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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 inﬂamma-

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 inﬂammation 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 inﬂammation （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 ampliﬁed 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 afﬁliated 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, 665, Kong Jiang Road, Shanghai, 200092 China.

（+86）-; （+86）-;

: dr.smilebao.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.

Ampliﬁcation 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-

ciﬁc primers and site-directed mutagenesis primer to

amplify gene fragment. The plasmid that contained the

target gene was puriﬁed and sequenced.

Expression of recombinant protein was induced by

isopropylthio-b-D-galactoside （IPTG）, and the

expressed product was puriﬁed 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. Brieﬂy, 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 brieﬂy 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 afﬁliated 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 signiﬁ-

cance were set at 0.05.

Results

Expression of WT IL-13 and IL-13R130Q

The WT and mutant IL-13 genes were ampliﬁed 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 signiﬁcant

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 signiﬁcant 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 signiﬁ-

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 signiﬁcance

[（0.16 ± 0.03） lgmL）1

vs. （0.47 ± 0.05） lgmL）1

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 puriﬁcation and

renaturation, however, no signiﬁcant 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

37ı. 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 37ı（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 signiﬁcance were set at 0.05. The ﬁgure show the

means ± SEs of results obtained from ﬁve 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 signiﬁcantly 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 inﬂuenc-

ing the risk of developing allergic inﬂammation 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 signiﬁcance 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 signiﬁcantly 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 reﬂects 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 signiﬁcantly

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 afﬁnity 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 afﬁnity 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 artiﬁcial agonist which bound

the IL-13 receptor with 5- to 10-fold improved afﬁn-

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 reﬂect unsus-

pected functional differences in the B-cell signalling

pathways that lead to IgE synthesis （Nu ¨ sslein et al.,

1992）. Our results conﬁrmed 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-speciﬁc 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 conﬁrmed

that IL-13 R130Q increased activity compared with

WT IL-13. The SNP identiﬁed 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.

Conﬂicts of interest

The authors declare that they have no competing

interests.

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