Download Innate Type 2 Immunity Is Associated with Eosinophilic Pleural

Innate Type 2 Immunity Is Associated with
Eosinophilic Pleural Effusion in Primary
Spontaneous Pneumothorax
Bo-In Kwon1*, Seokchan Hong1*z, Kihyuk Shin1, Eun-Hye Choi1,2, Jung-Joo Hwang1,3, and Seung-Hyo Lee1
1
Graduate School of Medical Science and Engineering, Biomedical Research Center, KAIST Institute for the BioCentury, Korea Advanced Institute of
Science and Technology, Daejeon, Korea; 2Eulji Medi-Bio Research Institute, Eulji University, Daejeon, Korea; and 3Department of Thoracic and
Cardiovascular Surgery, Eulji University Hospital, Daejeon, Korea
Rationale: Eosinophilic pleural effusion (EPE) is characterized by
greater than 10% eosinophilia and is frequently associated with air
and/or blood in the pleural cavity. Primary spontaneous pneumothorax (PSP), defined as the spontaneous presence of air in the pleural space, is one of the most common causes of EPE. Recent studies
have shown that type 2 immune responses play important roles in
eosinophilic airway inflammation resulting in pleural pathology.
Objectives: To determine the predominant immune responses associated with PSP in humans, and to examine whether IL-33, thymic
stromal lymphopoietin (TSLP), or type 2 innate lymphoid cell (ILC2)mediated immune responses are associated factors.
Methods: Eosinophil-associated cytokines were measured in the pleural fluid of patients with PSP and control subjects. Th2 cell and ILC2
responses in the pleural cavity and peripheral blood were also evaluated by in vitro restimulation and intracellular cytokine staining of
T cells and ILC2s in patients with PSP (n ¼ 62) and control subjects
(n ¼ 33). IL-33–mediated IL-5 production by ILC2s was also evaluated.
Measurements and Main Results: Significantly higher concentrations
of IL-5 and eotaxin-3 were detected in the pleural fluid of patients
with PSP, in addition to significantly higher concentrations of IL-33
and TSLP. Although IL-5 production was induced by IL-33 treatment
of ILC2s, other Th2 cell–mediated immune responses were not
detected.
Conclusions: Our results indicate that innate immune responses characterized by the production of IL-33, TSLP, and IL-5 are associated
with the development of EPE in PSP by an ILC2-dependent and Th2independent mechanism.
Keywords: pneumothorax; eosinophil; IL-33; IL-5; type 2 innate lymphoid cells
(Received in original form February 14, 2013; accepted in final form June 10, 2013)
* These authors made equal contributions to this work.
z
Current Address: Division of Rheumatology, Department of Internal Medicine,
University of Ulsan College of Medicine, Asan Medical Center, Seoul, Korea
Supported by the Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education (NRF2013R1A1A2010714), the Bio & Medical Technology Development Program
of the National Research Foundation funded by the Korean government
(2012M3A9C7050093), and a grant from Eulji Medi-Bio Research Institute
(2013EMBRIDJ0002).
Author Contributions: Conception and design, J.-J.H. and S.-H.L. Analysis and
interpretation, B.-I.K., S.H., K.S., and E.-H.C. Drafting the manuscript for important intellectual content, B.-I.K., S.H., J.-J.H., and S.-H.L.
Correspondence and requests for reprints should be addressed to Seung-Hyo Lee,
Ph.D., Cellular Immunology Laboratory, Graduate School of Medical Science and
Engineering, Korea Advanced Institute of Science and Technology, Daejeon, 305701, Korea. E-mail: [email protected]; or Jung-Joo Hwang, M.D., Ph.D.,
Department of Thoracic and Cardiovascular Surgery, Eulji University Hospital,
Daejeon, 302-799, Korea. E-mail: [email protected]
This article has an online supplement, which is accessible from this issue’s table of
contents at www.atsjournals.org
Am J Respir Crit Care Med Vol 188, Iss. 5, pp 577–585, Sep 1, 2013
Copyright ª 2013 by the American Thoracic Society
Originally Published in Press as DOI: 10.1164/rccm.201302-0295OC on July 1, 2013
Internet address: www.atsjournals.org
AT A GLANCE COMMENTARY
Scientific Knowledge on the Subject
Primary spontaneous pneumothorax (PSP) is one of the
most common causes of eosinophilic pleural effusion (EPE),
but its pathogenesis and associated immune responses are
unknown.
What This Study Adds to the Field
The present study shows that innate immune responses mediated by IL-33 and type 2 innate lymphoid cells are associated
with the development of EPE in PSP.
Eosinophilic pleural effusion (EPE), defined as greater than 10%
eosinophilia in the pleural fluid, is often associated with the presence of air and/or blood in the pleural space (1). Although
a common manifestation of various pleural diseases, the precise
mechanism underlying EPE is largely unknown (1). Eosinophils
are responsible for host protective immunity against parasitic
helminth and fungal infections (2, 3) and the pathogenesis of
allergic diseases through the production of various factors including IL-4, IL-5, chemokines, and lipid mediators (4, 5). IL-5 and
eotaxin, well-known regulators of eosinophils, have been implicated in the initiation and propagation of eosinophil recruitment in
EPE (6–9).
Primary spontaneous pneumothorax (PSP) is one of the common causes of EPE (1); however, the mechanisms and clinical
significance of eosinophil accumulation in PSP are not well understood (1, 10). One of the main issues in the management of
PSP is the high rate of recurrence (up to 52%) (11, 12). Inflammatory responses associated with cigarette smoking, an
independent risk factor for the recurrence of PSP, may be involved
in the pathogenesis of this disease (10, 13). Therefore, it is necessary to understand the mechanisms underlying development of
pleural inflammation characterized by eosinophilia in PSP.
Recently it has been shown that innate immune cells, such as
eosinophils and epithelial cells, play critical roles in inflammation
beyond simply functioning to link innate and adaptive immunity.
For example, IL-33 and thymic stromal lymphopoietin (TSLP)
produced by innate immune cells have been shown to mediate
airway inflammation through Th2 dependent and independent
mechanisms (14, 15). IL-33, a member of IL-1 super-family, is
expressed by multiple cell types including epithelial cells and
macrophages (14, 16). IL-33 binds to a multimeric receptor that
includes ST2 and IL-1 receptor accessory proteins and initiates
many inflammatory responses (17, 18). Recent studies have reported increased IL-33 in the blood and lungs during allergic
diseases and hypereosinophilia in human patients and in an
animal model (19–22). Moreover, a novel cell type, type 2
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AMERICAN JOURNAL OF RESPIRATORY AND CRITICAL CARE MEDICINE
innate lymphoid cells (ILC2s), which secrete IL-5 and IL-13 in
response to IL-33, have been discovered (23–27). Thus, IL-33
acts on innate immune cells to mediate type 2 inflammatory
responses through the production of IL-5 and IL-13.
In this study, we investigated whether type 2 adaptive and innate immunity is associated with PSP with EPE. We found significantly elevated IL-33 and TSLP in pleural fluid of patients
with PSP. Moreover, the concentration of IL-33 in pleural fluid
was positively correlated with IL-5 and eosinophilia. Finally,
we showed that ILC2s produce significant amounts of IL-5 when
incubated with IL-33 or pleural fluid from patients with PSP.
Together, our findings indicate that type 2 innate immunity is
strongly associated with the pathogenesis of EPE in PSP.
METHODS
VOL 188
2013
TABLE 2. CHARACTERISTICS OF STUDY SUBJECTS
IN CD4 T-CELL ASSAY
Sex, M/F
Age, yr
Height, cm
Weight, kg
BMI
Diagnosis
Pneumothorax (n ¼ 30)
Control Subjects (n ¼ 24)
28/2
18.7 6 3.2*
174.2 6 6.9*
60.7 6 8.3
20.0 6 2.3*
PSP (n ¼ 30)
12/12
45.4 6 21.2
163.5 6 9.2
64.8 6 12.1
24.2 6 3.5
Tbc (n ¼ 4)
Cancer (n ¼ 4)
Pneumonia (n ¼ 6)
Hyperhydrosis (n ¼ 10)
Definition of abbreviations: BMI ¼ body mass index; PSP ¼ primary spontaneous pneumothorax; Tbc ¼ tuberculosis.
Data are mean 6 SD.
* P , 0.001 compared with control subjects.
Patient Population
The study population consisted of 62 patients with PSP and 33 control
subjects whose clinical characteristics are listed in Table 1. Pleural fluid
lavage was performed on 30 patients with PSP and 24 control subjects
and was used to isolate CD4 T cells (Table 2). Peripheral blood mononuclear cells (PBMCs) were isolated from 21 patients with PSP and 12
control subjects (Table 3). PSP was confirmed by conventional chest
radiography.
Antibodies and Reagents
Monoclonal antibodies specific for human Pacific blue-CD3 (clone:
UCHT1), Pacific blue-CD4 (clone: RPA-T4), PE-Cy7-CD8 (clone:
RPA-T8), APC-Cy7-CD45 (clone: HI30), PE-Cy7-c-Kit (clone: 104D2),
PerCP-Cy5.5-CD294 (CRTH2, clone: BM16), PE-ST2 (clone: 2A5),
APC-Cy7-CD11b (clone: M1/70), PE-Siglec-8 (clone: 7C9), APC-IL-5 receptor a (clone: 26,815), V450-CD45 (clone: HI30), PE-IL-4 (clone: 8D4–
8), APC-IL-5 (clone: TRFK5), and fluorescein isothiocyanate–cocktail of
lineage marker (Lin: CD2 [RPA-2.10], CD3 [OKT3], CD14 [61D3],
CD16 [CB16], CD19 [HIB19], CD56 [CB56], and CD235a [HIR2]) were
purchased from BD Biosciences (San Diego, CA), BioLegend (San
Diego, CA), eBioscience (San Diego, CA), MBL International (Woburn,
MA), and R&D Systems (Minneapolis, MN). Recombinant IL-5 and
IL-33 was purchased from Peprotech (Rocky Hill, NJ) and polyclonal
antibody against IL-5 and IL-33 from R&D Systems.
Pleural Fluid Collection and Cytokine Measurement
Pleural fluids were centrifuged at 350 3 g for 10 minutes at 48 C, and
cell-free supernatants were stored at 2808 C. The concentrations of
IL-4 (eBioscience), IL-5 (BD Biosciences), eotaxin-3 (R&D Systems),
IL-13 (BD Biosciences), IL-33 (R&D Systems), and TSLP (R&D Systems) were determined by standard ELISA.
TABLE 1. CHARACTERISTICS OF STUDY SUBJECTS
IN CYTOKINE ASSAY
Sex, M/F
Age, yr
Height, cm
Weight, kg
BMI
Diagnosis
Pneumothorax (n ¼ 62)
Control Subjects (n ¼ 33)
57/5
18.7 6 3.2*
173.9 6 6.3*
59.6 6 8.2†
19.7 6 2.2*
PSP (n ¼ 62)
18/15
49.5 6 20.6
163.9 6 9.1
64.2 6 11.5
23.9 6 3.5
Tbc (n ¼ 7)
Cancer (n ¼ 9)
Pneumonia (n ¼ 7)
Hyperhydrosis (n ¼ 10)
Definition of abbreviations: BMI ¼ body mass index; PSP ¼ primary spontaneous pneumothorax; Tbc ¼ tuberculosis.
Patients with PSP had no history, nor clinical or radiographic signs of any other
lung disorder except pneumothorax.
*P , 0.001 compared with control subjects.
y
P , 0.05 compared with control subjects.
Collection of Pleural Lavage Fluid and Purification of Cells
Pleural fluid lavage was performed as described with minor modification (10, 28). Briefly, 200 ml of saline was injected into the pleural
space, and fluids were harvested during bleb resection or other surgical procedure. Collected fluids were filtered through 40-mm nylon
mesh and washed with Hanks’ balanced salt solution. CD41 and
CD1a/CD1421 cell populations were isolated using magnetic cell
sorting (autoMACSpro; Miltenyi Biotec Inc., Bergisch Gladbach,
Germany).
Cell Cultures
Cell cultures were performed as described previously (29). CD4 T cells
(2 3 105) cultured with or without irradiated antigen-presenting cells
(APCs; 2 3 105; 3,000 rad) were stimulated with 1 mg/ml of antiCD3/CD28 antibodies for 4 days, then supernatants were collected
and cytokines assessed using Milliplex MAP kit (Millipore, Bedford,
MA) and a Bioplex 200 system (Bio-Rad Laboratories, Hercules,
CA).
Flow Cytometry and Intracellular Cytokine Staining
Pleural fluid cells and PBMCs were incubated with PMA (50 ng/ml;
Sigma-Aldrich, St. Louis, MO) and ionomycin (500 ng/ml) for 5 hours
at 378 C in the presence of brefeldin-A (10 mg/ml). Cells were then fixed
using fluorescence-activated cell sorter lysing solution, permeabilized
with 0.5% saponin in phosphate-buffered saline, and incubated with
antibodies for surface markers or cytokines for 30 minutes at room
temperature. After washing, stained cells were analyzed by flow cytometry (LSRII; BD Biosciences).
For IL-33–mediated IL-5 production, pleural fluid cells and PBMCs
were treated with IL-33 with or without neutralizing antibody against
IL-33 for 5 hours. For IL-5/IL-33–mediated IL-4 production, pleural
fluid cells were treated with IL-5/IL-33 or pleural fluid with or without
neutralizing antibodies against IL-5 and/or IL-33.
TABLE 3. CHARACTERISTICS OF STUDY SUBJECTS IN PBMC ASSAY
Sex, M/F
Age, yr
Height, cm
Weight, kg
BMI
Diagnosis
Pneumothorax (n ¼ 21)
Control Subjects (n ¼ 12)
19/2
18.4 6 2.7*
175.5 6 6.7*
61.0 6 7.5
19.8 6 2.0†
PSP (n ¼ 21)
5/7
33.1 6 15.4
163.1 6 9.7
66.3 6 13.5
24.8 6 3.1
Cancer (n ¼ 3)
Hyperhydrosis (n ¼ 9)
Definition of abbreviations: BMI ¼ body mass index; PBMC ¼ peripheral blood
mononuclear cell; PSP ¼ primary spontaneous pneumothorax.
Data are mean 6 SD.
* P , 0.01 compared with control subjects.
y
P , 0.001 compared with control subjects.
Kwon, Hong, Shin, et al.: ILC2-Dependent IL-5 Production in Pneumothorax
Statistical Analysis
Comparisons were made by nonparametric Mann-Whitney U test using
GraphPad Prism Software V.5.0 (Graph Pad Software, San Diego,
CA). To assess correlations, Spearman correlation analysis was performed. P values (,0.05) were considered statistically significant.
RESULTS
IL-4, IL-5, IL-13, and Eotaxin-3 Concentrations Are Increased
in the Pleural Fluid of Patients with PSP
The demographic and clinical features of patients with PSP and
control subjects are described in Table 1. As shown, the percentages of eosinophils were significantly increased in the pleural
fluid of patients with PSP compared with control subjects (Figures 1A and 1B), consistent with the higher frequency of EPE in
patients with PSP (43 [72.9%] of 59 vs. 1 [3%] of 33). Previous
studies demonstrated that in pleural fluid, the levels of IL-5 and
eotaxin-3 are closely correlated with the number of eosinophils
suggesting these molecules may mediate the accumulation of
eosinophils (8, 9, 30). In agreement with these reports, we
detected a significant difference in IL-5 concentration in the
pleural fluids of patients with PSP compared with control
subjects (Figure 1C) (P , 0.0001). Similarly, eotaxin-3 was
detected at a much higher concentration in the pleural fluid of
PSP when compared with control subjects (Figure 1D) (P ,
0.0001). Given that eosinophilia and IL-5 production represent
some of the key features of Th2 cell-driven immune responses,
these findings point to an association between Th2 immunity
and EPE in PSP. To address this, we next measured the concentration of IL-4, a canonical cytokine of Th2 cells, and IL-13.
Significantly higher concentrations of IL-4 and IL-13 where detected
in patients with PSP compared with control subjects (Figures 1E and
1F). Therefore, in agreement with previous reports (31, 32) our
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findings indicate that the expression of Th2 cell-associated cytokines is increased in the pleural fluid of patients with PSP.
Th2 Cell–mediated Immune Responses Are Not Involved
in PSP
Based on our detection of Th2 cytokines and chemokines in the
pleural fluid of PSP, we next sought to determine the contribution of CD4 T cells to the production of the canonical Th2 cytokines IL-4, IL5, and IL-13 in pleural fluid. Therefore, we isolated
CD4 T cells from freshly obtained pleural lavage fluid using
a smaller representative of cohort of PSP subjects (Table 2)
as described previously (10). CD4 T cells were either directly
activated with anti-CD3/CD28 antibodies or cocultured with
irradiated autologous CD1a/CD141 cells as APCs in the presence of anti-CD3/CD28 antibodies. Because the basal levels of
cytokine production were highly variable, we expressed data as
fold differences in which the amount of cytokine produced by
antibody stimulation was expressed as a fraction of cytokine
levels without stimulation. However, significant differences in
the production of IL-4 and IL-5 between PSP and control
groups were not detected after in vitro stimulation (Figures
2A and 2B). We also could not detect significant differences
in the levels of IFN-g between in vitro stimulated PSP and
control CD4 T cells (Figure 2C). Similarly, no significant difference in the production of IL-4, IL-5, or IFN-g from in vitro–
stimulated blood CD4 T cells was detected (Figures 2D–2F). In
addition, because normalized values can be misleading and
background values could affect calculated results considerably,
we also presented the data as absolute values. Regardless of
how the data are presented, the results between groups were
not different except that cytokine production was enhanced
with antibody stimulation of blood CD4 T cells, but again these
effects were not different between control subjects and patients
Figure 1. Eosinophils, IL-5,
eotaxin-3, IL-4, and IL-13 levels
in the pleural fluid from patients
with primary spontaneous pneumothorax and control subjects.
(A) Percentage of eosinophils in
the pleural fluid of patients with
PSP or control subjects. (B) Representative Giemsa-stained cytospin slides are presented. The
levels of IL-5 (C), eotaxin-3 (D),
IL-4 (E ), and IL-13 (F ) were
quantified in pleural fluid from
patients with primary spontaneous pneumothorax or control
subjects. Each dot represents an
individual subject. Horizontal lines
represent the mean. Statistical
analysis was performed using
Mann-Whitney U tests. ***P ,
0.001. PF ¼ pleural fluid.
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Figure 2. Th1 (IFN-g) and Th2
(IL-4, IL-5) responses of CD4 T
cells from patients with primary spontaneous pneumothorax and control subjects.
After stimulation of pleural
fluid CD4 T cells from patients
with primary spontaneous
pneumothorax or control subjects with anti-CD3 and CD28
antibodies, IL-4 (A), IL-5 (B),
and IFN-g (C) production was
measured by ELISA. Stimulation of CD4 T cells from blood
was similarly performed and IL4 (D), IL-5 (E), and IFN-g (F)
production was measured. Each
dot represents an individual subject. Horizontal lines represent
the mean. Statistical analysis was
performed using Mann-Whitney
U tests.
with PSP (see Figure E1 in the online supplement). Because
CD4 T-cell stimulation in the presence of APCs may affect
overall results in which cytokine production by activated CD4
T cells can be regulated by APCs, we also stimulated CD4 T
cells with antibodies without APCs and still did not detect significant differences between groups (see Figure E2). Furthermore, we performed intracellular cytokine staining to address
the question of whether the secretion of cytokines in pleural
fluid was derived from CD4 T cells. Because expression of
CD4 is down-regulated after phorbol myristate acetate and ionomycin stimulation, surface staining for CD4 was not possible
(33). We thus used CD82/CD31 cells to select for CD4 T cells
as previously described (34) (Figure 3A). The frequency of IL-
4–producing CD4 T cells in pleural fluid was not significantly
increased in the PSP group (Figure 3B). Because a large number of CD8 T cells were also seen in pleural fluid of PSP and
control subjects, we compared the frequency of CD8 T cells
between groups and determined whether these cells produced
IL-4. Similar to the results from CD4 T cells, neither the numbers of CD8 T cells nor the frequency of IL-4–producing CD8
T cells in pleural fluid differed between control and PSP
groups (Figure 3C). In addition, we observed no IL-5 production from CD4 or CD8 T cells from both subjects (data not
shown). Finally, we measured Th1- and Th17-associated cytokines,
such as IFN-g and IL-17, and found that they were also not increased (data not shown). Collectively, these data indicate that
Figure 3. Flow cytometric detection of
intracellular cytokine production in CD4
and CD8 T cells obtained from pleural
fluid. (A) Gating method for the identification of T cells (left and center) and representative dot plots of the staining
strategy for IL-4– and IL-5–producing
CD4 (right bottom) and CD8 (right top)
T cells in pleural fluid. Percentages of IL4–producing CD4 (B) and CD8 (C) T cells
from pleural fluid after restimulation with
phorbol myristate acetate and ionomycin
are shown. Each dot represents an individual subject. Horizontal lines represent
the mean. Statistical analysis was performed using Mann-Whitney U tests.
Kwon, Hong, Shin, et al.: ILC2-Dependent IL-5 Production in Pneumothorax
581
neither classical Th2 immune responses nor CD8 T cell–mediated
responses are involved in the pathogenesis of PSP.
with the frequency of eosinophils and the concentrations of
IL-5 and eotaxin-3.
Increased IL-33 and TSLP Levels in Pleural Fluid
of Patients with PSP
Induction of IL-5 in ILC2s by Treatment with IL-33 or Pleural
Fluid of Patients with PSP
Our findings so far indicated that adaptive immune cells including CD4 and CD8 T cells in the pleural fluid are not a significant cellular source of Th2 cytokines and chemokines,
which are implicated in eosinophil recruitment in PSP. Recent
evidence suggests that innate immune cells, such as macrophages, eosinophils, and epithelial cells, have diverse and critical roles in the development and progression of inflammatory
diseases (35, 36). These cells can participate in allergic inflammation by producing several cytokines, chemokines, and
other effector molecules in response to various stimuli including antigens, infection, and mechanical stress, and they mediate complex immune responses not confined to classical Th2
immunity (35, 36). Moreover, it was discovered that IL-33
secretion could be elicited by mechanical stress in the absence
of cellular necrosis (37). Thus, we hypothesized that IL-33 and
TSLP, two recently discovered cytokines produced by epithelial cells, could be involved in the activation and recruitment
of eosinophils in PSP. To test this, we first measured the levels
of IL-33 and TSLP in the pleural fluid of patients with PSP
and found both were significantly higher than in control
subjects (Figures 4A and 4B). Next, we examined the correlation between the levels of IL-33 and the frequency of
eosinophils, and the levels of IL-5 and eotaxin-3 in pleural
fluid to assess the potential role of these cytokines in eosinophil recruitment. As shown in Figure 4C, there was a positive
correlation between the frequency of eosinophils and the concentration of IL-33 and a strong positive correlation between
the concentrations of IL-5 and IL-33, and eotaxin-3 and IL-33
in pleural fluid of patients with PSP (Figures 4D and 4E).
Thus, IL-33 and TSLP are induced in the pleural cavity of
patients with PSP, and IL-33 levels are positively correlated
Because it has been shown that IL-33 induces eosinophilia
by stimulating IL-5 secretion from several different cells
including CD41 Th2 cells and ILC2s (21, 25, 38), we investigated whether IL-33 detected in the pleural fluid of patients
with PSP could promote the production of IL-5 from ILC2s.
Immune cells were isolated from pleural fluid and the peripheral blood of patients with PSP and treated with either recombinant IL-33 or pleural fluid obtained from subjects with elevated
concentrations of IL-33. First, we selected ILC2s by examining
their specific cell surface molecule expression pattern identified
as CD451c-Kit1CRTH21Lin2 (Figure 5A, top), as described
previously (23–25). Furthermore, ILC2s express ST2, a receptor
for IL-33 (Figure 5A) (14). On the contrary, the number of
ILC2s in control subjects was too low to detect and significantly
lower than that of ILC2 in the pleural fluid of patients with PSP
(Figures 5A, bottom, and 5B). In agreement with previous
reports (19, 24), we detected the induction of IL-5 by pleural
fluid ILC2s from patients with PSP after IL-33 treatment, and
we found that IL-5 induction was ablated with IL-33 neutralizing antibody treatment (Figures 5C and 5D). Furthermore,
pleural fluid ILC2s showed enhanced production of IL-5 with
pleural fluid treatment and this effect was also inhibited by the
addition of anti–IL-33 antibody (Figures 5C and 5D). Because
not only ILC2 but also other cells, such as CD41 Th2 cells,
could produce IL-5 in response to IL-33, we examined IL-5
production by intracellular cytokine staining and showed that
only CRTH2 1 ILC2 produced IL-5 in the patients with
PSP (Figure 5E). Similar analyses from control subjects were
effectively impossible because of the lack of ILC2 (Figures 5A,
5B and 5F). We also quantified IL-33–stimulated IL-5 and IL-13
secretion by pleural fluid cells because ILC2s produce IL-5 and
Figure 4. Levels of IL-33 and
thymic stromal lymphopoietin
(TSLP) in patients with primary
spontaneous pneumothorax
(PSP) or control subjects, and
the correlation of IL-33 levels
with eosinophils frequency,
and IL-5 and eotaxin-3 levels.
The levels of IL-33 (A) and TSLP
(B) in pleural fluid collected
from patients with PSP and
control subjects were analyzed
by ELISA. (C) Correlation between IL-33 levels and the percentage of eosinophils in the
pleural fluid from patients with
PSP. Correlations of IL-33 with
IL-5 (D) and eotaxin-3 (E).
Each value represents a sample
from an individual subject. Statistical analysis was performed
using Mann-Whitney U tests to
compare the cytokine levels
among groups. ***P , 0.001.
Spearman correlation analysis
was performed to assess for
significant correlations between
quantitative variables.
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Figure 5. Induction of IL-5 by
IL-33, and pleural fluid (fluid)
treatment in type 2 innate lymphoid cells (ILC2s) of patients
with primary spontaneous
pneumothorax (PSP). (A) Gating method to identify ILC2s
that express CD45, c-kit,
CRTH2, and ST2 without lineage markers. (B) The frequency
of ILC2s in CD451 lineage2
cells between patients with
PSP and control subjects was
calculated. Representative flow
analysis to quantify IL-5 production induced by IL-33 and
pleural fluid is presented (C)
and cumulative data for IL-5
induction by IL-33 and pleural
fluid treatment (D). Twodimensional scatter plot analysis
for intracellular IL-5 production
by ILC2 in response to IL-33
and pleural fluid (E). Aggregate IL-5 (F) and IL-13 (G) secretion data as quantified by
ELISA from pleural fluid cells
treated with and without IL33. Statistical analysis was performed using Mann-Whitney U
tests. *P , 0.05, **P , 0.01,
***P , 0.001.
IL-13 in response to IL-33 (23–25), and found significantly
higher amounts of both cytokines from patients with PSP
relative to control subjects (Figures 5F and 5G). Because we
showed the increased levels of IL-4 in pleural fluid of the
patients with PSP (Figure 1E) and neither CD4 T cells nor
ILC2s produced IL-4 in response to IL-33, we tested a possibility
whether eosinophils produce IL-4 by a treatment with IL-33.
To so do, first we identified CD451CD11b1Siglec-81IL-5Ra
(CD125)1 eosinophils with fluorescence-activated cell sorter
analysis (Figure 6A) (39). After treatment with IL-33/IL-5 or
pleural fluid, eosinophils in the pleural fluid of patients with PSP
produced significant amounts of IL-4; however, we could not
detect IL-4 production in control subjects probably because of
the absence of eosinophils (Figures 6B and 6C). Lastly, we
investigated a possibility in which CD41 Th2 cells might secrete
IL-5 after stimulation with IL-33 because it has been shown
that IL-33–stimulated Th2 cells could be activated and produce
effector cytokines including IL-5 (38). Similar to anti-CD3/
CD28 antibody stimulation, we could not detect IL-5 production in CD4 T cells in the pleural fluid of patients with PSP
treated with either IL-33 or pleural fluid (see Figure E3). Together, these results demonstrate that the levels of IL-33 were
increased in the pleural cavity of patients with PSP and that this
cytokine directly promoted the production of IL-5 from ILC2s,
and specifically not Th2 cells, ultimately resulting in EPE.
DISCUSSION
In this study, we addressed the question of whether innate and/or
adaptive immune responses are involved in the process of EPE in
Kwon, Hong, Shin, et al.: ILC2-Dependent IL-5 Production in Pneumothorax
583
Figure 6. Eosinophils of patients
with primary spontaneous pneumothorax (PSP) produce IL-4.
(A) Gating method to identify
eosinophils that express CD45,
CD11b, Siglec-8, and IL-5 receptor a chain (CD125) without lineage markers. (B) Representative
flow histogram analysis to quantify IL-4 induced by IL-5/IL-33
and cumulative results are presented. The IL-4 production levels were decreased by treatment
with anti–IL-5/IL-33 antibodies.
(C) Representative flow analysis
and cumulative data for IL-4 induction by pleural fluid (fluid)
treatment under the indicated
conditions. Statistical analysis
was performed using MannWhitney U tests. *P , 0.05,
***P , 0.001.
PSP. We have shown that eosinophil-associated cytokines and
chemokines, IL-5, eotaxin-3, IL-4, and IL-13, are significantly elevated in pleural fluid from patients with PSP compared with
that of control subjects. Furthermore, the levels of IL-33 and
TSLP, two recently discovered cytokines that mediate various
inflammatory diseases through the recruitment and activation
of eosinophils and other immune cells, are also significantly
higher in the pleural fluid of patients with PSP. Although we
were unable to detect any significant difference in Th2 immune
responses between pleural fluid cells of patients with PSP and
control subjects, we did find significant induction of IL-5 by
ILC2s treated with IL-33. Therefore, our study suggests that innate immune responses characterized by the production of IL5, IL-33, and TSLP participate in the development of EPE in
PSP through a Th2-independent and ILC2-dependent mechanism.
Our results are consistent with previous reports that IL-5 and
eotaxin-3 are closely correlated with EPE in post–coronary artery bypass graft pleural effusions, suggesting that these cytokines play a critical role in the development of eosinophilia in
pleura (8, 9). Another study also demonstrated that pleural fluid
in EPE after PSP has higher levels of several inflammatory
cytokines including IL-5, IL-6, and tumor necrosis factor-a.
IL-4, which is the canonical cytokine of Th2 immunity, is
a potent inducer of eotaxin expression, and eosinophilic inflammation is one of the main features of Th2-mediated inflammation (2, 35). In contrast to our findings, IL-4 was not
detectable in the pleural fluid of patients with EPE associated
with coronary artery bypass graft, malignancy, or trauma (31,
40). Taken together, these findings strongly suggest that eosinophilic immune responses play an important role in the
pathogenesis of EPE, but classical Th2-mediated responses
may not be involved.
We were unable to detect a significant increase in CD41 Th2associated immune responses in pleural fluid of patients with
PSP. For example, there was no significant increase of IL-4–
producing CD4 T cells in the pleural fluid of patients with
PSP in two independent experiments. Thus, these data suggest
that Th2-mediated adaptive immunity is not implicated in the
eosinophilic inflammation associated with pneumothorax. Indeed, this is not surprising considering that adaptive immunity
is characterized by delayed response through antigen recognition by specific T-cell receptors, but PSP occurs rapidly, before
adaptive immunity is likely to become fully activated. In addition, a study involving an animal model of pneumothorax provides evidence that conventional Th2-associated immunity may
not be involved in pleural eosinophilia (41). This same study
also demonstrated that pneumothorax-associated EPE in
mice is abolished in IL-5–deficient, but not in IL-13 knock-out
mice (41). Furthermore, recent studies have suggested that innate immune cells, including airway epithelial cells and ILC2s,
play a role in the initiation and propagation of airway inflammation and their mechanism of action is not confined to the
classical Th2 immune response (36). Thus, our present findings
indicate that innate eosinophilic immune responses, but not Th2
immunity, are significantly involved in the pathogenesis of PSP.
Indeed, our data clearly show that significantly increased numbers of ILC2s exist in the pleural fluid of patients with PSP.
Furthermore, ILC2s have been shown to respond to IL-33 with
the expression of its receptor, ST2, and the production of IL-5
and IL-13. However, we could not find significantly increased
584
AMERICAN JOURNAL OF RESPIRATORY AND CRITICAL CARE MEDICINE
production of IL-5 from blood ILC2s (data now shown). Therefore, it is reasonable to hypothesize that mechanical stress may
cause the release of IL-33, and ILC2s responding to IL-33 secrete IL-5 during eosinophilic inflammation over the course
of PSP.
IL-33, produced by hematopoietic and nonhematopoietic
cells, such as dendritic cells, macrophages, epithelial cells, and
endothelial cells, was originally discovered as a ligand for Th2
cell-associated receptor, ST2. It has been shown that IL-33/
ST2 signaling participates in the pathogenesis of various inflammatory diseases, such as allergic asthma, rheumatoid
arthritis, and inflammatory bowel disease (18). IL-33 can activate eosinophils to produce CXCL8 and superoxide, and it enhances adhesion and survival of these cells (42, 43). In addition,
IL-33 can activate bone marrow–derived mast cells and basophils to produce proinflammatory cytokines and chemokines
including IL-6; IL-13; regulated upon activation, normal; and
monocyte chemoattractant protein-1 (44, 45). Recent evidence
suggests that IL-33 has a role in the development of airway
inflammation by a T cell–independent mechanism (46). This
study showed that exposure of house dust mite allergen can
directly induce production of IL-33 from airway epithelium
through a Toll-like receptor-mediated signaling pathway (46).
We detected significantly higher levels of IL-33 and TSLP in the
pleural fluid from patients with PSP, which is to our knowledge
the first report describing this phenomenon. Because T cell–
mediated adaptive immune responses were not apparent, we
conclude that ILC2s play an important role in the course of
EPE in PSP through immediate secretion of inflammatory
cytokines including IL-5 and/or IL-13, resulting in inflammation, such as tissue eosinophilia. The cells responsible for the
production of these cytokines were not defined; however, given
that the epithelial cell lining of airways play critical roles in the
initiation and progression of diverse inflammatory diseases, it is
likely that mesothelial cells, which express markers for epithelial
cells, and lining of the pleura contribute to eosinophilic inflammation in EPE.
By definition, PSP occurs in subjects without underlying lung
disease, and subpleural bullae are found in 76–100% of patients,
as compared with 20% of control subjects (12). The mechanisms
of bulla formation remains uncertain but a respectable explanation is that degradation of elastic fibers in lung occurs, induced by influx of inflammatory cells. Eosinophils have long
been related to fibrosis because they secrete such products as
inflammatory and fibrogenic mediators including lipid mediators, chemokines, and cytokines (47). After bullae have formed,
inflammation-induced obstruction of the small airway increases
alveolar pressure, resulting in an air leak, consequently causing
pneumothorax. Furthermore, the relatively high recurrence rate
of pneumothorax is a major problem in the management of
PSP, and several reports suggest that PSP is associated with
poor lung function (11, 12). EPE in PSP can cause continuous
inflammation through their products and these could be related
to the high recurrence rate of pneumothorax. Cigarette smoking
is a risk factor for the development and recurrence of PSP, and
some studies suggest that smoking can evoke eosinophilic inflammation in the lung (48, 49). Indeed, one report clearly demonstrates that cigarette smoke is a major etiologic factor for the
development of acute eosinophilic pneumonia, using the provocation test (49). Further studies are needed to address the
effects of smoking on the development of EPE in PSP.
In conclusion, we have found that levels of IL-5, eotaxin-3,
IL-33, and TSLP are significantly increased in patients with
PSP. However, enhancement of Th2-mediated adaptive immune
responses was not detected in CD4 T cells from the pleural fluid
of the patients with PSP. On the contrary, our results suggest that
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2013
innate immune responses including IL-33 and TSLP secretion
are likely to play an important role in the pathogenesis of EPE
in PSP by a Th2-independent mechanism. More importantly, our
results show that IL-33–treated ILC2s can produce IL-5, and this
production is inhibited by treatment with a neutralizing antibody
against IL-33. Therefore, our study suggests that innate immune
responses characterized by the production of IL-5, eotaxin-3, IL33, and TSLP, participate in the development of EPE in PSP
through an ILC2-dependent mechanism.
Author disclosures are available with the text of this article at www.atsjournals.org.
Acknowledgment: The authors thank Drs. David Corry and Farrah Kheradmand
and all the members of the Hye Hwa Forum for helpful comments on the manuscript.
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