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 A common variant associated with asthma, interleukin 13 R130Q,

promotes the production of IgE
Y. Chu, L. Hua, Q. Liu & Y. Bao
Summary
Interleukin (IL)-13 plays an important role in the
pathogenesis of asthma. A polymorphic variant of
human IL-13 R130Q, results in substitution of an
arginine with a glutamine was shown to be associated
with asthma in Chinese Han nationality. We examined
the functional consequences of this variant in vitro to
investigate whether this variant enhanced functional
activity compared with wild type IL-13. The wild-type
and mutant IL-13 genes were amplified from the plasmid
of pET22b-hIL-13 by PCR and site-directed mutagenesis
PCR. Both the PCR product and the vector pET28a(+)
were digested by the NdeI and BamHI. Then the PCR
product was cloned in the prokaryotic expression vector
of pET28a(+). The plasmids were constructed and
transformed into E. coli BL21(DE3).The positive clones
were selected, and tested by sequencing. Peripheral
blood mononuclear cells (PBMCs) from healthy partici-
pants were isolated and cultured with increasing con-
centrations of recombinantWT IL-13 and IL-13 R130Q.
IgE was detected with ELISA kit in the supernatants.
Recombinant WT IL-13 and IL-13 R130Q were
successfully expressed into the prokaryotic expression
system and their biological activity was consistent with
standard protein. Our results show that IL-13 R130Q is
more active than WT IL-13 in inducing hydrocortisone-
dependent IgE synthesis. There were statistical signifi-
cances between them. IgE induction by physiologic
concentrations was obviously increased. IL-13 R130Q
has increased activity compared with wild type IL-13
in vitro. And IL-13 R130Qmay be used for new target of
asthma for diagnosis and therapy in the future.
Introduction
Asthma is one of the most common chronic in?amma-
tory lung diseases worldwide. It is well known that
asthma is an immune-mediated disease and associated
with an excessive T-helper type 2 (Th2) immune
response (Busse & Lemanske, 2001; Umetsu et al.,
2002).
Interleukin (IL)-13 is produced by Th2 cells in
response to antigen receptor engagement (Fattouh &
Jordana, 2008) and can induce IgE synthesis in cul-
tured B cells (Punnonen et al., 1993). The IL-13 gene
is located in the chromosome 5q31q33 region, and
its role in the genetics of allergic diseases, such as
asthma, has already been widely investigated (Punnon-
en et al., 1993). Furthermore, some animal models of
allergic lung in?ammation have provided compelling
evidence that IL-13 plays a pivotal role in the develop-
ment of cardinal features of allergic asthma including
airway hyper-responsiveness, remodelling and eosino-
philic in?ammation (Wills-Karp, 2004).
The IL-13 gene contains a block of common single-
nucleotide polymorphisms (SNPs) in virtually com-
plete linkage disequilibrium (LD), which span the
third intron (+1923CT), the fourth exon (+2044GA)
and the 3¢ untranslated region of the gene (+2525GA,
+2580CA, 2749CT) (Graves et al., 2000). IL-13
A2044G is expected to result in the nonconservative
replacement of arginine (130R) with glutamine (130Q)
and is associated with bronchial asthma (Heinzmann
et al., 2000), atopic dermatitis (He et al., 2003) and
increased IgE levels (Graves et al., 2000).
Previously, we have shown that a polymorphic vari-
ant of human IL-13, R130Q, increased activity com-
pared to wild-type (WT) IL-13 using statistical
analysis in children of Chinese Han nationality (Li
et al., 2009). The aim of this study was to investigate
whether this variant has enhanced functional activity
in vitro. Thus, we examined the effects of WT IL-13
and IL-13 R130Q priming on IgE synthesis.
Materials and methods
Expression of recombinant IL-13
The WT and mutant IL-13 genes were ampli?ed from
the plasmid pET22b-hIL-13 by PCR and site-directed
mutagenesis PCR. The primers were designed as follow-
ings: IL-13 forward-primer:5¢-TATACTCGCATATGG
GCCCTGTGCCTCCCTCTA-3¢; IL-13 reverse-primer:
5¢-TATGGATCCTTATCAGTTGAACCGTCCCTCGC
Department of Pediatrics, Xin Hua Hospital af?liated to Shanghai
Jiaotong University School of Medicine, Shanghai, China
Received 3 July 2011; revised 26 December 2011; accepted 4 Janu-
ary 2012
Correspondence: Yixiao Bao, Department of Pediatrics, Xin Hua
Hospital, NO.1665, Kong Jiang Road, Shanghai, 200092 China.
Tel: (+86)-021-25078999; Fax: (+86)-021-65030840;
E-mail: dr.smilebao@yahoo.com.cn
ª 2012 Blackwell Publishing Ltd
International Journal of Immunogenetics, 2012, 00, 1–6 1
doi: 10.1111/j.1744-313X.2012.01091.x-3¢;IL-13 mutant reverse-primer: 5¢-TATGGATCCTTA
TCAGTTGAACTGTCCCTCGCG-3¢. The primers con-
tain an NdeI and a BamHI site (underlined) at their 5¢
and 3¢ ends.
Ampli?cation of IL-13 was performed in a 50-lL
total volume reaction containing 50 pmol lL)1
of each
primer, 10 · PCR buffer, 25 mM dNTPs, plasmid
pET22b-hIL-13 and PfuI DNA polymerase. Samples
were denatured for 5 min at 95C and then cycled 23
times through the following steps: 30 s at 95C, 45 s
at 68C and 1 min at 72C. The PCR products were
subjected to electrophoresis on 2% agarose gel and
visualized by ethidium bromide staining. The PCR
product was then digested at 37C for 12 h using
NdeI and BamHI restriction enzymes (MBI Fermentas,
Glen Burnie, ML, USA). The digests were cloned into
the pET28a(+) expression vector to construct the
expression plasmids that were transformed into E. coli
BL21(DE3). To identify positive clones, we used spe-
ci?c primers and site-directed mutagenesis primer to
amplify gene fragment. The plasmid that contained the
target gene was puri?ed and sequenced.
Expression of recombinant protein was induced by
isopropylthio-b-D-galactoside (IPTG), and the
expressed product was puri?ed through a Ni column
(Ni-NTA). TF-1 erythroleukemia cells proliferate in
response to IL-13, so we analysed the bioactivity of
expressed WT IL-13 and IL-13 R130Q using an 3-
(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bro-
mide (MTT) assay to measure the proliferative
response of TF-1 cells to WT IL-13 and the R130Q
variant.
MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-
diphenyltetrazolium bromide] assay
TF-1 cells exhibited a strong proliferate response to
granulocyte–macrophage colony-stimulatory factor
(GM-CSF), so we used rhGM-CSF as standard protein
in this study. Cells were cultured with various concen-
trations of WT IL-13, the R130Q variant and rhGM-
CSF for 24 h, and then 2 · 103
cells ⁄well were seeded
into 96-well plates in triplicate. Brie?y, 10 lL of MTT
[5 lgmL)1
in phosphate buffered saline (PBS)] was
added to each well, and the cells were incubated at
37C for 4 h. Cell culture medium was then removed,
and 100 lL DMSO was added to the wells. Plates
were brie?y shaken at 60 rpm for 5 min to dissolve
the precipitate and remove the bubbles and then read
at 490 nm using a microplate reader. The cellular pro-
liferation curve was delineated by the mean absor-
bency at different concentrations. Cells that were
cultured with PBS were used as the negative control.
Peripheral blood mononuclear cells (PBMCs) culture
and IgE determination
The participants (n = 6) were normal nonatopic stu-
dents from the Medical School of Shanghai Jiaotong
University. The participants ranged from 22 to
26 years of age (50% men and 50% women).
Approval for human studies protocol was obtained
from Xinhua Hospital af?liated to Shanghai Jiaotong
University, and informed consent was obtained from
all participants. PBMCs isolated through Ficoll-
Hypaque density centrifugation were washed and
responded (3.0 · 106
cells per mL) in complete
RPMI-1640 medium (GIBCO, Grand Island, NY,
USA) containing 100U mL)1
penicillin and 100U mL)1
streptomycin (GIBCO) supplemented with 10% foetal
bovine serum (Hyclone, Glen Burnie, ML, USA) and
maintained at 37C in 5%CO2. Cells were resus-
pended and stimulated with increasing concentrations
of WT IL-13 or IL-13 R130Q in the presence of
hydrocortisone (HC, 1 lM). Culture supernatants were
harvested after 7 days and assessed for IgE concentra-
tion by ELISA kit (CUSABIO BIOTECH).
Statistical analysis
All statistical analyses were performed using SPSS ver-
sion 13.0, and all results were expressed as
means ± SEs obtained from more than three replicates.
A 2-tailed paired Student’s t-test was used to compare
bioactivity of recombinant IL-13 as well as response
to WT IL-13 and IL-13 R130Q. P values for signi?-
cance were set at 0.05.
Results
Expression of WT IL-13 and IL-13R130Q
The WT and mutant IL-13 genes were ampli?ed from
the plasmid of pET22b-hIL-13 by PCR and site-directed
mutagenesis PCR. The products are shown in Fig. 1.
This shows that the genes were inserted and there was
no mutation or the reading frame shift when we
sequenced the recombinant plasmid using universal pri-
mer. The sequences were in accordance with the IL-13
Marker A B
1200 bp
900 bp
700 bp
500 bp
300 bp
100 bp
Figure 1. PCR products of IL-13 and IL-13R130Q. A, the products
of IL-13; B, the products of mutant IL-13.
2 Y. Chu et al.
ª 2012 Blackwell Publishing Ltd
International Journal of Immunogenetics, 2012, 00, 1–6cDNA, which were published by Genbank (Genbank
accession number AC000137.1). The recombinant IL-
13 and IL-13R130Q were successfully expressed in the
form of inclusion bodies; the polypeptide showed a rela-
tive molecular mass of about 14.4 kDa by SDS-PAGE
(Fig. 2) in accordance with the design.
The recombinant proteins of WT IL-13 and IL-13 R130Q
have biological activity and are consistent with
standard protein
TF-1 cells were cultured in the presence of various
concentrations of IL-13, R130Q variant or standard
rhGM-CSF. Subsequent cell proliferation was detected
by MTT assay. The results demonstrated that TF-1
cell proliferation in response to the recombinant pro-
teins WT IL-13 or R130Q variant was comparable to
standard rhGM-CSF (Fig. 3). The proliferation of TF-
1 cells was promoted after cultured with recombinant
WT IL-13, IL-13 R130Q and rhGM-CSF. The biologi-
cal activity of WT IL-13 or IL-13 R130Q was consis-
tent with rhGM-CSF. There was no signi?cant
difference of biological activity between the recombi-
nant WT IL-13 and IL-13 R130Q [OD = (0.17 ±
0.01) vs. OD = (0.18 ± 0.02), P > 0.05] at the concen-
tration of 0.01 ng mL)1
. The values of OD of WT
IL-13 at the concentration of 0.02, 0.04, 0.1, 0.2 and
0.4 were 0.24 ± 0.03, 0.30 ± 0.02, 0.34 ± 0.02, 0.40 ±
0.02 and 0.54 ± 0.03, respectively. The values of OD
of IL-13 R130Q at the concentration of 0.02, 0.04,
0.1, 0.2 and 0.4 were 0.26 ± 0.02, 0.32 ± 0.01, 0.36 ±
0.02, 0.42 ± 0.01 and 0.56 ± 0.01, respectively. There
was also no signi?cant difference between two recom-
binant proteins at various concentrations (P > 0.05).
IL-13 R130Q is more active than WT IL-13 in inducing
hydrocortisone-dependent IgE synthesis
PBMCs from nonallergic donors (n = 6) were isolated
through Ficoll-Hypaque density centrifugation and
incubated with increasing concentrations of WT IL-13
or IL-13 R130Q in the presence of hydrocortisone
(HC). Figure 4 shows that IgE synthesis was signi?-
cantly increased in response to IL-13 stimulation, par-
ticularly with IL-13 R130Q. Substantial IgE synthesis,
in response to IL-13 R130Q [(0.16 ± 0.02)lgmL)1
vs.
(0.49 ± 0.07)lgmL)1
, P < 0.001], was detected in cul-
tures simulated with 90 pg mL)1
of variant. The dif-
ference in the response to a higher IL-13 concentration
(500 pg mL)1
) approached statistical signi?cance
[(0.16 ± 0.03) lgmL)1
vs. (0.47 ± 0.05) lgmL)1
,
0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
0 0.1 0.2 0.3 0.4 0.5
Concentration (ng per well)
A490 nm
rhGM-CSF
IL-13
IL-13 R130Q
Negative control
Figure 3. Bioactivity analyses of recombinant proteins. In vitro, bio-
assays using a factor-dependent human erythroleukaemic cell line
(TF-1 cells) showed that plant rhIL-13 retained the biological func-
tions of the authentic hIL-13 protein (Wang et al., 2008). The recom-
binant proteins were biological active after puri?cation and
renaturation, however, no signi?cant difference was observed
between them about the biological activity (P > 0.05).
Figure 2. SDS-PAGE result of expression products. Both the recombi-
nant plasmid of pET28a(+)-rhIL-13(B and C) and pET28a(+)-rhIL-13m(E)
expressed the recombinant protein after the induction of IPTG under
37?. Line A and D show the recombinant plasmid of pET-28a(+)-rhIL-
13(A) and pET-28a(+)-rhIL-13m(D) before the IPTG induction respec-
tively. F for the empty plasmid of pET-28a(+) (F)before IPTG induction,
G and H for the products induced by IPTG under 37?(G and H) respec-
tively. The size of the products (14.4 kDa) is indicated on the right.
0.0000
0.0200
0.0400
0.0600
0.0800
0.1000
30 pg mL–1
90 pg mL–1
500 pg mL–1
2000 pg mL–1
IgE synthesis (μg mL–1)
WT IL-13
IL-13 R130Q
* *
Figure 4. IgE production in response to IL-13. IL-13 R130Q is more
active than WT IL-13 in inducing hydrocortisone-dependent IgE
synthesis. Normal human PBMCs were stimulated with increasing
concentrations of WT IL-13 (black bars) or IL-13 R130Q (gray bars) in
the presence of HC (1 lM) for 7 days. Supernatants were then har-
vested and assessed for IgE concentration by ELISA kit. Responses
to the IL-13 variants were compared using a 2-tailed paired Student’s
t test. P values for signi?cance were set at 0.05. The ?gure show the
means ± SEs of results obtained from ?ve experiments. *P < 0.000.
IL-13 R130Q promotes the production of IgE 3
ª 2012 Blackwell Publishing Ltd
International Journal of Immunogenetics, 2012, 00, 1–6P < 0.001]. IgE induction by IL-13 concentrations (30
and 2000 pg mL)1
) was not signi?cantly different in
the two variants. When no IL-13 was added, the level
of IgE was 0.10 ± 0.01 lgmL)1
.
Discussion
Asthma is a common clinical syndrome resulting from
several factors such as immunity, environment and
heredity. The importance of genetic factors in in?uenc-
ing the risk of developing allergic in?ammation is well
known (Cookson & Moffatt, 2000; Vercelli, 2003).
Numerous studies have revealed that IL-13 R130Q, a
common variant encoded by the IL-13 A2044G poly-
morphism, markedly increases the risk of developing
asthma (Kim et al., 2006; Hosseini-Farahabadi et al.,
2007; Llanes et al., 2009; Park et al., 2009). How-
ever, such data provide evidence for the signi?cance of
the WT IL-13 and IL-13 R130Q variant by way of
statistical analysis, while our experimental results
show that IL-13 R130Q is signi?cantly more active in
elevating serum IgE in vitro.
As mentioned earlier, elevated IgE has been proved
to be associated with a higher risk of allergic disease
(Sears et al., 1991; Douglass & O’Hehir, 2006). IL-
13-induced IgG4 and IgE synthesis re?ects immuno-
globulin isotype switching and is not because of a
selective outgrowth of new B cells committed to IgG4
or IgE production. In the current study, PBMCs from
nonallergic donors were incubated with increasing
concentrations of WT IL-13 or IL-13 R130Q in the
presence of HC, and IgE synthesis was signi?cantly
increased. IgE induction by physiologic concentrations
was obviously increased, but when we used supraphys-
iologic concentrations to stimulate PBMCs, the IgE
induction was decreased. In contrast, (Tollerud et al.,
1991) have found a strong association between ele-
vated IL-13 levels and IgE production in vivo in a
group of children. So whether this association between
IL-13 and IgE production did occur still needed to be
proved in vitro.
The mechanism accounting for the observed
increased activity of IL-13 R130Q variant is not yet
clear. As we know, the biological activity of IL-13 is
regulated via type 1 and type 2 IL-13 receptors. The
type 1 receptor is a heterodimer of the IL-4R alpha
chain (a) and IL-13Ra1; the type 2 receptor is com-
posed of the IL-13Ra1 and IL-13Ra2 chains (Ly
et al., 2005; Finkelman et al., 2010). The IL-13Ra2
chain binds IL-13 with high af?nity and internalizes
after binding to ligand without involvement of other
chains (Donaldson et al., 1998). In addition, IL-13
appears to regulate the expression of IL-13Ra2, sug-
gesting that ligand and receptor may cross-regulate
one another (Kioi et al., 2008). have shown that IL-
13Ra2 may act to modulate the effects of IL-13
in vivo in various ways; IL-13Ra2 could enhance IL-
13 activities by increasing the strength of IL-13 signal-
ling or attenuate IL-13 effects by negative signalling
or simply as a molecular decoy. Currently, many stud-
ies have suggested that IL-13Ra2 was a key negative
regulator of IL-13 in vivo (Wood et al., 2003). A
computer modelling study suggested that Arg130 was
directly involved in the interaction with IL-13 recep-
tor and that charge-changing variants were likely to
display different biological properties. Alanine scan-
ning mutagenesis revealed 130R to be important for
IL-13 binding to IL-13Ra2 (Graves et al., 2000).
(Mitchell et al., 2010) suggested that the variant
showed a lower af?nity with the IL-13Ra2 and an
enhanced stability in both human and mouse plasma.
Substitution of 130R with the negatively charged
aspartic acid created an arti?cial agonist which bound
the IL-13 receptor with 5- to 10-fold improved af?n-
ity, so the 130Q changes the activity of the molecular
and mutant IL-13 enhanced signal transduction. It
was also found that among asthmatic children, sub-
jects homozygous for Gln130 (130Q) had higher lev-
els of serum IL-13 than those homozygous for Arg130
(Arima et al., 2002). The structural IL-13 change led
to functional change, so IL-13 R130Q as a functional
genetic factor could elevate synthesis of IgE as shown
in our results.
It has been shown that hydrocortisone (HC) and its
synthetic derivatives are able to potentiate in vitro IL-
4-induced IgE production by PBMCs (Nu ¨ sslein et al.,
1994; Noguchi et al., 2001), but the interaction
between HC and IL-13 has not been investigated.
There is a positive crosstalk between IL-13 and HC in
earlier research, and this crosstalk may re?ect unsus-
pected functional differences in the B-cell signalling
pathways that lead to IgE synthesis (Nu ¨ sslein et al.,
1992). Our results con?rmed this view by using WT
IL-13 and IL-13 R130Q to stimulate PBMCs in the
presence of HC. As one of the agents of pharmaco-
therapy of asthma, HC could control acute asthma
(Vladich et al., 2005). Cho et al., (2002) suggested
that HC could enhance allergen-speci?c IgE produc-
tion by PBMCs from atopic patients. On the other
hand, Klebl et al., (1994) suggested that glucocorti-
coid treatment does not give rise to a substantially
enhanced risk for increased IgE synthesis and the
development of sensitizations in nonallergic persons
receiving steroids. So further studies should be per-
formed in vivo or in vitro.
IL-13 has been proposed as a therapeutic target for
bronchial asthma because it plays crucial roles in the
pathogenesis of the disease. Choi et al., (2009) have
developed an in vitro test system measuring transcrip-
tional downregulatory activities on IL-13 as a primary
screening method to select drug candidates from
natural products.
In conclusion, our results reported here con?rmed
that IL-13 R130Q increased activity compared with
WT IL-13. The SNP identi?ed in this study may be
used to develop markers to assess the risk of asthma.
IL-13 R130Q may be used for new target of asthma
for diagnosis and therapy in the future.
4 Y. Chu et al.
ª 2012 Blackwell Publishing Ltd
International Journal of Immunogenetics, 2012, 00, 1–6Acknowledgement
This work was supported by a grant from the
National Science Foundation of China (grant number
30872805 and 30972750). Yi Chu and Li Hua con-
tributed equally to this work.
Con?icts of interest
The authors declare that they have no competing
interests.
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