group (Table 4).
In patients with asthma, total IgE concentration was significantly lower in individuals with TT
and CT genotype of the CD14 gene than in the
group with CC genotype (median levels of IgE, respectively: 22.95 U/ml, 65.4 U/ml, 208 U/ml; p <
Table 1. The frequency of CD14 and TLR4 gene polymorphisms in patients with asthma and in control group
Tabela 1. Częstość występowania polimorfizmów w genach CD14 i TLR4 w grupie pacjentów chorych na astmę
i osób zdrowych
Gene (Gen)
Genotype (Genotyp)
Asthma (Astma)
n (%)
Control group (Grupa kontrolna)
n (%)
Total (Razem)
n (%)
TLR4
AA
94 (88.7)
142 (89.3)
236 (89.0)
AG
12 (11.3)
17 (10.7)
29 (11.0)
CT
53 (50.0)
73 (45.9)
126 (47.5)
TT
21 (19.8)
33 (20.8)
54 (20.4)
CC
32 (30.2)
53 (33.3)
85 (32.1)
CD14
Table 2. The frequency of CD14 and TLR4 gene polymorphisms in patients with atopic and non-atopic asthma
Tabela 2. Częstość występowania polimorfizmów w genach CD14 i TLR4 w grupie pacjentów chorych na astmę atopową
i nieatopową
Gene (Gen)
TLR4
CD14
Genotype (Genotyp)
Asthma (Astma)
non-atopic (nieatopowe)
n (%)
atopic (atopowe)
n (%)
AA
51 (100)
43 (78.2)
AG
0 (0.0)
12 (21.8)
CT
30 (58.8)
23 (41.8)
TT
16 (31.4)
5 (9.1)
CC
5 (9.8)
27 (49.1)
CT + TT
46 (90.2)
28 (50.9)
CC
5 (9.8)
27 (49.1)
TT
16 (31.4)
5 (9.1)
CC+CT
35 (68.6)
50 (90.9)
CT
30 (58.8)
23 (41.8)
TT+CC
21 (41.2)
32 (58.2)
χ2 value p value
χ2 = 10.47
p < 0.001
χ2 = 11.08
p < 0.005
χ2 = 17.56
p < 0.00005
χ2 = 6.93
p < 0.01
χ2 = 2.42
p = 0.1199
418
T. Zaborowski et al.
Table 3. The frequency of different genotypes of polymorphism CD14 and TLR4 genes in patients with atopic asthma
and in control group
Tabela 3. Częstość występowania różnych form polimorfizmów w genach CD14 i TLR4 w grupie pacjentów
z astmą atopową i osób zdrowych
Gene (Gen)
Genotype (Genotyp)
Atopic astma (Astma atopowa)
n (%)
Control group
(Grupa kontrolna)
n (%)
χ2 value
p value
TLR4
AA
43 (78.2)
142 (89.3)
AG
12 (21.8)
17 (10.7)
χ2 = 4.32
p = 0.0377
CT
23 (41.8)
73 (45.9)
TT
5 (9.1)
33 (20.8)
CC
27 (49.1)
53 (33.3)
CD14
χ2 = 5.99
p = 0.049
Table 4. The frequency of different genotypes of polymorphism CD14 and TLR4 genes in patients with non-atopic asthma
and in control group
Tabela 4. Częstość występowania różnych form polimorfizmów w genach CD14 i TLR4 w grupie pacjentów chorych
na astmę nieatopową i u osób zdrowych
Gene (Gen)
Genotype (Genotyp)
Non-atopic astma (Astma nieatopowa)
n (%)
Control group
(Grupa kontrolna)
n (%)
χ2 value
p value
TLR4
AA
50 (100)
142 (89.3)
AG
0 (0)
17 (10.7)
χ2 = 4.48
p < 0.05
CT
30 (58.8)
73 (45.9)
TT
16 (31.4)
33 (20.8)
CC
5 (9.8)
53 (33.3)
CD14
0.01). There was no relationship between CD14
gene polymorphism and the percentage and number of peripheral blood eosinophiles. In patients
with AG genotype in the TLR4 gene, the level of
total IgE in serum was significantly higher than
among those with the AA genotype (respectively
median IgE concentrations: 320 U/ml and 65.4 U/
ml, p < 0.05). There was no correlation between
the presence of polymorphic forms of the TLR4
gene and the percentage and number of eosinophiles in blood.
Discussion
The results obtained of polymorphism –159C
< T CD14 gene frequency are comparable to those
observed in other populations. CC genotype was
found in 32.1% of respondents, CT in 47.5% and
TT in 20.4%. Böttcher et al. studying two groups
of 115 healthy Swedish children and children
with bronchial asthma found the presence of CC
genotype in 37%, CT in 42% and TT in 21% of
respondents [3]. In turn, Baldini et al., among 481
randomly selected children discovered the CC
genotype in 29.4%, CT in 49.4% and TT in 21.3%
χ2 = 2.42
p = 0.12
[2]. For comparison, the study of polymorphism
–159C < T CD14 gene in the Japanese population
yielded similar results: genotype CC in 32.4%, CT
in 44.1% and TT in 23.5% of healthy adults [9].
In the Turkish population, similar studies have
shown the presence of CC genotype in 21.1%, CT
in 52.8%, and TT in 26.1% among children with
bronchial asthma [28].
The incidence of polymorphism 1187A < G in
the TLR4 gene did not significantly differ from
the values previously reported. Genotype AA occurred in 89%, and the AG genotype in 11% of
respondents. One of the largest studies, conducted in Austria and Germany among 609 children
– healthy and with bronchial asthma, showed
the prevalence of genotype AA in 92.1% while
the AG genotype was diagnosed in only 7.9% of
subjects [7]. In the case of the Swedish population
of asthmatic children, AA genotype was found in
90% and the AG genotype in 10% of patients [3].
Among 334 adults with asthma surveyed in Germany, the incidence of AA genotype was 91.7%,
and genotype AG, 8.3% [31 = 23].
Comparison of the distribution of polymorphisms in the group of respondents with diagnosed
asthma and in healthy individuals did not show
CD14 and TLR4 Polymorphisms in Asthma
the existence of statistically significant differences
in the incidence of various polymorphisms of the
CD14 and TLR4 genes. However, after isolating
a group of people with the atopic form of bronchial asthma, it turned out that the CC genotype
in position –159 of the CD14 gene and AG in position 1187 of the TLR4 gene occurred significantly
more frequently in those patients.
The study presented is another one that shows
the relationship between gene polymorphisms
of receptors for endotoxin and the incidence of
atopic asthma.
Koppleman et al. studied 159 patients with
atopic asthma, and 158 of their healthy spouses
[14]. Among patients with asthma, 32.1% were
CC homozygotes in the CD14 gene, 47.8% CT
heterozygotes, and 20.1% TT homozygotes. In the
control group, CC genotype was found in 19.6%,
CT in 53.8%, TT in 26.6% of individuals [14]. In
the group of individuals with CC genotype, atopic
asthma occurred significantly more often. In the
study of O’Donnell et al. conducted among 718
Australian children and young adults, it was found
that in patients with genotype CC in the CD14
gene, the incidence of atopy is significantly greater
than in children with CT and TT genotypes. [24].
The frequency of the given genotypes in 8-yearold atopic children were: CC in 37%, CT in 45%
TT in 18% of patients. In the group of children
without atopy, the prevalence of genotypes was as
follows: CC in 23%, CT in 50% and TT in 27% of
respondents. Böttcher et al. studied 115 Swedish
children and found a four times higher incidence
of atopic asthma in children with genotype AG in
the TLR4 gene as compared to children with genotype AA [3].
Werner et al. studied 334 adults suffering
from asthma for the presence of polymorphism of
1187A < G in the TLR4 gene and endotoxin exposure [32]. It turned out that among individuals
with AA genotype of the TLR4 gene, higher endotoxin levels in house dust samples were linked
with a higher incidence of bronchial asthma. By
contrast, in patients with genotype AG, a reverse
relationship between the level of LPS in house dust
and the occurrence of overactive bronchitis was
discovered.
There are also studies that have not confirmed
the link between polymorphisms of the tested genes
and the occurrence of allergic diseases. Kedda et
al., who studied 568 patients with asthma in Australia, did not confirm the relationship between
the presence of CD14 gene polymorphism and
the incidence and type of asthma and the severity of its course [13]. Similar results were obtained
by Shibasaki et al. in the population of Japanese
children with atopic asthma [30]. Two major stud-
419
ies conducted in Germany showed no relationship
between the polymorphisms –159C < T of the
CD14 gene and IgE level and the incidence of allergic diseases [7, 29]. In the case of 336 patients of
Caucasian origin, Yang et al. did not confirm the
link between the occurrence of polymorphic forms
of the TLR4 gene and the severity of the course of
bronchial asthma [36]. What is more, in the population of “Huts”, T allele in position 159 was positively associated with positive skin tests [23].
Only a few cited studies evaluated the concentration of endotoxin in the environment of
the respondents. The divergence of results may
therefore result from different levels of endotoxin
whose protective impact on the development of
asthma may depend on both receptor affinity and
concentration in the environment. Another reason for the difference in results may be the different ages of patients in which they were exposed to
LPS. Most authors believe that the endotoxin may
have a protective effect only in childhood, when by
promoting the Th1 response it impedes the emergence of diseases associated with Th2 response.
The mechanism explaining the association of
different polymorphic forms of the CD14 gene and
TLR4 gene with a frequency of asthma occurrence
has not been clearly described yet. It is assumed
that the rare occurrence of atopic asthma in individuals with TT genotype in position –159 of
the CD14 gene and in people with genotype AA in
position 896 of the TLR4 gene may be associated
with the increased response to endotoxin. Arbor
and colleagues found a significantly smaller fall of
FEV1 after inhalation of endotoxin in 83 healthy
patients with genotype AA of the TLR4 gene [1].
Michel et al. also showed that individuals with AG
genotype with inhaled endotoxin were characterized by a smaller systemic response to LPS [21].
Evidence of that were the lower levels of C-reactive
protein and a smaller increase of the number of
leukocytes in the blood of people from this group.
In turn, Levan et al., examining the relationship
of polymorphism –159C < T in the CD14 gene
in response to LPS [17], found, among patients
with TT genotype, a significantly lower increase of
sCD14 in serum. Landmann et al. have shown that
the primary stimulant of the secretion of a soluble
form of CD14 receptor in endotoxin assumed that
TT genotype is associated with a weaker response
to inhaled endotoxin [15].
Other evidence of the existence of a relationship between polymorphisms of genes for receptors for endotoxin and atopic asthma are the significantly higher levels of IgE in patients with CC
genotype of the CD14 gene observed in our study.
Koppelman et al. found that among people with
positive skin tests, CC homozygotes had signifi-
420
T. Zaborowski et al.
cantly higher levels of IgE [14]. This relationship
was also confirmed by research carried out in
a group of 481 randomly selected children in Italy
[2]. The relationship between IgE in the serum and
the polymorphism –159C < T of the CD14 gene
was also investigated by Gao et al. (a group of
300 healthy British and 200 healthy Japanese
people) [9]. British individuals showed that people with TT and CT genotype had significantly
lower values of total IgE compared to those with
genotype CC. The authors observe reasons for the
higher levels of IgE in a group of individuals with
genotype CC as a weaker impact of endotoxin on
these people, and thus less stimulation of cells associated with Th1 response. This hypothesis is
confirmed by results of a study of the relationship
between the polymorphisms of 1187A < G in the
TLR4 gene and the level of total IgE [9].
The original results of this study led to the
conclusion that there is a greater risk of non-atopic
asthma occurrence in patients with genotypes AA
in the TLR4 gene and TT in the CD14 gene.
This proves the differences in the etiology
of both diseases at a molecular level. Non-atopic
asthma occurs at a later age than atopic asthma,
and has many features in common with chronic
obstructive pulmonary disease. Th1 lymphocytes
may play a role in its formation, and endotoxin
may be an additional risk factor for the occurrence
of this disease. Therefore, high concentrations of
LPS, as well as changes in the affinity of receptors
for endotoxin, can cause non-atopic asthma.
Summarizing, the effect of the protective actions of endotoxin remains debatable, and it is
assumed that it can occur only if the appropriate
dose of stimulation is applied and it is done within
a reasonable period of time before the onset of the
disease. On the basis of the results of this work it
can also be assumed that this effect is strongly dependent on CD14 and TLR4 genes polymorphism.
The investigated population does not differ
substantially from other studied Caucasian populations in terms of the different polymorphisms of
the CD14 and TLR4 genes. TT genotype in the position 159 of CD14 and AA genotype in the position 1187 of TLR4 are present significantly less frequently in atopic asthma and are associated with
the lower concentration of IgE in blood serum in
individuals with asthma. CC homozygotes in the
CD14 gene and AG heterozygotes in the TLR4
gene have a higher risk of atopic asthma, while the
TT genotype of the CD14 gene and AA genotype
of the TLR4 gene could promote the formation of
non-atopic asthma.
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Address for correspondence:
Paweł Krawczyk
Department of Pneumonology, Oncology
and Allergology, Medical University
Chodźki 4a
20-950 Lublin
Poland
Tel.: +48 81 756 48 17
E-mail: [email protected], [email protected]
Conflict of interest: None declared
Received: 10.02.2011
Revised: 21.03.2011
Accepted: 1.08.2011