Pulmonary IL-17E (IL-25) Production and IL-17RB?Myeloid
Cell-Derived Th2 Cytokine Production Are Dependent upon
Stem Cell Factor-Induced Responses during Chronic Allergic
Vladislav Dolgachev,* Bryan C. Petersen,* Alison L. Budelsky,†Aaron A. Berlin,*
and Nicholas W. Lukacs2*
In the present studies local neutralization of allergen-induced stem cell factor (SCF) leads to decreased production of Th2
cytokines, a reduction in inflammation, allergen-specific serum IgE/IgG1, and attenuation of severe asthma-like responses.
The local blockade of pulmonary SCF also resulted in a significant reduction of IL-17E (IL-25). Sorted cell populations from
the lung indicated that IL-25 was produced from c-kit?cells, whereas Th2 cytokine production was primarily from c-kit?
cell populations. SCF stimulated c-kit?eosinophils produced IL-25, whereas bone marrow-derived mast cells did not. Using
4get mice that contain a IL-4-IRES-eGFP that when transcribed coexpress GFP and IL-4, our studies identified cells that
comprised a CD11b?, GR1?, Ly6C?/?, c-kit?, CD4?, CD11c?, MHC class IIlowcell population as a source of IL-4 in the
lung after chronic allergen challenge. In the bone marrow a similar cell was identified with approximately a third of the
IL-4?cells also expressing c-kit?. The pulmonary and bone marrow IL-4?cell populations were significantly reduced upon
local pulmonary anti-SCF treatment. Subsequently, when IL-25R was examined during the chronic allergen responses the
expression was found on the IL-4?myeloid cell population that expressed CD11b?GR1?. Interestingly, the IL-25R?cells in
the bone marrow were also all CD11b?GR1?, similar to the lung cells, but they were also all c-kit?, potentially suggesting
a maturation of the bone marrow cell once it enters the lung and/or is stimulated by SCF. Overall, these studies suggest a
complex relationship between SCF, bone marrow-derived IL-25-responsive myeloid cells, Th2 cytokines, and chronic allergic
The Journal of Immunology, 2009, 183: 5705–5715.
allergic responses by multiple investigators (1–11). Importantly,
SCF and c-kit have been shown to be highly up-regulated in air-
ways of asthmatics (12) as well as in nasal polyps in aspirin-sen-
sitive asthmatics (13), and circulating levels of SCF have been
correlated to the severity of asthmatic disease (14). Investigators
have also begun examining the role of SCF on eosinophil accu-
mulation and activation. In particular, the most recent studies have
demonstrated that SCF mediates eosinophil-induced degranula-
tion, cytokine production, and survival (15–17). Additionally, us-
ing in vivo models of chronic allergen-induced response results
demonstrated that SCF was involved in driving Th2 cytokine pro-
duction in the lung, as well as mucus overexpression and airway
tem cell factor (SCF)3-induced mast cell generation and
activation during allergen-induced airway responses has
been identified as an important mechanism for promoting
remodeling (16, 18). In those latter studies use of an inhibitor that
blocked c-kit receptor tyrosine kinase activation, imatinib, reca-
pitulated a similar effect as anti-SCF treatment in mice. Taken
together, these studies were striking and suggested that SCF or its
receptor may be an effective target for therapeutic intervention in
IL-25 (IL-17E) is a member of the IL-17 family of cytokines
and binds to a receptor, IL-17RB, that is shared with IL-17B (19).
Original studies on IL-25 demonstrated that it could directly in-
duce Th2 cytokine responses when injected in vivo into naive mice
(20). In these initial studies the results indicated that IL-25 pre-
dominantly induced Th2 cytokine in an Ag-independent manner
via a CD11b?/MHC class II?cell population. This latter obser-
vation is also reflected in the results for the present study. Since the
initial studies, researchers have identified that IL-25 is induced
during allergic and parasitic responses and that by using IL-25?/?
mice or Ab inhibition, IL-25 has a significant role in the generation
of severe airway inflammation (21–25). The exact mechanism of
how IL-25 induces enhanced Th2 responses has not been clearly
identified. However, IL-25 has been suggested to affect several cell
populations. The effect of IL-25 on T cells was recently demon-
strated to enhance differentiation into Th2-type cells by promoting
GATA3 expression as well as enhancing NFATc1 and JunB (26).
Additionally, IL-25 may synergize with other important Th2 as-
sociated factors such as TSLP to further enhance the skewing of
responses that would exacerbate the pathology in allergic disease
(27). Furthermore, the role of IL-25 for induction of Th2 cytokines
has also been suggested to directly associate with non-T cell pop-
ulations, including mast cells, basophils, and eosinophils (24, 25,
*Department of Pathology, University of Michigan Medical School, Ann Arbor, MI
48109; and†Department of Inflammation, Amgen, Seattle, WA 98119
Received for publication May 27, 2009. Accepted for publication September 1, 2009.
The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked advertisement in accordance
with 18 U.S.C. Section 1734 solely to indicate this fact.
1This research was supported in part by National Institutes of Health Grants
HL059178 and AI036302.
2Address correspondence and reprint requests to Dr. Nicholas W. Lukacs, Depart-
ment of Pathology, University of Michigan Medical School, 4059 BSRB, 109 Zina
Pitcher, Ann Arbor, MI 48109. E-mail address: firstname.lastname@example.org
3Abbreviations used in this paper: SCF, stem cell factor; CRA, cockroach allergen;
AHR, airway hyperresponsiveness.
Copyright © 2009 by The American Association of Immunologists, Inc. 0022-1767/09/$2.00
The Journal of Immunology
28). Interestingly, a recent study indicated that during parasitic
infection a bone marrow-derived cell population was directly
activated by IL-25, leading to Th2 type cytokine responses (25).
These latter studies identified a myeloid-derived cell that was not
a T cell, mast cell, or basophil. This particular IL-25-responsive
cell population increased in the bone marrow and subsequently
was recruited to the site of Th2 inflammation. Furthermore, a re-
cent study demonstrated that during persistent viral infection a
CD11b?macrophage population produced IL-13 in a CD1-depen-
dent mechanism (29). A recent publication has outlined the role of
IL-25 for direct induction of Th2 cytokines when instilled into the
lungs of naive mice by blocking the responses using an IL-25R
blocking Ab (30). Thus, a non-T cell source of Th2 cytokines may
also act to enhance allergic inflammation. This novel area may
help explain how chronic asthmatics continue to have significant
problems in the face of immune modulatory treatment protocols
directed at T cell responsiveness.
The data presented in these studies indicate that when SCF is
neutralized in the lung there is a significant reduction of inflam-
matory cells that is accompanied by a reduction of IL-25 produc-
tion. Additionally, blocking SCF also has a significant effect on
IL-25 responsiveness in the lung and the bone marrow. The results
further indicate that the reduction in Th2 cytokines after anti-SCF
treatment predominantly alters a Th2 cytokine-producing myeloid
cell and has a significant impact on chronic disease phenotypes.
The expression of IL-25R corresponds to the IL-4?myeloid cells.
These data give further insight into the source of Th2 cytokines
that is dependent upon SCF/c-kit and IL-25 for driving the severity
Materials and Methods
Animals and allergen model
BALB/c/J wild-type and 4get mice were purchased from The Jackson Lab-
oratory and used at 6–8 wk of age. The allergen we used was a clinical
grade skin test cockroach allergen (CRA) that has little endotoxin contam-
ination (?20 ng/ml). Our chronic model has been set up as outlined pre-
viously and as depicted below (18). Briefly, animals are immunized sys-
temically by i.p. sensitization emulsified with IFA and after 14 days they
are given four intranasal exposures of allergen (1.5 ?g/10 ?l) 4 days apart
followed by two intratracheal administrations (5 ?g/mouse) 4 days apart.
The intratracheal challenge allowed the administration of the allergen di-
rectly into the airways to induce the most intense disease exacerbation. We
analyzed the animals histologically and biochemically for changes in air-
way responses as listed below. This methodology provides a chronic and
consistent exacerbating airway model with severe mucus overproduction
and peribronchial fibrotic response, a difficult aspect to initiate in mice.
Measurement of airway hyperreactivity
Airway hyperreactivity was measured using mouse plethysmography,
which is specifically designed for the low tidal volumes (Buxco Research
Systems), as previously described (3, 31). Briefly, the mouse to be tested
was anesthetized with sodium pentobarbital and intubated via cannulation
of the trachea with an 18-gauge metal tube. The intubated mice were ven-
tilated at a volume of 200 ?l at a rate of 120 breaths/min. The airway
resistance was measured in the closed plethysmograph by directly assess-
ing tracheal pressure and comparing the level to corresponding box pres-
sure changes. These values were monitored and immediately transformed
into resistance measurements using computer-assisted calculations. Once
baseline levels had stabilized and initial readings were taken, a methacho-
line challenge was given via the cannulated tail vein. After determining a
dose-response curve (0.001–0.5 mg), an optimal dose was chosen (0.250
mg of methacholine). This dose was used throughout the rest of the ex-
periments in this study. After the methacholine challenge, the response was
monitored and the peak airway resistance was recorded as a measure of
Collagenase dispersed lungs and bone marrow cell extraction
Lungs and lung draining mediastinal lymph nodes were isolated and dis-
persed using 1 mg/ml collagenase A (Roche Applied Science) in RPMI
1640 (Invitrogen) with 5% FCS (Atlas Biologicals) and 2 U/ml DNase
(Sigma-Aldrich) at 37°C for 45 min. Cells were dispersed to a single-cell
population by flushing tissue through a 16-gauge cannula. After lysis of
RBC, cells were counted and numbers used in the calculation of specific
cell populations were based upon differential staining in flow cytometry.
In vivo neutralization of SCF
Neutralization of SCF was conducted using a polyclonal rabbit anti-murine
SCF Ab developed in our laboratory (2). The protein A column purified
anti-SCF or control Ab was administered intratracheally with CRA at the
time of the final two intratracheal challenges along with intranasal treat-
ment daily given between the final two allergen challenges (200 ?g/mouse)
for a total of five daily administrations of Ab.
Quantification of cytokines
RNA was isolated from the upper right lobes of lung, lymph nodes, and
bone marrow using TRIzol (Invitrogen). Levels of mRNA were assessed
using quantitative PCR analysis (TaqMan) with predeveloped primers and
probe sets from Applied Biosystems. Quantification of the genes of inter-
ests were normalized to GAPDH and expressed as fold increases over the
negative control for each treatment at each time point. Protein levels of
cytokines were quantified using a Bio-Plex bead-based (Luminex) cytokine
assay purchased from Bio-Rad Laboratories.
Flow cytometry analysis
Analyses of pulmonary and bone marrow cell populations by flow were
assessed using previously established standard techniques. Briefly, Dul-
becco’s PBS (DPBS) with 1% FBS and 0.09% sodium azide were used in
all of our staining procedures. A total of 1 ? 106cells was used with
viability staining with the Live/Dead staining kit (Invitrogen) along with
the Fc receptors blocked using mouse Fc block (1 ?g/100 ?l; BD Phramin-
gen) for 20 min on ice. Diluted fluorescent Abs specific for the different
surface markers or control Abs were incubated for 10 min at 4°C. Cells
were washed and the pelleted cells were resuspended in DPBS containing
1% FBS and 0.09% sodium azide and analyzed by flow cytometry on the
same day to avoid loss of fluorescence intensity. In some experiments we
also performed intracellular staining of formalin-fixed cells. The following
monoclonal mAbs were obtained from BD Biosciences: CD11b-PE-Cy7,
CD8a-PE-Cy5, streptavidin-allophycocyanin-Cy7, and MHC II(I-Ad)-PE;
from BioLegend: Ly6C-PE, Gr-1-AF700, and streptavidin-allophycocya-
nin-Cy7; and from eBioscience: CD8a-PE-Cy5, FceRI ?-AF647, c-kit-al-
lophycocyanin, c-kit-PE-Cy7, Gr-1-PB, and anti-IL-4-PE. Data were col-
lected in an LSR II BD Biosciences flow cytometer and analyzed using
FlowJo software (Tree Star). The mAb to IL-17RB was biotinylated and
used for flow cytometer.
Ag-elicited peritoneal eosinophil purification
Eosinophils were elicited by injection of thioglycolate plus soluble egg Ag
into the peritoneum of Schistosoma mansoni-infected mice. The injection
of soluble egg Ag into infected mice induces a pool of circulating eosin-
ophils recruited into the peritoneum in an Ag-specific manner. After 48 h,
the mice were lavaged peritoneally. The initial population contained peri-
toneum and included ?50% eosinophils, only 2–5% neutrophils, and 35–
45% mononuclear cells (lymphocytes and macrophages). Adherent cell
populations were removed by plastic adherence. The nonadherent cells
were washed and resuspended in PBS/BSA and eosinophils were purified
by negative selection using the MACS system. The Abs used were anti-
Thy1 (for T cells), anti-B220 (for B cells), and anti-class II (for APCs).
After the plate adherence and MACS separation, the population of cells
contained ?97% eosinophils contaminated with neutrophils (?1%) and
mononuclear cells (1–2%).
Bone marrow-derived mast cells
The generation of mast cells was accomplished as previously described (1)
by culturing bone marrow with rSCF (50 ng/ml) and rIL-3 (50 ng/ml) for
8 days. The cells were evaluated for FceR1 by flow cytometry to establish
mast cell phenotype along with cytospin analysis. The cells were then used
for evaluation of SCF-induced IL-25 and IL-25R expression.
Aanalysis of IgG1 and IgE levels in plasma
Anesthetized animals were bled by cardiac puncture. The syringes were
placed in ice and blood was allowed to coagulate. Samples were centri-
fuged for 18 min at 850 ? g and the supernatant was recentrifuged at
26,800 ? g for 30 min. The resulting serum was used to determine IgG and
IgE levels. IgG and IgE capturing and detection Abs were from R&D
Systems. The specific level was determined by direct ELISA on CRA-
coated 96-well plates as previously described using appropriate goat
5706SCF-INDUCED IL-25 IN ALLERGIC LUNG DISEASE
anti-mouse secondary Abs and reported as the mean absorbance at OD492
for comparison (32).
Data were evaluated by one-way ANOVA and, where appropriate, further
evaluated with the parametric Student-Newman-Keuls test for multiple
comparisons or the nonparametric Mann-Whitney rank-sum test.
Inhibition of SCF regulates Th2 cytokines and IL-25
responsiveness in the lung
In previous studies, blocking SCF in the airway demonstrated a
significant attenuation of airway hyperresponsiveness (AHR), eo-
sinophil accumulation, mucus overproduction, and collagen depo-
sition (2–4, 15, 16, 18). To better define these responses we ex-
amined the expression of Th2 cytokines and found the anti-SCF
treatment significantly reduced the mRNA and protein expression
of IL-4, IL-5, and IL-13 in the lungs of allergic mice (Fig. 1, A and
B). Interestingly, in these same mice the lymph node response was
also examined and no alteration of T cell-associated Th2 cytokines
was observed in animals treated with anti-SCF (data not shown).
We suggested that the anti-SCF treatment was altering locally re-
cruited cell populations that could influence or directly produce
Th2 cytokines. In this model of chronic allergen responses we
found no significant allergen-induced increase in IL-17 or IFN, and
no change was observed with the anti-SCF treatment (data not
shown). The data in Fig. 1C illustrate that during chronic allergen-
induced disease the leukocyte numbers increase within the lung,
and when animals were treated with anti-SCF the number of spe-
cific leukocyte subsets in the lung were significantly reduced, in-
cluding the c-kit?populations. Surprisingly, the number of T cells
was not significantly altered. In contrast, when we examined the
myeloid lineage cells, CD11b?mononuclear and granulocytic
(primarily eosinophils) populations, there was a significant reduc-
tion in these subsets. Examination of the histology demonstrated a
significant decrease in leukocyte accumulation in the lung (Fig.
1D), especially eosinophils, along with reduced intensity of mucus
and goblet cell presence as previously described (18). Finally, cor-
responding to the reduction in severity and Th2 cytokines, when
allergen-specific IgE and IgG1 was examined there was a signif-
icant reduction with allergic animals that were treated with anti-
SCF (Fig. 1E).
A recent set of studies has demonstrated that IL-25 can influence
both T and non-T cell populations to produce significant levels of
Th2 cytokines (25) and therefore may not entirely depend upon
Ag-driven responses. In the present studies the expression of IL-25
was investigated in lungs of chronic allergen-challenged animals.
There was a significant up-regulation of IL-25 during allergic re-
sponses in the lungs of challenged mice (Fig. 2), but not in drain-
ing lymph nodes during our studies (data not shown). When we
depleted SCF by local airway administration of neutralizing Ab we
observed a significant reduction in IL-25 expression, suggesting
that one of the consequences leading to attenuation of disease with
anti-SCF may be reduced IL-25-mediated responses. However,
these results were not clear on whether SCF directly or indirectly
drove IL-25 production.
Given the above data suggesting that blocking SCF locally
would reduce Th2 cytokines and IL-25 production, our studies
sought to verify that this was a direct mechanism in the lung. To
more definitively identify what cell population was producing the
cytokines, we sorted c-kit?and c-kit?cells from allergen-chal-
lenged mice. The mRNA was immediately isolated from the sorted
cells and it was determined whether they were the source of IL-25
and Th2 cytokines. The data clearly indicate that while the c-kit?
cell population was the source of IL-25, the c-kit- cell population
was the predominant source of the Th2 cytokines (Fig. 2B). Two
cell populations that primarily express c-kit and can play a central
role in promoting chronic allergic disorders are mast cells and
eosinophils. In particular, c-kit?eosinophils have been shown to
respond to SCF closely associated with the induction of Th2 cy-
tokines (15, 33). To further assess the possibility that eosinophils
are a source of IL-25, purified eosinophils (?97% pure) were stim-
ulated with SCF and the expression of IL-25 was assessed by real-
time PCR. Eosinophils significantly up-regulated IL-25 expression
when exposed to as little as 1 ng/ml SCF. We also grew mast cells
from bone marrow and assessed whether these cells expressed
IL-25 after SCF stimulation (Fig. 2D). No increase in IL-25 ex-
pression was observed after SCF stimulation in the mast cell cul-
tures. Additionally, when mast cells or eosinophils were incubated
with IL-25, no increase in Th2 cytokines, IL-4, IL-5, or IL-13, was
observed, and no surface expression of IL-17RB could be detected
by flow cytometry or by quantitative PCR (data not shown). To-
gether, these studies begin to outline the activation pathway re-
quired for SCF-mediated IL-25 generation in eosinophils and sug-
gest that they may be the predominant source of IL-25, but do not
respond to IL-25, during allergen-induced disease.
IL-25-induced Th2 cytokines in lungs and bone marrow of
We next assessed whether there were increases in IL-25-respon-
sive cells in the lungs of the chronic allergen-challenged mice
vs cells from naive mice, as previous studies have suggested
that IL-25 can directly induce Th2 cytokines in an Ag-indepen-
dent manner (20, 25). To understand this aspect, our experi-
ments used enzymatically dispersed lungs from naive or chronic
allergen-challenged mice as above. The dispersed cells were
then exposed to rIL-25 (10 ng/ml; R&D Systems) in the absence
of allergen, and the expression of Th2 cytokines was assessed.
Interestingly, when we examined IL-25-induced Th2 cytokines
there was a significant increase in the expression of Th2 cyto-
kines, IL-4, IL-5, and IL-13, in lung cells from chronic aller-
gen-challenged mice compared with cells from lungs of naive
animals (Fig. 3A). Isolated cells from lungs of allergic animals
treated with anti-SCF demonstrated a significant reduction in
the Th2 cytokines IL-5 and IL-13 compared with allergic ani-
mals treated with control Ab. However, the response was not
completely diminished in the anti-SCF-treated animals. An im-
portant issue to address is whether the IL-25-responsive cells
that are found in the lung during allergic responses are locally
or peripherally derived. A previous publication described that
IL-25-responsive cells can be identified in the bone marrow
(25). To examine whether the bone marrow was also altered
during the chronic allergic response in the lung, we isolated
cells from the long bones of the hind legs of the mice and
characterized their responsiveness to IL-25. The data in Fig. 3B
illustrate that when isolated bone marrow cells were stimulated
by IL-25 (10 ng/ml per 3 ? 106cells), there was a significant
up-regulation of IL-4 and IL-13 in the bone marrow from al-
lergic mice but not from naive mice. Additionally, when bone
marrow from allergic mice treated with anti-SCF as above was
stimulated with IL-25, significantly less IL-4 and IL-13 pro-
duction was observed. These data suggest that the allergic re-
sponse in the lungs altered the generation of IL-25-responsive
cells in the bone marrow and was dependent in part on the
production of pulmonary SCF. When we examined the plasma
level of SCF in chronic allergen-challenged mice, we observed
a significant increase in SCF levels compared with naive mice
that were significantly reduced when mice were treated with
5707 The Journal of Immunology
anti-SCF into the airway (34), further supporting a feedback
mechanism. Overall, these data thus far illustrate several im-
portant points: (1) IL-25 is up-regulated in the lung after
chronic allergen challenge in a SCF-dependent manner, (2) IL-
25-responsive cells are increased in the lung and bone marrow
during chronic allergic responses, (3) the IL-25-induced Th2
cytokine production could be mediated in an Ag-independent
manner, (4) IL-25 and Th2 cytokines are derived from two different
allergen-induced pulmonary disease. Chronic allergen-sensitized and -challenged mice were given control or anti-SCF purified IgG Abs into the airways
during the final two allergen challenge periods on a daily basis. Twenty-four hours after the final allergen challenge the lungs were harvested from the mice.
The lungs were processed for mRNA isolation and cytokines were assessed by real-time PCR analysis with fold increase calculated in comparison to levels
in lungs from naive mice (A). Cytokine protein levels were measured in lung homogenates processed using a lysis solution consisting of protease inhibitors
and subsequently subjected to Bioplex analysis (B). Twenty-four hours after the final chronic allergen challenge lungs from control Ab or anti-SCF-treated
animals were dispersed by collagenase to a single-cell suspension. Total cells counted and individual cell populations were characterized by flow cytometry
analysis using specific cell subset markers. Total numbers of individual subsets were calculated using the flow cytometry analysis and the total numbers
counted from individual mice (C). Lungs of individual mice were processed for histology, stained with H&E, and representative airways were photographed
at ?400 and ?1000 magnification (D). In correspondence to the reduction of Th2 cytokines allergen-specific serum IgE and IgG1 were significantly
reduced in anti-SCF-treated animals (E). ?, p ? 0.05 comparing anti-SCF-treated to control Ab-treated animals with five to seven mice per group.
Neutralization of SCF attenuates IL-4, IL-5, and IL-13 expression in the lungs (A) and significantly reduces inflammation (B) during chronic
5708SCF-INDUCED IL-25 IN ALLERGIC LUNG DISEASE
cell populations, and (5) eosinophils are potentially a significant SCF-
induced source of IL-25.
IL-25 exacerbates asthmatic disease and reconstitutes anti-SCF
attenuation of local responses
To further describe the relationship between SCF and IL-25, we
have recapitulated the model previously published where rIL-25
instilled into the airway can enhance the disease response (22,
23). In these studies rIL-25 (100 ng/mouse) was instilled into
the airways of allergic mice upon the final challenges of aller-
gen along with anti-SCF. The data demonstrate that when we
added IL-25 into allergic control mice, we observed the ex-
pected exacerbation of pathophysiologic disease. Additionally,
IL-25 administration reconstituted the AHR response in mice
that received anti-SCF, or an alternative view may be that the
anti-SCF treatment reduced IL-25-induced effects (Fig. 4A).
Analysis of the cytokine expression levels during the allergen-
induced responses demonstrated that administration of rIL-25
into the airways of the anti-SCF-treated animals reconstituted
the Th2 cytokine responses compared with control Ab-treated
allergic animals (Fig. 4B). Finally, when histology of the tissue
was examined, IL-25 clearly enhanced the disease based upon
chronic allergen challenge, and anti-SCF-treated animals (A). To identify the source of SCF-induced IL-25 in the lung chronic allergen-challenged animals
had dispersed lung cells separated by MACS cell sorting for c-kit 24 h postchallenge. Immediately after the sort, c-kit?or c-kit?populations were processed
for mRNA analysis for designated cytokines (B). Eosinophils were isolated from S. mansoni-infected mice (see Materials and Methods) and stimulated with
SCF and assessed for IL-25 expression by quantitative PCR (C). Bone marrow-derived mast cells were grown in vitro with rSCF (50 ng/ml) and rIL-3 (50
ng/ml) for 2 wk and stimulated with various doses of SCF (D). ?, p ? 0.05.
SCF regulates pulmonary IL-25 production in c-kit?eosinophils. Assessment of IL-25 expression levels was assessed in lungs of naive,
5709The Journal of Immunology
inflammation and mucus staining, while the coadministration of
IL-25 with anti-SCF showed an altered exacerbating response
(Fig. 4C). Taken together, these studies have further linked IL-
25-induced disease with the local overexpression of SCF within
the lung and together appear to play a significant role in the
progression of the disease.
Identification of IL-4-producing cells in the lungs of allergic
mice using 4get mice and expression of IL-25R
To further investigate the Th2 cytokine producing population in
the lung, we utilized 4get mice that possess a bicistronic expres-
sion of GFP along with IL-4 (IL-4-IRES-eGFP; 4get). Upon flow
cytometry characterization of GFP?cells in lungs of allergic an-
imals, distinct subsets were positive for GFP/IL-4 (Fig. 5). Rep-
resentative flow cytometry histograms demonstrate the distribution
of total lung cells (Fig. 5A, left panels) and IL-4?/GFP?lung cells
(Fig. 5A, right panels). The lower right panel clearly depicts IL-
4?CD11b?cells as the main source of IL-4 in lung of chronic
allergen-challenged animals. In our initial studies we compared a
cells. To begin to identify the direct target of IL-25, we isolated lungs cells
from naive or allergic animals treated them with rIL-25 in vitro (A). In
initial studies we examined the IL-25-induced Th2 cytokine expression
from the lung cells. The protein expression data were assessed using Bio-
plex proteomic analysis. Data represents means ? SE from five to six mice
per group. ?, p ? 0.05 comparing allergic to naive lung cells; #, p ? 0.05
comparing anti-SCF-treated to control Ab-treated animals. To assess the
global effect of SCF expression in lungs of allergic animals on IL-25-
induced Th2 cytokine responses bone marrow was assessed (B). Femurs
from naive, chronic allergen-challenged mice, or animals treated with anti-
SCF were flushed with PBS and isolated cells (2 ? 106cells/ml) were
stimulated in vitro with IL-25 (10 ng/ml). After 4 h mRNA expression was
assessed by quantitative PCR. Data represent means ? SE from four to five
mice per group. ?, p ? 0.05.
IL-25 induces Th2 cytokines directly from dispersed lung
tutes AHR responses (A) and the Th2 cytokine expression (B) in anti-SCF-
treated animals. Chronic allergen-challenged animals were given anti-SCF
and/or IL-25 during the final two allergens. AHR was measured at 24 h after
final challenges. In separate experiments, animals were assessed for Th2 cy-
tokines by isolating whole lung mRNA and performing quantitative PCR.
Histologic examination of H&E-stained lungs from allergen-treated mice in-
dicates a significant alteration in the inflammation in anti-SCF treated animals
that was enhanced and reconstituted by exogenous rIL-25 treatment (C). Stud-
ies were performed with five to eight mice per group. ?, p ? 0.05 comparing
anti-SCF treated animals to control Ab-treated group in the IL-25 expression
data; #, p ? 0.05 in rIL-25 plus anti-SCF compared with anti-SCF alone;
??, p ? 0.05 of IL-25 treatment compared with allergen only.
Exogenous administration of rIL-25 (100 ng/ml) reconsti-
5710 SCF-INDUCED IL-25 IN ALLERGIC LUNG DISEASE
chronic allergen-challenged animal with naive 4get mice and sur-
prisingly found that most of the cells that were IL-4?were
CD11b?, while only a minority of the cells were CD4?(Fig. 5B).
Subsequent experiments, illustrated in Fig. 6, were used to further
characterize the IL-4?, CD11b?myeloid cell population and il-
lustrate that none of the CD11b?cells was c-kit?, while half were
Ly6C?and nearly half were GR1?(?45%). Finally, when we
gated on the CD11b?/IL-4?population, ?25% of cells were
GR1?/Ly6C?(Fig. 6B). The chronic allergen-sensitized and
-challenged animals displayed a significant increase in GFP/IL-4?
cells in the lungs compared with naive animals, and these were
significantly reduced upon anti-SCF treatment (Fig. 6, A and C).
As demonstrated by the quantitative analysis in Fig. 6C, anti-SCF
significantly reduced the IL-4?cell populations in the lung with
every marker we examined. To assure ourselves that the GFP re-
porter expression driven by the IL-4 promoter correlates directly in
this model with IL-4 protein production, we utilized intracellular
staining for IL-4 of the cells from the lungs of chronic allergen-
treated animals (Fig. 6, D and E). The protein expression data
correlate extremely well with the IL-4/GFP reporter assessment.
Because earlier literature had implicated basophils and mast cells
as a significant source of Th2 cytokines, our analysis included
Fc?RI cells that would comprise basophils and mast cells (data not
shown). Only a small number of the IL-4?cells were Fc?RI?, and
they were not altered in the anti-SCF-treated animals, and none of
the IL-4? cells were c-kit?. Additional markers that were exam-
ined included CD11c and MHC class II, but neither of these mark-
ers displayed any correlation to the IL-4/GFP?population in the
lungs of allergic animals (data not shown). Additionally, the IL-
4?CD4?T cells were not reduced upon anti-SCF treatment in the
animals (data not shown). Overall, there appear to be several
CD11B?IL-4?populations in the lung based upon additional sur-
face marker staining.
Since our previous data had suggested that chronic allergen
challenges resulted in increased IL-25 responsiveness, we exam-
ined the expression of IL-17RB (IL-25R) using a recently de-
scribed specific mAb (30). The data illustrate that all of the IL-4?
IL-17RB?cells were CD11B?, with most expressing GR1 and
approximately half expressing Ly6C (Fig. 6F). When animals
were treated with anti-SCF, the total numbers of IL-17RB?cells
were significantly reduced (Fig. 6G), reflecting the apparent re-
sponsiveness to IL-25 restimulation above. These data further sug-
gest a correlation between chronic Th2 cytokine production, SCF,
and IL-25 responsiveness.
Because we also identified that there were IL-25-responsive
Th2 cytokine-producing cells in the bone marrow of allergic
animals, the expression patterns of these cells were also exam-
ined. The data in Fig. 7 indicate that compared with naive 4get
animals there was an up-regulation of IL-4 production in cells
from the bone marrow of allergic mice. Additionally, the ani-
mals treated with anti-SCF into the airway have a decreased
expression of the IL-4?cells, with the wild-type mice having
?5% and anti-SCF-treated mice having ?2% IL-4/GFP?cells
within the bone marrow (Fig. 7A). Analysis of the individual
markers again demonstrated a similar phenotype as in the lung,
with nearly all of the IL-4?cells being CD11B?(?95%), most
GR1?(?80%), some Ly6C?(?25%), and, unlike the lung,
many were c-kit?(?60%), perhaps suggesting a maturing phe-
notype on its way to losing c-kit expression once it migrates
into the lung (Fig. 7A). Additionally, when we examined Fc?R1
very little expression was observed in the IL-4?population,
similar to the lung cell data (data not shown). Thus, it appears
that a similar cell population is also increased in the bone mar-
row and that neutralization of SCF in airways of allergen-chal-
lenged mice reduces the number of the cell population. We also
examined the expression of IL-17RB?on the bone marrow-
derived cells and found that ?8% of the overall IL-4?cells in
the bone marrow expressed IL-17RB?, again at a lower per-
centage than those found in the lung (Fig. 7B). However, the
IL-17RB?cells were CD11B?, GR1?cells similar to the IL-
17RB?cells in the lung, but were also c-kit?, suggesting that
perhaps the cells mature before reaching the lung or once they
migrate into the lung. These data support the findings in Fig. 3B
demonstrating IL-25-induced Th2 cytokines in bone marrow of
chronic allergen-challenged mice. Overall, these data are strik-
ing, and while the maturation of this cell population is unclear,
it likely demarcates an important and potentially pathogenic
cell that can contribute to the allergic inflammation.
Previous studies have established a significant role for stem cell
factor during the development of allergic airway inflammation that
was associated with severity and disease progression (12, 16, 18,
35, 36). During allergic airway responses SCF appears to impact
the lungs of allergic mice. Using 4get (IL-4-GFP reporter) mice to identify
the cells that express IL-4, isolated cell populations from lungs of allergen-
challenged mice were assessed by flow cytometry in naive and chronic
allergen-challenged mice (A). The total cells that were positive for IL-4
were quantitated from individual mice (n ? 5 mice/group) and assessed for
significance in allergic compared with naive animals (B). Data represent
representative graphs from flow cytometry (A) or means ? SE from five
mice per group (B). ?, p ? 0.05 of allergen compared with naive animals.
Identification of IL-4?-responsive CD11b?myeloid cell in
5711The Journal of Immunology
mast cell activation, eosinophil accumulation and activation, as
well as having an effect on the airway remodeling and physiologic
responses. In the present studies the focus was on the cellular
mechanism of how SCF altered the ongoing responses in a ther-
apeutic model. While the IL-25 expression by eosinophils in re-
sponse to SCF may provide a pivotal disease-enhancing effect
creased in allergic mice and attenuated by
pulmonary neutralization of SCF. Further in-
vestigation for the Th2 cytokine-producing
myeloid cells was performed by gating on
IL-4?cells in dispersed lungs from chronic
allergen-challenged 4get animals. Most of
the IL-4?cells were CD11b along with a
subset of Ly6C?and GR1?cells as shown
in representative histograms (A). The quan-
titation of individual cells demonstrates that
a significant number of the IL-4?cells were
reduced by anti-SCF treatment (B). The enu-
meration of the IL-4?subsets of cells in
chronic allergen-treated animals was compared
with those in the anti-SCF-treated animals (C).
Expression of IL-4 protein examined by intra-
cellular cytokine staining correlates with GFP/
IL-4 expression from the GFP reporter upon
chronic allergen challenge as revealed by in-
tracellular staining (IL-4/PE Production) with
anti-IL-4 Abs (D). The enumeration of the
GFP reporter-positive cells and intracellular
IL-4 staining revealed equivalent numbers (E).
Further analyses of the chronic allergen-chal-
lenged IL-4?cells in 4get animals using an
IL-25R (IL-17RB)-specific Ab identified a
subset of cells that were primarily CD11B?
GR1?with a smaller subset of Ly6C?cells
(F). Quantitation of the cells within the lung
demonstrated that when chronic allergen-chal-
lenged mice were treated with anti-SCF a sig-
nificant decrease in the IL-25R?cells was ob-
served (G). The data in C and E represent
means ? SE from five mice per group.
IL-4?myeloid cells are in-
5712 SCF-INDUCED IL-25 IN ALLERGIC LUNG DISEASE
locally in the lungs during an allergen challenge, the most in-
teresting results pertain to the IL-4?myeloid cells that respond
to IL-25, resulting in Th2 cytokine production. Previous studies
have identified eosinophils as a potential source of IL-25 (27).
SCF-stimulated eosinophils have enhanced binding to VCAM-1
and fibronectin (33) and enhanced survival (17), and SCF also
can induce profibrotic and chemotactic mediators in eosinophils
(15) and now IL-25 production. In the present research a link
was made establishing a specific role of SCF/c-Kit activation
for the generation of IL-25, leading to the progression and se-
verity of chronic allergic airway disease. While a number of
studies have recently been published that demonstrate that
IL-25 has a significant role in the production of Th2 cytokines
and influences the severity of inflammatory Th2-associated dis-
ease models, the mechanism of activation and progression is not
completely clear (21–23, 27, 37). Studies have demonstrated
that IL-25 induces enhanced Th2 cytokine production through
at least two different cell populations. An Ag-specific T cell
response is enhanced by IL-25 and induces additional Th2 cy-
tokines to be produced, as well as augments Th2 cell polariza-
tion and memory (23, 27). A second bone marrow-derived
non-T cell population also appears to respond to IL-25 and pro-
duce cytokines (25). These latter data help to define the mech-
anism of how IL-25 directly induced Th2 cytokines when in-
jected in vivo in the original studies (20) and in a recent study
when IL-25 was directly injected into the airway of mice (30).
The recognition that SCF influences the production of Th2 cy-
tokines in the lung is reflected by previous studies using locally
delivered anti-SCF for alteration of severe asthma-like re-
sponses (16, 18, 35). This latter aspect is also highlighted by
our findings indicating that anti-SCF does not alter lymph node-
associated Th2 cytokine production (data not shown), a finding
in the lymph node that was also demonstrated when IL-25
blockade was used (21).
gic 4get mice have increased IL-4?
myeloid cells compared with naive
animals with a similar phenotype as
those found in the lung and also ex-
press c-kit (A). IL-25R?cells in the
bone marrow are CD11B?GR1?c-
kit?cells with some also expressing
Ly6C (B). Femurs from chronically
challenged animals were flushed with
saline and cells were assessed by flow
cytometry for the expression of cellu-
lar markers to distinguish cellular
subsets. Histograms are representative
of three to four mice per group and
reflect differences in IL-4?cells in al-
lergic compared with naive animals.
Quantitation of the bone marrow cells
is expressed as percentage of total
based upon specific staining (C). Data
represent means ? SE from three to
four mice per group.
Bone marrow of aller-
5713 The Journal of Immunology
While we have not compared the relative level of Th2 cyto-
kines that each IL-4?cell population produces, most of the
IL-4?cells within the lung appear to be CD11B?myeloid cells
of varying phenotype based upon surface protein expression
assessed by flow cytometry. The use of an IL-25R-specific Ab
(30) identified a subset of the myeloid cell population that can
produce IL-4 with a discrete cell population that was CD11B?
GR1?and c-kit?in the lung after chronic allergen challenge. A
striking result was the downstream effects found in the bone
marrow during allergic responses where the generation of the
IL-25-responsive cells was reduced when SCF was blocked in
the lung. These cells were phenotypically similar to those found
in the lung except that they also expressed c-kit. Thus, the ef-
fects of SCF appear to be global in nature, even though the
protein appears to be made locally in response to allergen chal-
lenge. These results are consistent with the generation of IL-
25-responsive cells in the bone marrow during helminth-in-
duced Th2 responses that demonstrated a c-kit?myeloid cell
capable of producing IL-4 (25). We would suggest that in the
previous publication, which utilized a very immunogenic, com-
paratively acute parasitic response, that the c-kit?population
from the bone marrow accumulated in tissue, whereas our
chronic allergen challenge led to enhanced SCF production lo-
cally in the lung, leading to a more mature cell phenotype. It
might be intriguing to examine whether longer treatment pro-
tocols might help to eliminate this population that persisted in
the lung that is likely derived from the c-kit?bone marrow
population. A recent study has identified an IL-13-producing
myeloid cell that is MHC class II?during persistent viral in-
fections (29), while another finding indicated that c-kit?(SCF
receptor) dendritic cells promote Th2 and Th17 cytokine asso-
ciated with allergic disease (38, 39). It is tempting to speculate
that these are related populations to the one we have identified
in a differentially activated immune environment. While bone
marrow-derived cells express Th2 cytokines in response to IL-
25, we would suggest that the cells depend upon SCF to allow
maturation and/or skewing toward Th2 cytokine producing
cells. It would follow that when SCF was blocked locally in the
lung that there would be reduced local Th2 cytokines due to a
reduction in IL-25.
The intriguing finding that pulmonary generated SCF influ-
ences the development of an IL-25-responsive cell population
in the bone marrow may demonstrate an important feedback
mechanism. While we have not thoroughly examined other po-
tential signals, it may be that the generation of the IL-25-re-
sponsive cell populations depend upon additional cytokines,
such as IL-4 or IL-25 itself, that are also needed to establish the
proper immune environment. Since SCF also influences the
generation of chemokines during allergen-induced responses
(16, 17, 40), the recruitment of the IL-25-responsive cell from
the bone marrow to the lungs may also be altered upon local
blockade of SCF. A previous study identified that an IL-25-
responsive cell population that produced Th2 cytokines was
CCR2- and CCR3-positive (25), corresponding to chemokines
that are highly up-regulated during Th2-type responses, that is,
CCL2 and CCL11 family chemokines. These data along with
the finding that many of these cells have a CD11b?GR1?
(Ly6C?/?) “inflammatory” monocyte phenotype suggest that
perhaps this cell population may be present in multiple inflam-
matory sites, including the gut and bladder (41, 42). This phe-
notypic designation would predict that they should respond to
CCR2 ligands for migration into the lungs as originally defined
(43). Whether additional maturation signals are needed for the
cell to obtain the ability to respond to IL-25 and/or produce Th2
cytokines is an area for further investigation.
Most studies have examined T cells as a focus of IL-25 re-
sponsiveness. Studies have suggested that IL-25 augments the
skewing of Th2 cells by not only direct effects on the T cell but
also by indirect effects on the APC (21, 22, 23, 27). This re-
sponse has been coordinated with other factors that appear to
affect the nature of the response, such as TSLP and IL-13. In-
terestingly, the most profound effects of IL-25 are found in the
presence of APC and specific Ag, further supporting both direct
and indirect effects on T cells. In the present studies, our anal-
ysis of CD4 T cells indicated that a relatively small percentage
of the lung CD4 cells were also IL-4?, which may represent
differentiated Th2-type cells (27). Although the source of IL-25
has not been fully elucidated, it can be produced by a number
of cell populations. While we did not detect IL-25 in bone mar-
row-derived mast cells stimulated with SCF, they have been
described to produce IL-25 by other stimuli (44). A recent study
has suggested that epithelial cells produce IL-25 in response to
innate signals from allergens (22). This latter observation may
be especially relevant during respiratory allergen and virus-in-
duced exacerbations within a Th2 environment to activate IL-25
production, leading to subsequent exacerbation by Th2-produc-
ing myeloid cells. How these responses correlate to the findings
in the present studies with blockade of SCF is unclear, but they
suggest that there are likely multiple sources of IL-25 that could
exacerbate the Th2 environment. Interestingly, the inhibition
observed by SCF blockade could be overcome by the addition
of rIL-25 into the airway. While the numbers of IL-4?T cells
were not significantly altered with anti-SCF and the number of
IL-4/GFP?cells did not increase as significantly as did the
myeloid cell population during chronic allergen challenge, the
T cells likely produce much higher levels of Th2 cytokine on a
per cell basis. It may be that during exacerbations the increased
number of Th2 cytokine-producing myeloid cells adds a signif-
icant level of additional cytokine, resulting in the most severe
Taken together, these studies demonstrate an important mecha-
nistic role for SCF that correlates to observations in human asthma
where an increased expression of SCF in the lung and in serum
after allergen challenges was observed and correlated to the se-
verity of disease (11–14). Thus, a complex network of activation
may be emerging with SCF as an important initiating cytokine
locally in the lung with its ability to activate IL-25 production
during allergic responses as well as contribute to the maturation of
the IL-17RB?cells in the lung and/or bone in a Th2 immune
environment. A key feature is production of IL-25 by SCF-stim-
ulated eosinophils, which are only present during allergic disease.
While there appear to be multiple populations of IL-4-producing
cells in the lung, the IL-17RB?cells appear to be CD11b?GR1?.
This overall concept supports the role of SCF for local lung as well
as bone marrow-associated effects influencing the overall re-
sponses. It is now reasonable to speculate that targeting SCF in the
airway during allergic asthma could provide an effective treatment
for reducing IL-25 and Th2 cytokines and therefore allergic asth-
The authors acknowledge the outstanding artwork of Robin Kunkel.
The authors have no financial conflicts of interest.
5714SCF-INDUCED IL-25 IN ALLERGIC LUNG DISEASE
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5715The Journal of Immunology