Articles in PresS. Am J Physiol Lung Cell Mol Physiol (January 16, 2015). doi:10.1152/ajplung.00305.2014
Increased IL-33 expression in chronic obstructive pulmonary disease
Jie Xia, Junling Zhao, Jin Shang, Miao Li, Zhilin Zeng, Jianping Zhao, Jianmiao
Wang, Yongjian Xu, Jungang Xie
Department of Respiratory and Critical Care Medicine, Tongji Hospital, Tongji
Medical College, Huazhong University of Science and Technology, Wuhan 430030,
Running title: IL-33 and COPD
Corresponding Author: Jungang Xie, MD, PhD
Department of Pulmonary and Critical Care Medicine, Tongji Hospital, Tongji
Medical College, Huazhong University of Science and Technology, 1095 Jiefang
Avenue, Wuhan 430030, China. Tel.: 86-27-83663596; Fax: 86-27-83662898. Email:
15 [email protected]
Contributions of authors: Conception: XJ. Design: XJ, XY. Data collection and
analysis: XJ, ZJ, SJ, LM, ZZ. Drafting the manuscript for important intellectual
content: XJ, XJ, ZJ, WJ, XY.
19 20 21 22
Copyright © 2015 by the American Physiological Society.
Chronic obstructive pulmonary disease (COPD) is an inflammatory lung disease
characterized by inflammatory cell activation and the release of inflammatory
mediators. Interleukin-33 (IL-33) plays a critical role in various inflammatory and
immunological pathologies, but evidence for its role in COPD is lacking. This study
aimed to investigate the expression of IL-33 in COPD and to determine whether IL-33
participates in the initiation and progression of COPD. Levels of serum IL-33 and its
receptors were measured by ELISA, and serum levels of IL-33, ST2 and interleukin-1
receptor accessory protein (IL-1RAcP) were elevated in patients with COPD
compared to control subjects. Flow cytometry analysis further demonstrated an
increase in peripheral blood lymphocytes (PBLs) expressing IL-33 in COPD patients.
Immunofluorescence analysis revealed that the main cellular source of IL-33 in lung
tissue was bronchial epithelial cells (HBEs). Cigarette smoke extract (CSE) and
lipopolysaccharide (LPS) could enhance the ability of PBLs and HBE to express
IL-33. Furthermore, PBLs from patients with COPD showed greater IL-33 release in
response to the stimulus. Collectively, these findings suggest that IL-33 expression
levels are increased in COPD and related to airway and systemic inflammation.
Therefore, IL-33 might contribute to the pathogenesis and progression of this disease.
Key words: chronic obstructive pulmonary disease; Interleukin-33; human bronchial
epithelial cells; peripheral blood lymphocytes
Chronic obstructive pulmonary disease (COPD) is a leading public health problem,
and the incidence of COPD has recently increased—it is projected to rank third
worldwide for mortality (37). COPD is typically characterized by irreversible and
slowly progressive shortness of breath upon exertion, which is caused by airway
obstruction resulting from airway inflammation. Bronchial biopsies from COPD
patients show infiltration with increased numbers of neutrophils, T cells and
macrophages (6, 7, 13), which induce the production of various cytokines in the
neutrophil-associated cytokines (5, 16). These cytokines are associated with different
patterns of inflammation in the airways, and eventually cause chronic bronchitis and
emphysema by inducing mucus production, alveolar destruction and remodeling of
IL-33 is a new member of the IL-1 family that has attracted great attention because of
its function in immune responses. It is mainly expressed by a variety of cells and
tissues in healthy and inflamed tissues. Immune cells, macrophages and dendritic cells
can also produce IL-33 in response to specific stimuli (46). IL-33 is known to be a
ligand for ST2 and plays an important role in the immune response and inflammatory
diseases. Apart from Th2-type immune response, recent studies indicate that IL-33 has
a remarkable effect on Th1-type responses by promoting the secretion of Th1-type
cytokines by NK and NKT cells (48, 55, 56). IL-1 receptor-related protein ST2 and
IL-1 receptor accessory protein (IL-1RAcP) are required for IL-33-mediated signal
transduction because these molecules form parts of its receptor complex (14).
It has been reported that changes in the serum concentration of IL-33 and ST2 occur
in allergic and heart diseases (11, 32, 50). Additionally, high expression levels of
IL-33 have been detected in lung tissues, which play an important role in respiratory
diseases. IL-33 expression levels are significantly elevated in bronchial asthma and
bronchial epithelium cells are an important source of IL-33 (36, 41, 42). Although
elevated sST2 levels have been reported in patients with COPD (17), the relationship
and function of the IL-33 pathway in the pathogenesis of this disease remain
In order to investigate the role and source of IL-33 in COPD, we measured the serum
levels of IL-33, and the receptors ST2 and IL-1RAcP in COPD patients and controls,
and the intracellular expression levels of IL-33 in peripheral blood and lung tissues.
IL-33 and its soluble receptors were increased in COPD, and the cellular sources of
IL-33 in the peripheral blood and lung tissues included peripheral blood lymphocytes
(PBLs) and human bronchial epithelial (HBE) cells. Moreover, we found that IL-33
production by these cells could be stimulated by CSE and LPS, which were major risk
factors for COPD.
Materials and Methods
A total of 112 patients with COPD and 115 sex, age and race matched controls were
chosen for inclusion in our study. The clinical characteristics of these populations are
described in detail in Table 1. COPD was diagnosed according to the American
Thoracic Society criteria and the Global Initiative for COPD (GOLD) criteria (57)
(post-bronchodilator forced expiratory volume in 1 s (FEV1)/forced vital capacity
ratio 20. Plasma cotinine concentrations were measured by a direct
ELISA kit (Immunalysis, Pomona, CA) to confirm smoking status. The sensitivity of
the cotinine assay is 1 ng/ml. Plasma cotinine levels for COPD patients and controls
were all above the recognized minimum criterion of active smoke exposure
(9.5 ng/ml)(23). Subjects who suffered from asthma or other obstructive lung diseases,
or who exhibited disease exacerbation or respiratory infection in the four weeks prior
to recruitment were excluded from this study. Patients receiving inhaled
corticosteroids or oral theophylline, anti-inflammatory therapy or oral steroids for
chronic inflammatory diseases during the previous 4 weeks were also excluded.
In these COPD and healthy individuals, lung tissues from some patients were
collected during surgical resection of solitary pulmonary nodules and lung cancer was
excluded by pathology after surgery. A total of 12 lung specimens were collected,
including 6 COPD and 6 control subjects. In these subjects, the 12 participants who
supplied lung tissue specimens also provided venous blood simultaneously, and others
only provided a blood sample.
All study subjects were recruited from Tongji Hospital, Tongji Medical College,
Huazhong University of Science and Technology in Wuhan (Hubei, China) and signed
a consent form. Our study was approved by the Research Ethics Committee of
Huazhong University of Science and Technology.
ELISA was used to measure the protein concentrations of IL-33, ST2 and IL-1RAcP
in serum or cell culture supernatants. First, an ELISA plate was coated with capture
antibody (R&D Systems, Minneapolis, MN, USA). Then, the serum or sample
supernatants were added to the plate, and horseradish peroxidase (HRP)-conjugated
secondary mAb was added. After a washing step, tetramethylbenzidine was added in
the dark. To stop the reaction, 2 mol/L H2SO4 was added to each well. Absorbance
was determined at 490 nm and a reference wavelength of 570 nm using a
spectrophotometer. Concentrations were calculated based on a standard curve. The
limits of detection for the IL-33, ST2 and IL-1RAcP ELISA kits were 1.65, 13.5 and
31.2 pg/mL, respectively.
Flow cytometry was used to investigate the cellular source of IL-33 in peripheral
blood. An aliquot of 100 µL whole blood was added to a 6-well plate containing 800
µL RPMI-1640 Medium (GIBCO Laboratories, Grand Island, NY). Biolegend
Brefeldin A Solution (Becton Dickinson, Franklin Lakes, NJ, USA) was added to the
mixed cell cultures, which were incubated for 4 h. After red blood cells were lysed
using lysis solution (Becton Dickinson), cytofix/cytoperm solution (BectonDickinson)
was added to the tube for incubation for 20 min. Then, the cell suspension was
divided into 2 tubes. In the dark, monoclonal anti-human IL-33- phycoerythrin
(IL-33-PE; R&D Systems) and IgG2B isotype control-PE (R&D Systems) were added
to the cells separately. After one final wash, cells were resuspended in 2%
paraformaldehyde and assessed using a Becton Dickinson LSR flow cytometer. Data
were analyzed using CellQuest software.
Formalin-fixed and paraffin-embedded lung tissue was obtained from 6 COPD
patients and 6 healthy control subjects. Then, 5 μm sections mounted onto slides were
used for immunofluorescence detection of IL-33. After deparaffinazition in methanol
and rehydration in a graded alcohol series, the sections were washed in TBS and
boiled in sodium citrate buffer for 20 min in a microwave oven for epitope retrieval.
Slides were incubated with 10% donkey serum for 1 h at room temperature, and then
were incubated overnight with mouse anti-human IL-33 antibody (1:100, Alexis
Biochemicals, Lausen, Switzerland), mouse anti-human ST2 antibody (1:100, Alexis
Biochemicals) rabbit anti-human IL-1RAcP antibody (1:200, GeneTex Inc, San
Antonio, TX, USA) or matched isotype controls (Becton Dickinson) at 4°C. Sections
were incubated with Alexa Fluor 488-donkey anti-mouse, Alexa Fluor 594-goat
anti-mouse Alexa Fluor 488- donkey anti-rabbit secondary antibody. Finally, slides
were visualized using laser scanning confocal microscopy (Zeiss, Oberkochen,
Total RNA extraction, reverse transcription (RT) and quantitative real-time PCR
Total RNA from cells and tissue was extracted using TRIzol reagent (Invitrogen,
Carlsbad, CA). The cDNA was prepared from 500 ng total RNA using Prime Script
RT Master Mix (Takara, Dalian, China) following the manufacturer’s instructions.
Real-time PCR was performed to quantify mRNA levels using an ABI Prism 7500
sequence detection system with SYBR Premix Ex Taq (Takara, Dalian, China). The
PCR parameters were 95ºC for 30 s, followed by 40 cycles of 95ºC for 5 s and 60ºC
for 34 s. Results were expressed as 2–ΔΔCT, normalized to levels of
glyceraldehyde-3-phosphate dehydrogenase (GAPDH) in untreated cells or control
subjects. Speciﬁc primers used in this study were designed using the program
BatchPrimer3 v1.0 and were as follows: IL33, GAAGAACACAGCAAGCAAAGC
and TACCAAAGGCAAAGCACTCC; GADPH, ACCACAGTCCATGCCATCAC
Cell culture and treatments
HBEs were purchased from ATCC (Manassas, VA, USA) and PBLs were isolated
from human peripheral blood samples obtained from study subjects. The cell culture
medium consisted of DMEM (GIBCO) or RPMI 1640 Medium, 10% fetal bovine
serum (GIBCO), 100 U/ was recently found in ASMCs bundles penicillin and 100
μg/ml streptomycin (KeyGEN, Nanjing, China). When cells reached 80% confluence,
HBEs were digested with 0.25% trypsin and the cell concentration was adjusted to
106/ml. Meanwhile, PBLs were also suspended in culture medium at a concentration
of 106/ml. Then, cells and medium with or without irritant (as a control) were added to
CSE (Murty Pharmaceuticals, Inc., Lexington, KY, USA) and LPS (Sigma–Aldrich,
USA) were 10 μg/ml and 100 ng/ml respectively. After incubation in a humidiﬁed
incubator (95% air, 5% CO2, 37°C) for 24 h, supernatants were stored at –20ºC for
ELISA analysis and cells were collected and stored at –80ºC in TRIzol for mRNA
extraction. All experiments on HBEs were performed in triplicate and were repeated
at least 3 times.
Cell apoptosis assay
Cell apoptosis was quantified using Annexin V/PI Apoptosis Detection Kit (KeyGen,
Nanjing, China). After treatment, HBEs and PBLs were collected and resuspended in
500μl binding buffer and then incubated with a mixture of 5μl FITC-labeled
Annexin-V and 5μl propidium iodide (PI) for 15 min at room temperature in the dark.
Samples were analysed by flow cytometry using a Becton Dickinson LSR flow
cytometer. The data were analyzed with CellQuest software. Annexin V labeled the
apoptotic cells, whereas PI labeled the late apoptotic and necrotic cells with
membrane damage. Undamaged cells remained negative for both parameters.
DNA extraction and genotyping
Genomic DNA was extracted from 5 mL whole peripheral blood using a DNA
extraction kit (Qiagen; Hilden, Germany) according to the standard protocol. PCR
primers were designed using Primer 3 Input (version 0.4.0) and were as follows:
rs4986790 and rs49867901, TGTATTCAAGGTCTGGCTGGT and TGTTTCAAAT
TGGAATGCTGGA; rs11536889, CTGGGATCCCTCCCCTGTA and TTTCCCTG
ATGACATCCTGA. PCR was performed using the mix of 100 ng genomic DNA, 1.5
mM MgCl2, 0.2 mM dNTPs, and 1× PCR buffer, 0.5 ul Taq polymerase, and 1 ul of
the forward and reverse primers in a total of 50 μL. The PCR conditions were as
follows: 94°C for 1 min, 58°C for 50 sec, 72°C for 90 sec for 40 cycles, 72°C for 10
min, then followed by 4°C until the reaction mixtures were removed from the cycler.
PCR products were sequenced using an ABI3730xl sequencer (Applied Biosystems,
Foster City, Calif).
Data are presented as means ± SD. Statistical analyses were performed using SPSS
19.0 (SPSS, Chicago, IL, USA) and Prism 5 software (GraphPad Software Inc., San
Diego, CA, USA). Student’s t-test, Mann-Whitney test, one-way ANOVA test,
Kruskal-Wallis test or Friedman’s test followed by Dunn’s test and χ2 test was used as
appropriate. All statistical tests were two-tailed and P < 0.05 was considered to
indicate a statistically significant difference.
Levels of IL-33 and its receptors are elevated in serum from COPD patients
Protein levels of IL-33, sST2 and IL-1RAcP in the serum were measured in COPD
patients and controls. Significantly higher levels of IL-33 in serum were detected in
COPD patients (319.24±77.47 pg/ml) than control subjects (222.60±26.11 pg/ml,
Figure 1A). ST2 and IL-1RAcP are receptors for IL-33. Because of different ST2
mRNA splice variants, there are two ST2 protein isoforms (10). The form that we
detected in serum was sST2, which has been widely studied in allergic and heart
disease as an inflammatory marker (11, 32, 50). A significant difference in ST2 levels
was observed between COPD patients (3.13±0.95 ng/ml) and control subjects
(0.93±0.66 ng/ml, Figure 1B). Additionally, we found that the soluble IL-1RAcP
levels in serum from subjects with COPD (2.85±0.10 ng/ml) was higher than that in
healthy subjects (0.98±0.05 ng/ml, Figure 1C). Thus, our data indicated that COPD
patients showed increased levels of IL-33 and its soluble receptors in serum compared
to healthy subjects.
IL-33 expression in peripheral blood cells, including PBLs and neutrophils
Significantly higher levels of serum IL-33 protein could be observed in COPD
patients. To corroborate the ELISA findings and characterize the cellular sources of
IL-33 in peripheral blood, we also measured IL-33 expression in PBLs and
neutrophils from 20 COPD patients and 20 controls by flow cytometry. We found that
IL-33-positive cells were detectable in the lymphocyte group and the frequency of
IL-33-positive cells was higher in COPD patients than in control patients (11.62 ±
8.848 vs. 2.238 ± 4.490, respectively, Figure2). A low frequency of IL-33-positive
cells was detected in the neutrophil group and there was no significant difference
between COPD and control patients.
IL-33 is highly expressed in the lungs of COPD patients
We next investigated whether IL-33 expression was increased in the lungs of COPD
patients. Immunofluorescence staining showed expression of IL-33 in HBEs, but there
was little signal detected in airway smooth muscle cells (ASMCs; Figure 3A–B). In
agreement with previous reports that IL-33 expression could be observed in nuclei of
endothelial cells (2, 36, 51), IL-33 was detected in the nuclei of epithelial cells.
Additionally, the IL-33 staining intensity of COPD was obviously higher than that of
the control groups (Figure 3C). Finally, we discovered that ST2 and IL1-RAcP were
expressed in the cytoplasm of HBEs, and IL-1RAcP was also expressed in ASMCs
(Figure 3D). However, the expression levels of ST2 and IL-1RAcP were not
significantly different between COPD and control patients. The isotype controls were
negative in the epithelial cells.
IL-33 expression is up-regulated by CSE and LPS in HBEs and PBLs
Based on our observations that HBEs and PBLs were the cellular sources of IL-33, we
further characterized the regulatory factors involved in controlling IL-33 expression,
including CSE and LPS, which are risk factors for the development of COPD. The
protein levels of IL-33 in supernatants of HBEs cultured with or without stimulation
were measured by ELISA. IL-33 secretion was markedly increased after CSE
stimulation compared to untreated cells (Figure 4A). We further assessed the effect of
LPS treatment on IL-33 expression by HBEs and found that there was also a
significant difference between cells treated with or without LPS. A stronger response
was induced in cultured HBEs when those cells were treated with a mixture of LPS
and CSE. This finding indicated that that a mixture of LPS and CSE might
synergistically stimulate IL-33 expression in HBEs.
Expression of IL-33 in cultured PBLs was examined in cells from 20 COPD patients
and 20 healthy controls with or without stimulation. After treatment with CSE, LPS
alone or together, the expression level of IL-33 in PBLs from COPD patients was
significantly increased compared to that of untreated cells (Figure 4B). Notably, a
stronger response was induced in cultured PBLs from COPD patients when those cells
were stimulated with the mixture of CSE and LPS. These data were similar to those
obtained using CSE and LPS-treated HBEs. However, there were no statistical
differences in IL33 gene expression levels between treated and untreated PBLs from
healthy controls (P = 0.174, Friedman’s test). Importantly, after stimulation with the
mixture, IL33 gene expression levels in PBLs from COPD patients were significantly
higher than those from healthy controls. These findings indicated that the responses of
cells from COPD patients and healthy controls to these stimuli were different.
In addition, when cells were double stained with Annexin V and PI, no apparent
differences were observed in the cell death (apoptosis and necrosis) rates between the
cells incubated with CSE and LPS and those incubated with media alone (Figure 4C),
which suggested that IL-33 was actively secreted from HBEs and PBLs without
apparent cell damage.
TLR4 Polymorphisms Analysis in COPD Patients and Controls
rs4986790, rs4986791 and rs11536889 polymorphisms of Toll-like receptor 4 (TLR4)
gene have been detected in 20 COPD patients and 20 controls. The genotype
distribution of TLR4 SNPs in these subjects was summarized in Table 2. In the 40
samples, no heterozygous or homozygous variant genotypes of the rs4986790 and
rs49867901 polymorphism were detected. Our data showed that there were no
significant differences in the rs11536889 genotype distribution between COPD
patients and controls (P >0.05).
There is increasing evidence implicating IL-33 in various inflammatory diseases, such
as rheumatoid arthritis (33), atopic dermatitis (21), ulcerative colitis (39), and asthma
(30, 42, 46); however, no study to date has investigated the role of IL-33 in COPD.
Herein, we show that levels of IL-33 expression are elevated in COPD, and confirm
that the HBEs in the airway and PBLs in the peripheral blood were the cellular
sources of IL-33. IL-33 could be induced by CSE and LPS, which are risk factors for
COPD, and we found that the sensitivity of PBLs from COPD subjects to stimuli was
much higher than that of healthy subjects.
Our study is the first to provide evidence that the expression of IL-33 and its receptors
are increased in the serum of COPD patients. A recent study showed that the
concentrations of serum IL-33 in patients with COPD during acute episodes were
significantly lower than those in individuals with healthy controls and patients at the
stable phase of COPD (53). However, as an alarmin, it has been demonstrated that
IL-33 could be released when cells sense inflammatory signals or undergo necrosis
(35). Increased serum levels of IL-33 was found to be associated with numerous
inflammatory diseases, including systemic lupus erythematosus (34), systemic
sclerosis (59), inflammatory skin disorders (59) and Crohn's disease (9). Moreover,
previous study in animal model showed that the serum level of IL-33 was elevated in
COPD (43), which is consistent with the detection of increased serum IL-33 levels in
COPD patients in our study. Additionally, immunofluorescence staining showed that
the expression of IL-33 was also increased in lung tissues of COPD patients,
indicating that IL-33-upregulation was probably related to systemic and airway
inflammation in COPD. Furthermore, both serum sST2 and IL-1RAcp levels showed
a striking increase in patients with COPD compared to those in healthy subjects.
Because a beneficial effect of the high levels of soluble IL-1RAcp and ST2 is to
antagonize the pro-inflammatory function of IL-33 (58), the enhanced levels of these
molecules demonstrated increased IL-33 expression and release in COPD.
Recent studies have implicated IL-33 in the pathogenesis of asthma. In asthma, IL-33
can significantly enhance inflammation and cause pathological changes in the airways
by recruiting inflammatory cells and inducing the release of inflammatory cytokines
(40, 49, 52). Interestingly, a similar pattern of inflammation also exists in COPD. In
IL-33-treated mice, the most obvious histopathological changes in the lungs were
epithelial lining hypertrophy, goblet cell hypertrophy and mucus hypersecretion (46),
which are the prominent pathophysiological features of COPD patients. By activating
macrophages to secrete cytokines or by direct stimulation, IL-33 can promote
neutrophil maturation (56). The activation and recruitment of neutrophils to the
airway has been strongly linked to the inflammatory response in COPD. In IL-33
transgenic mice, increased IL-8 was detected in the BALF (61), which is a potent
chemotactic cytokine for neutrophils and is thought to play a central role in COPD (8).
Combined with the finding of higher IL-33 expression in peripheral blood and
airways from COPD patients in our study, we speculate that IL-33 may play an
important role in the pathogenesis of COPD.
Furthermore, we characterized the sources of IL-33 in COPD. We made the novel
observation that PBLs and HBEs in the airways produced IL-33 cytokine. We
detected IL-33 protein immunoreactivity in HBEs from lung tissue sections from
COPD and control subjects, and the expression of IL-33 was significantly higher in
COPD patients than in controls. However, we failed to observe immunostaining of
ASMCs. Furthermore, our flow cytometry results showed that PBLs were the major
source of IL-33 in the serum, particularly in COPD patients. By contrast,
IL-33-positive neutrophils were scarcely detectable in either patients or healthy
Although IL-33 was initially found in the endothelial cells of high endothelial venules
(4), previous reports confirmed the expression of IL-33 in various cell types,
including epithelial cells, smooth muscle cells, keratinocytes, fibroblasts, dendritic
cells and macrophages (28, 36, 46, 54). IL-33 expression has also been detected in
venules from chronically inflamed tissues from patients with Crohn’s disease or
rheumatoid arthritis. Moreover, IL-33 expression levels were correlated with
inflammation and tissue damage, which can lead to apoptosis and the release of IL-33
protein. Several studies have demonstrated that IL-33 is highly expressed in epithelial
cells from many tissues, including the skin (18), gastrointestinal tract (29) and airway
(27), which make contact with allergens, pathogens, tobacco smoke and other
common environmental agents (36). Recent studies of epithelial cells from asthma
patients reported significantly increased expression and secretion levels of IL-33
compared to those from controls (42). In our study, IL-33 was mainly derived from
airway epithelial cells and PBLs, which may account for the airway and systemic
inflammation observed in COPD.
Another important finding of our study is that CSE and LPS significantly enhanced
IL-33 expression in both PBLs and HBEs. It has been reported that IL-33 and ST2 can
be induced in CS-exposed mice (43). However, our study is the first to provide
evidence that IL-33 expression in HBEs and PBLs could be induced by CSE and LPS,
which are risk factors for the development of COPD. Moreover, low levels of IL-33
were detected in the supernatants of cultured ASMCs, irrespective of whether they
were stimulated, which was consistent with the negative immunostaining of ASMCs
in lung tissue. More importantly, we noted a significantly higher release of IL-33 for
COPD patients compared to healthy controls after PBLs were stimulated with the
mixture of CSE and LPS.
As an alarm signal, IL-33 is released to activate the immune system in response to
inflammatory signals or tissue injury caused by factors such as toxins, high free fatty
acids, inflammation or oxidative stress (4, 31). Increased IL-33 has been reported to
be detected in synovial fibroblasts and keratinocytes after activation with IL-1β and
TNF-α (26, 47). It has been speculated that IL-33 protein could be released when cell
damage occurs to alert the immune system to ‘danger’ (60). Cigarette smoking is the
key cause of the initiation of COPD, and LPS plays an important role in the
progression of COPD. Both of stimuli can activate inflammatory cells and enhance
cytokine release (15, 20). Consequently, we surmised that CSE and LPS are related to
the high expression of IL-33 in COPD, and they may play an important role in the
progression of COPD through modulating the release of IL-33.
In order to explore the mechanism of the high expression of IL-33 in COPD after CSE
and LPS stimulating, rs4986790, rs4986791 and rs11536889 polymorphisms of TLR4
gene were detected. TLR4 is a transmembrane protein that plays an essential role in
detecting LPS. After LPS binding to TLR4, the intracellular signaling events could
affect the production of proinflammatory cytokines (1). TLR4 SNPs have been
described in associating with greater susceptibility to various inflammatory diseases,
including rheumatoid arthritis (44), atherosclerosis (24), asthma (12) and COPD (45).
The rs4986790 and rs4986791 polymorphisms of TLR4 gene have been reported to be
strongly associated with endotoxin hyporesponsiveness to LPS in Caucasians (3) by
affecting the extracellular domain of TLR4. Another study showed that the
rs11536889 polymorphisms in the TLR4 gene were likely associated with COPD in
Japanese subjects (22). However, in the present study, we observed no rs4986790 and
rs4986791 polymorphisms in our samples, which is consistent with previous studies
on Chinese Han populations (19), Korean (25) and Japanese (38). Moreover, the
analysis of rs11536889 polymorphisms showed that there was no significant
difference between COPD patients and controls, which means TLR4 SNPs may not be
related to LPS hyporesponsiveness in our study.
In conclusion, this study provides compelling evidence for elevated IL-33 expression
in COPD, including in the airways and peripheral blood. IL-33 is mainly produced by
airway epithelial cells in airway inflammation and PBLs in systemic inflammation.
Moreover, CSE and LPS, which are risk factors for COPD, could both promote IL-33
secretion. Our study supports the importance of IL-33 signaling in the pathogenesis of
COPD, but the target cells and mechanism of action remain to be elucidated.
Therefore, more studies should be carried out to achieve a more comprehensive
understanding of the role of IL-33 in COPD and to determine the therapeutic value of
anti-IL-33 therapy in COPD patients.
Present address of J. Xie: Department of Respiratory and Critical Care Medicine,
Tongji Hospital, Tongji Medical College, Huazhong University of Science and
Technology, Wuhan, China
This study was supported by the National Natural Science Foundation of China (No.
81370145, 81370156, 81470227 and 81200029), The National Support Programme of
the Twelfth five-year plan: clinical translational research on respiratory diseases (No.
No conflicts of interest, financial or otherwise, are declared by the authors.
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Table 1. The characteristics of COPD patients and controls. COPD
Smoking (pack year)
Serum Cotinine (ng/ml)
FEV1 % pred
FEV1, forced expiratory volume in 1 second; FVC, forced vital capacity; FEV1 %
pred, FEV1 percent predicted. Data shown as means ± SD.
653 654 655 656 657 658 659 660 661 662 663 664 665
Table 2. Distribution of the TLR SNPs in COPD patients and controls. Genotype
COPD, n (%)
Control, n (%)
667 668 669 670 671 672 673 674 675 676 677 678 679 680
Figure 1. The levels of IL-33, sST2 and IL-1RAcP in plasma samples from patients
with COPD were significantly higher than those in controls. Each molecule was
separately measured by ELISA using plasma from study subjects. A, The levels of
IL-33 in plasma from subjects. B, The levels of sST2 in plasma from subjects. C, The
levels of IL-1RAcP in plasma from subjects. Data are presented as means ± SD. ****