Mast cells can promote the development of multiple features of chronic asthma in mice.
ABSTRACT Bronchial asthma, the most prevalent cause of significant respiratory morbidity in the developed world, typically is a chronic disorder associated with long-term changes in the airways. We developed a mouse model of chronic asthma that results in markedly increased numbers of airway mast cells, enhanced airway responses to methacholine or antigen, chronic inflammation including infiltration with eosinophils and lymphocytes, airway epithelial goblet cell hyperplasia, enhanced expression of the mucin genes Muc5ac and Muc5b, and increased levels of lung collagen. Using mast cell-deficient (Kit(W-sh/W-sh) and/or Kit(W/W-v)) mice engrafted with FcRgamma+/+ or FcRgamma-/- mast cells, we found that mast cells were required for the full development of each of these features of the model. However, some features also were expressed, although usually at less than wild-type levels, in mice whose mast cells lacked FcRgamma and therefore could not be activated by either antigen- and IgE-dependent aggregation of Fc epsilonRI or the binding of antigen-IgG1 immune complexes to Fc gammaRIII. These findings demonstrate that mast cells can contribute to the development of multiple features of chronic asthma in mice and identify both Fc Rgamma-dependent and Fc Rgamma-independent pathways of mast cell activation as important for the expression of key features of this asthma model.
- SourceAvailable from: Riccardo Sibilano[Show abstract] [Hide abstract]
ABSTRACT: Mast cells (MCs) are cells of hematopoietic origin that normally reside in mucosal tissues, often near epithelial cells, glands, smooth muscle cells, and nerves. Best known for their contributions to pathology during IgE-associated disorders such as food allergy, asthma, and anaphylaxis, MCs are also thought to mediate IgE-associated effector functions during certain parasite infections. However, various MC populations also can be activated to express functional programs-such as secreting preformed and/or newly synthesized biologically active products-in response to encounters with products derived from diverse pathogens, other host cells (including leukocytes and structural cells), damaged tissue, or the activation of the complement or coagulation systems, as well as by signals derived from the external environment (including animal toxins, plant products, and physical agents). In this review, we will discuss evidence suggesting that MCs can perform diverse effector and immunoregulatory roles that contribute to homeostasis or pathology in mucosal tissues.Mucosal Immunology advance online publication, 11 February 2015; doi:10.1038/mi.2014.131.Mucosal Immunology 02/2015; · 7.54 Impact Factor
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ABSTRACT: Mast cells in tissues are developed from mast cell progenitors emerging from the bone marrow in a process highly regulated by transcription factors. Through the advancement of the multicolor flow cytometry technique, the mast cell progenitor population in the mouse has been characterized in terms of surface markers. However, only cell populations with enriched mast cell capability have been described in human. In naïve mice, the peripheral tissues have a constitutive pool of mast cell progenitors. Upon infections in the gut and in allergic inflammation in the lung, the local mast cell progenitor numbers increase tremendously. This review focuses on the origin and development of mast cell progenitors. Furthermore, the evidences for cells and molecules that govern the migration of these cells in mice in vivo are described.Molecular Immunology 03/2014; · 3.00 Impact Factor
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ABSTRACT: Although mast cells have long been known to play a critical role in anaphylaxis and other allergic diseases, they also participate in some innate immune responses and may even have some protective functions. Data from the study of mast cell-deficient mice have facilitated our understanding of some of the molecular mechanisms driving mast cell functions during both innate and adaptive immune responses. This review presents an overview of the biology of mast cells and their potential involvement in various inflammatory diseases. We then discuss some of the current pharmacological approaches used to target mast cells and their products in several diseases associated with mast cell activation.Pharmacology [?] Therapeutics 01/2014; · 7.75 Impact Factor
? The?Journal?of?Clinical?Investigation http://www.jci.org Volume 116 Number 6 June 2006
Mast cells can promote the development
of multiple features of chronic asthma in mice
Mang Yu,1 Mindy Tsai,1 See-Ying Tam,1 Carol Jones,1 James Zehnder,1 and Stephen J. Galli1,2
1Department of Pathology and 2Department of Microbiology and Immunology, Stanford University School of Medicine, Stanford, California, USA.
increased?levels?of?lung?collagen.?Using?mast?cell–deficient?(KitW-sh/W-sh and/or KitW/W-v)?mice?engrafted?with?
Asthma is a complex inflammatory disorder that is becoming
increasingly prevalent, particularly in the developed world (1–3).
Once thought to be mainly a disorder of airway smooth muscle
reactivity, asthma now is recognized to be a syndrome associat-
ed with persistent chronic inflammation of the airways (1–3). It
has also become apparent that long-standing asthma can result
in changes in the airways themselves, called “airway remodeling”
(1–5). Thus, the spectrum of findings in this disorder is thought to
include increased numbers of mucus-secreting goblet cells in the
airway epithelium, associated with excessive production of mucus,
and increased deposition of collagen in the lung as well as airway
hyperresponsiveness (AHR) to immunologically nonspecific ago-
nists of bronchoconstriction and chronic inflammation of the air-
way mucosa, with cellular infiltrates of lymphocytes, eosinophils,
and other inflammatory leukocytes (1–8).
In many patients, asthma has an allergic or atopic component,
characterized by allergic sensitivity to environmental allergens and
increased serum levels of antigen-specific and total IgE antibodies
(1–3). Accordingly, attempts to understand the pathophysiology of
asthma often focus on the effector cells of allergic inflammation,
including the mast cell (MC). MCs are derivatives of hematopoietic
progenitors that differentiate and mature locally in most tissues,
especially those that are exposed to the external environment, such
as the airways (9–11). Moreover, some studies have reported that
MCs are present in increased numbers in the airways of patients
with asthma (8, 12, 13).
While the mechanisms responsible for the increased numbers
of MCs in the airways of some asthmatic patients have not been
defined, there is no doubt that MCs have the potential to influence
multiple aspects of the pathology and pathophysiology of asthma.
For example, MCs express the high affinity receptor for IgE (i.e.,
FcεRI) on their surface and therefore can bind IgE (14–16). Upon
exposure to di- or multivalent antigen recognized by FcεRI-bound
IgE, these FcεRI undergo aggregation, initiating a complex process
of signal transduction that culminates in the secretion of a broad
spectrum of proinflammatory mediators, cytokines, and chemo-
kines (11, 14–16). In the mouse, the FcR γ chain (FcRγ) is shared by
FcεRI and FcγRIII (17), and MCs can be activated to release media-
tors via Ag-, IgG1-, and FcγRIII-dependent mechanisms (17–22)
as well as by Ag-, IgE-, and FcεRI-dependent mechanisms (14–16).
These MC-derived products can have diverse effects in the air-
ways, including those that might, at least in part, contribute to
multiple features of this disorder (1, 8, 13, 23, 24). Thus, individual
MC mediators, cytokines, or chemokines can promote AHR and
chronic allergic inflammation, goblet cell hyperplasia, enhanced
mucin gene expression and mucus secretion, and collagen depo-
sition (1, 8, 13, 23, 24). Moreover, patients with active asthma
or asthma patients challenged via the airways with allergens to
which they have sensitivities exhibit evidence of MC activation,
such as increased levels of MC-derived products in bronchoalveo-
lar lavage (BAL) fluid (13, 24). Finally, histological signs of airway
MC degranulation in vivo can be identified in biopsy specimens
derived from asthmatic patients (6, 13, 24).
Yet it is not at all clear to what extent MCs and IgE-dependent
MC activation actually are important for the long-term pathol-
ogy of asthma. First, a number of other inflammatory cells,
including T lymphocytes, macrophages, eosinophils, basophils,
and neutrophils, have also been implicated as potential effector
cells in asthma (6, 8, 25–28). However, in humans, it can be dif-
ficult to ascertain the individual contributions of each of these
potential effector cells to the pathology of the disorder. Second,
while attempts to treat asthma by reducing levels of IgE with the
humanized anti-IgE antibody omalizumab have produced clinical
benefit in some patients, the magnitude of such benefit often is
Nonstandard?abbreviations?used: AHR, airway hyperresponsiveness; BAL, bron-
choalveolar lavage; BMCMC, bone marrow–derived cultured mast cell;
BMCMCs→KitW/W-v, BMCMC-engrafted KitW/W-v mice; Cdyn, dynamic compliance;
MC, mast cell; Penh, enhanced respiratory pause; RL, lung resistance.
Conflict?of?interest: The authors have declared that no conflict of interest exists.
Citation?for?this?article: J. Clin. Invest. 116:1633–1641 (2006). doi:10.1172/JCI25702.
1634? The?Journal?of?Clinical?Investigation http://www.jci.org Volume 116 Number 6 June 2006
limited (29, 30). The extent to which the relatively modest clinical
results obtained with omalizumab reflect an inability to reduce
IgE levels to sufficiently low levels, as opposed to the presence of
mechanisms of disease pathogenesis that operate independently
of IgE, is not clear.
Studies of asthma models in mice can exploit the power of genet-
ic approaches in analyzing how the presence or absence of poten-
tial effector elements can influence the expression of the various
features of the models (23, 31). Such work has implicated MCs in
the expression of certain features that develop in relatively short-
term models of asthma, including AHR and eosinophil-containing
inflammatory infiltrates in the lung (32–34). However, in other
short-term models of asthma, which typically involve sensitization
with antigen administered together with an adjuvant such as alu-
minum hydroxide, AHR and eosinophil infiltration can be elicited
in animals that genetically lack MCs (23, 25, 33, 35).
Even though humans with allergic asthma are typically exposed
to allergens repeatedly over long periods of time, only a few stud-
ies of mice have investigated protocols of allergen challenge that
attempt to mimic this pattern of exposure (36–38). Moreover,
all of the latter mouse studies used sensitization protocols that
included artificial adjuvants. We developed a long-term protocol
for i.n. antigen challenge of mice that had been sensitized without
artificial adjuvant and that?results in the development of many
of the features of chronic asthma. We then used that model to
investigate the extent to which MCs and MC activation by FcRγ-
dependent mechanisms are required for optimal development of
multiple features of chronic asthma in mice.
Airway responses to antigen or methacholine are largely MC dependent. In
our chronic asthma model, OVA-sensitized wild-type (Kit+/+) mice
exhibited increased enhanced respiratory pause (Penh) respons-
es to aerosolized methacholine administered 24 hours after the
eighth OVA challenge compared with responses in PBS-treated
WBB6F1-Kit+/+ mice or OVA- or PBS-treated MC-deficient WBB6F1-
KitW/W-v mice (Figure 1A). However, WBB6F1-KitW/W-v mice that
had been selectively engrafted with WBB6F1-Kit+/+ bone marrow–
derived cultured MCs (BMCMCs) (Kit+/+ BMCMCs→KitW/W-v mice)
exhibited responses that were statistically indistinguishable from
those in Kit+/+ mice (Figure 1A). Very similar results were obtained
when such experiments were repeated using C57BL/6-Kit+/+,
C57BL/6-KitW-sh/W-sh, and C57BL/6-Kit+/+ BMCMCs→KitW-sh/W-sh
mice (Supplemental Figure 1A; supplemental material available
online with this article; doi:10.1172/JCI25702DS1).
Thus, in this model, airway responses to methacholine appeared to
be largely or entirely MC-dependent in either WB × C57BL/6 F1 (i.e.,
WBB6F1) or C57BL/6 mice. Indeed, the role of MCs in the enhance-
ment of Penh responses to methacholine in this chronic model
appeared to be even greater than that which we reported in a more
acute model of asthma, which also was elicited in mice that had been
sensitized to OVA in the absence of artificial adjuvant (33).
We also measured airway responses to antigen or PBS. Individ-
ual OVA-sensitized Kit+/+ or Kit+/+ BMCMCs→KitW/W-v mice exhib-
ited a biphasic response to the ninth OVA challenge, with peaks
of responsiveness at approximately 3 or 6 hours and at approxi-
mately 9 or 12 hours; the responses were substantially reduced by
24 hours after OVA challenge (Supplemental Figure 2). When data
from individual mice were pooled, the biphasic response to OVA
was still evident in OVA-sensitized Kit+/+ mice but not in OVA-sen-
sitized Kit+/+ BMCMCs→KitW/W-v mice (Figure 1B). However, peak
airway responses in these 2 groups were quite similar in magnitude.
In contrast, there were no detectable responses to OVA in OVA-sen-
sitized KitW/W-v mice, nor did any group of PBS-treated mice exhibit
a response to PBS challenge (Figure 1B). Very similar results were
obtained when the same experiments were performed in C57BL/6-
Kit+/+, C57BL/6-KitW-sh/W-sh, and C57BL/6-Kit+/+ BMCMCs→KitW-sh/W-sh
mice (Supplemental Figure 1B).
Thus, airway responses induced by OVA challenge in OVA-sensitized
KitW/W-v or KitW-sh/W-sh mice, like Penh responses to methacholine after
OVA challenge in such mice, were largely or entirely MC dependent.
To assess specifically airway responses distal to the trachea, we
also performed invasive measurements of lung resistance (RL) and
dynamic compliance (Cdyn) in mice treated with aerosolized metha-
choline 24 hours after the ninth challenge with OVA or PBS. OVA-
sensitized Kit+/+ or Kit+/+ BMCMCs→KitW/W-v mice exhibited simi-
lar levels of AHR after OVA challenge whereas the OVA-sensitized
MC-deficient KitW/W-v mice showed responses that were statistically
indistinguishable from those of the PBS-treated control groups
(Figure 1, C and D). Thus, our findings using invasive measure-
ments of lung function to assess AHR in anesthetized, tracheosto-
mized mice are basically in accord with those we obtained by using
Penh to assess airway responses in conscious mice.
Airway responses following i.n. OVA challenge in a mouse model of
chronic asthma. (A) Penh responses to aerosolized methacholine 24
hours after the eighth OVA or PBS challenge. (B) Penh measured 1
hour before and 1, 3, 6, 9, 12, 15, 18, 21, and 24 hours after the ninth
OVA or PBS challenge. (C) Changes in RL and (D) lung Cdyn after
aerosolized methacholine administered 24 hours after the ninth OVA
or PBS challenge. Data are from OVA-sensitized/challenged WBB6F1-
Kit+/+ (filled circles), WBB6F1-KitW/W-v (open circles), and WBB6F1-Kit+/+
BMCMCs→KitW/W-v (gray circles) mice and PBS-treated WBB6F1-Kit+/+
(filled squares), WBB6F1-KitW/W-v (open squares), and WBB6F1-Kit+/+
BMCMCs→KitW/W-v (gray squares) mice. *P < 0.05 versus correspond-
ing PBS-treated controls; †P < 0.05 versus OVA-sensitized/challenged
KitW/W-v group. n = 8 per group (A and B); n = 4 per group (C and D).
? The?Journal?of?Clinical?Investigation http://www.jci.org Volume 116 Number 6 June 2006
Antibody responses in this model are MC independent. MCs can be
activated by antigen to secrete mediators by both IgE/FcεRI- and
IgG/FcγRIII-dependent mechanisms (14–22). Such secreted prod-
ucts include cytokines that?can regulate the production of IgE and
other antibodies (23, 39, 40). MCs can also express other functions
that have the potential to influence the development of adaptive
immune responses (23, 39, 40). Accordingly, it has been proposed
that MCs might function in part to enhance levels of antibody pro-
duction during some acquired immune responses (23, 39, 40).
We found that OVA sensitization resulted in the development of
significant antigen-specific IgE and IgG1 responses, but the lev-
els of these antibodies measured at the end of our experiments
in groups of OVA-sensitized Kit+/+, KitW/W-v, or Kit+/+ BMCMCs→
KitW/W-v mice were statistically indistinguishable (Supplemental
Figure 3).?Thus, the differences in airway responses to OVA or in
the AHR to methacholine among OVA-sensitized Kit+/+, KitW/W-v, or
Kit+/+ BMCMCs→KitW/W-v mice cannot be explained by differences
in levels of OVA-specific IgE or IgG1 antibodies.
This asthma model is associated with increased numbers of MCs and MC-
dependent lung inflammation. OVA treatment resulted in significant
increases in the numbers of MCs in the lungs of the WBB6F1-Kit+/+
mice, including the appearance of intraepithelial MCs (Figures 2A
and 3A); similar findings were observed in the C57BL/6-Kit+/+ mice
(Supplemental Figure 4A and data not shown). These findings have
also been reported in some patients with asthma (13). OVA treatment
also induced other features of allergic inflammation, such as promi-
nent infiltrates of leukocytes, including lymphocytes and eosinophils
(Figures 2, D and G, and 3, B and D); again, similar findings were
observed in C57BL/6-Kit+/+ mice (Supplemental Figure 4, B and D).
In certain settings, KitW/W-v mice can develop MC populations
by mechanisms that do not require normal signaling via the c-Kit
receptor (11). However, the lungs of OVA-treated
KitW/W-v mice (Figures 2B and 3A) or KitW-sh/W-sh mice
(Supplemental Figure 4A) remained completely
devoid of MCs. In contrast, lung MC numbers
in OVA- or PBS-treated Kit+/+ BMCMCs→KitW/W-v
mice were statistically indistinguishable from
those in Kit+/+ mice (Figures 2C and 3A); simi-
lar findings were observed in Kit+/+ BMCMCs→
KitW-sh/W-sh mice (Supplemental Figure 4A).
In OVA-sensitized/challenged WBB6F1-Kit+/+
mice, the ninth OVA challenge resulted in a rapid
rise in the levels of histamine detectable in the
serum, with levels remaining significantly elevated over baseline
(i.e., pre-OVA challenge) values even 24 hours after OVA challenge
(Supplemental Figure 5 and Figure 3C). In OVA-sensitized/chal-
lenged mice, serum histamine measured 24 hours after the last
OVA challenge was not only significantly elevated in Kit+/+ mice,
but also, albeit to a lesser extent, in Kit+/+ BMCMCs→KitW/W-v mice;
in contrast, only very low and statistically indistinguishable levels
of serum histamine were detected in OVA- or PBS-treated KitW/W-v
mice (Figure 3C); similar findings were observed in C57BL/6-
Kit+/+, C57BL/6-KitW-sh/W-sh, and C57BL/6-Kit+/+ BMCMCs→
KitW-sh/W-sh mice (Supplemental Figure 4C). The corresponding
values (serum histamine 24 hours after challenge) for OVA-
sensitized, PBS-challenged WBB6F1-Kit+/+, WBB6F1-KitW/W-v, or
WBB6F1-Kit+/+ BMCMCs→KitW/W-v mice were 31.4 ± 4.2, 6.0 ± 1.0,
and 28.7 ± 4.6 nM, respectively. In naive WBB6F1-Kit+/+, WBB6F1-
KitW/W-v, or WBB6F1-Kit+/+ BMCMCs→KitW/W-v mice, serum hista-
mine levels were 25.9 ± 5.1, 5.1 ± 1.2, and 23.6 ± 3.4 nM, respec-
tively. These results indicate that, in this setting, serum histamine
is derived solely or largely from MCs and/or is derived from other
cellular sources in an MC-dependent manner.
Kit+/+ and Kit+/+BMCMCs→KitW/W-v mice were also very similar in
measurements of the inflammation associated with responses to
OVA sensitization and challenge, with markedly increased numbers
of inflammatory cells in the lungs versus only minimal responses in
KitW/W-v mice (Figures 2, D–I, and 3, B and D). OVA-treated Kit+/+ or
Kit+/+ BMCMCs→KitW/W-v mice also exhibited significant elevations
in BAL fluid monocytes, macrophages, neutrophils, eosinophils, and
lymphocytes (Figure 3D). While there were some differences in the
number of individual types of leukocytes in the BAL fluid of antigen-
challenged Kit+/+ versus Kit+/+ BMCMCs→KitW/W-v mice, taken togeth-
er, the data in Figures 2, D–I, and 3, B and D, demonstrate that MCs
Histology of lungs of OVA-sensitized/challenged mice
24 hours after the ninth OVA challenge. (A–C) Tolu-
idine blue stain demonstrating MCs (MCs within the
epithelium are indicated by arrows in A and C; a sub-
mucosal MC is indicated by an arrowhead in A). Scale
bar: 10 μm. (D–F) H&E staining showing perivascu-
lar and peribronchial infiltrates of inflammatory cells.
Scale bar: 50 μm. (G–I) Congo red stain showing
eosinophils (some indicated by arrows) and lympho-
cytes in the inflammatory infiltrates. Scale bar: 20 μm.
(J–L) Masson trichrome stain showing hyperplasia of
mucin-secreting goblet cells and subepithelial fibrosis.
Black arrows indicate goblet cells, open arrows indi-
cate collagen (stained blue), and arrowheads indicate
airway smooth muscle. Scale bar: 15 μm.
1636? The?Journal?of?Clinical?Investigation http://www.jci.org Volume 116 Number 6 June 2006
are largely responsible for the antigen-induced leukocyte recruitment
and chronic inflammation in this model. The same conclusion was
supported when the same experiments were repeated using C57BL/6-
Kit+/+, C57BL/6-KitW-sh/W-sh, and C57BL/6-Kit+/+ BMCMCs→KitW-sh/W-sh
mice (Supplemental Figure 4, B and D).
Activation of MCs via IgE or, in the mouse, IgG1 antibodies
requires signaling through the FcRγ chain (FcRγ) that is shared by
FcεRI and FcγRIII (14, 17). We found that levels of FcRγ mRNA in
lungs obtained 24 hours after the last challenge with OVA or PBS
were highly upregulated in OVA-treated Kit+/+ or Kit+/+ BMCMCs→
KitW/W-v mice but not in MC-deficient KitW/W-v mice (Figure 4A); sim-
ilar results were obtained in C57BL/6-Kit+/+, C57BL/6-KitW-sh/W-sh,
and C57BL/6-Kit+/+ BMCMCs→KitW-sh/W-sh mice (Supplemental Fig-
ure 6). Although FcRγ is not restricted to MCs (41), these results
are in accord with our findings that levels of tissue MCs and other
hematopoietic cells are much more markedly increased in the
lungs of OVA-challenged as opposed to PBS-challenged wild-type,
Kit+/+ BMCMCs→KitW/W-v, or Kit+/+ BMCMCs→KitW-sh/W-sh mice than
in OVA-challenged MC-deficient KitW/W-v or KitW-sh/W-sh mice (Fig-
ures 2, A–I, and 3, A, B, and D; Supplemental Figure 4, A, B, and
D). We also quantified levels of a transcript which is thought to
be restricted to T cells, i.e., T cell–associated GTPase (42), and found
that levels of this mRNA were also highly upregulated in the lungs
of OVA-treated Kit+/+ or Kit+/+ BMCMCs→KitW/W-v mice but not in
MC-deficient and KitW/W-v mice (Figure 4B).
Taken together, these quantitative RT-PCR results are consistent
with our histological findings, which showed that OVA treatment
was associated with increased numbers of MCs (Figures 2, A and C,
and 3A; Supplemental Figure 4A) and striking lung infiltrates of
lymphocytes, as well as of other leukocytes (Figure 2, D, F, G, and
I, and data not shown).
MC-dependent enhancement of airway goblet cell hyperplasia, mucin
gene expression, and collagen deposition. Quantitative RT-PCR analysis
demonstrated a highly MC-dependent upregulation of expression
of genes encoding mucins 5AC and 5B, which are the major com-
ponents of mucus secreted by goblet cells and submucosal glands,
respectively (7, 43) (Figure 5, A and B; Supplemental Figure 7, A
and B). These findings are consistent with our histological obser-
vations, which revealed that the striking increases in numbers of
mucus-secreting goblet cells in the airway epithelium of OVA-
treated mice were largely or fully MC-dependent (Figures 2, J–L,
and 5C; Supplemental Figure 7C).
Features of allergic inflammation in this
chronic asthma model 24 hours after the
ninth OVA or PBS challenge. (A and B) Num-
bers of lung MCs (A) and eosinophils (B).
(C) Serum histamine concentration. (A–C)
White bars, PBS-treated group; black bars:
OVA-sensitized/challenged group. **P < 0.01
or ***P < 0.001 versus corresponding PBS
controls (n = 6); ††P < 0.01 or †††P < 0.001
versus group indicated (n = 6 per group). ND,
not detected. (D) Numbers of leukocytes in
BAL fluid from the right lungs of WBB6F1-
Kit+/+ (black bars), WBB6F1-KitW/W-v (white
bars), and Kit+/+ BMCMC→KitW/W-v (gray
bars) mice following OVA sensitization/chal-
lenge (O) or PBS treatment (P). **P < 0.01 or
***P < 0.001 versus corresponding PBS con-
trols (n = 8 per group); †P < 0.05, ††P < 0.01,
or †††P < 0.001 versus group indicated (n = 8
per group). MON, monocytes; MAC, macro-
phages; PMN, neutrophils; EOS, eosinophils;
Lung mRNA levels of genes encoding (A) FcRγ chain and (B) T cell–
specific GTPase 24 hours after the ninth OVA or PBS challenge. White
bars, PBS-treated group; black bars, OVA-sensitized/ challenged group.
***P < 0.001 versus corresponding PBS controls (n = 6 per group);
†††P < 0.001 versus group indicated (n = 6 per group). Eq, equivalents.
? The?Journal?of?Clinical?Investigation http://www.jci.org Volume 116 Number 6 June 2006
Masson trichrome staining revealed that OVA treatment resulted
in enhanced deposition of collagen in the airways of Kit+/+ or Kit+/+
BMCMCs→KitW/W-v mice but had little or no such effect in the air-
ways of KitW/W-v mice (Figure 2, J–L); similar results were observed in
C57BL/6-Kit+/+, C57BL/6-KitW-sh/W-sh, and C57BL/6-Kit+/+ BMCMCs→
KitW-sh/W-sh mice (data not shown). Much of this enhanced collagen
deposition was localized in the airways immediately below the air-
way epithelium, a location that also exhibited what appeared to be
an increased amount of airway smooth muscle (Figure 2, J and L).
Quantification of lung hydroxyproline in WBB6F1 mice confirmed
that the OVA-induced increase in lung collagen in this model was
predominantly MC-dependent (Figure 5D).
MC expression of FcRγ is required for optimal expression of enhanced
airway responses, numbers of airway MCs, airway inflammation, and
mucin gene expression. MCs can be activated to release media-
tors in response to ligand-dependent engagement of multiple
distinct cell surface receptors and signaling pathways (10, 11,
39). However, antigen-dependent activation of MCs via either
IgE/FcεRI or IgG1/FcγRIII requires signaling mediated by the
γ chain (FcRγ) common to both of these receptors (14, 17). To
examine the extent to which FcεRI/FcγRIII-mediated MC acti-
vation is required for expression of our chronic asthma model,
we compared the expression of various features of our model in
KitW/W-v or KitW-sh/W-sh mice that had been engrafted with C57BL/6-
FcRγ–/– versus C57BL/6–FcRγ+/+ BMCMCs.
MC expression of FcRγ in BMCMCs→KitW/W-v mice had no
detectable effects on the levels of total or antigen-specific IgE or
IgG1 antibodies (Supplemental Figure 8). However, OVA treat-
ment induced little or no enhancement of Penh responses to
methacholine in FcRγ–/– BMCMCs→KitW/W-v mice, but substantial
enhancement of the responses was observed in FcRγ+/+ BMCMC→
KitW/W-v mice (Figure 6A). Similarly, OVA challenge of OVA-sen-
sitized mice induced a much stronger airway response in FcRγ+/+
BMCMC→KitW/W-v mice than in FcRγ–/– BMCMCs→KitW/W-v mice
(Figure 6B). Very similar results were obtained in C57BL/6-FcRγ+/+
versus C57BL/6-FcRγ–/– BMCMCs→KitW-sh/W-sh mice (Supplemen-
tal Figure 9), except that the FcRγ–/– BMCMCs→KitW-sh/W-sh mice,
in contrast to the FcRγ–/– BMCMCs→KitW/W-v mice, exhibited no
detectable enhancement of Penh in response to OVA challenge
(compare with Supplemental Figure 9B and Figure 6B).
In accord with these results, we found that expression of FcRγ by
adoptively transferred MCs significantly enhanced the ability of
such MCs to orchestrate most of the other features of this chronic
asthma model in the recipient KitW/W-v mice (Figures 7 and 8; Sup-
plemental Figures 10 and 11). Indeed, OVA-induced increases in
numbers of lung MCs appeared to be entirely dependent on the
expression of FcRγ by the adoptively transferred MCs (Figure 7A
and Supplemental Figure 10A). These results strongly suggest
that an effect mediated through MC-FcRγ, perhaps including the
ability of IgE antibodies to promote MC survival or proliferation
even in the absence of known antigen (44–48), contributed to the
increased numbers of MCs observed in this setting.
However, in some other features of the response that were strong-
ly expressed in FcRγ+/+ BMCMCs→KitW/W-v or FcRγ+/+ BMCMCs→
KitW-sh/W-sh mice, OVA treatment also induced significant, albeit
relatively modest, responses in the FcRγ–/– BMCMCs→KitW/W-v or
FcRγ–/– BMCMCs→KitW-sh/W-sh mice. For numbers of lung eosino-
phils (Figure 7B and Supplemental Figure 10B), numbers of most
types of leukocytes present in the BAL fluid 24 hours after OVA
challenge (Figure 7D and Supplemental Figure 10D), and levels
of mRNA of genes encoding mucin 5AC or 5B (Figure 8, B and
C; Supplemental Figure 11, B and C), FcRγ, and T cell GTPase
(Supplemental Figure 12, A and B), the responses in the FcRγ–/–
Airway goblet cell numbers and mucin gene expression in this chronic
asthma model 24 hours after the ninth OVA or PBS challenge. (A and
B) Lung mRNA levels of genes encoding mucin 5AC and mucin 5B.
(C) Numbers of goblet cells along the airway epithelium. (D) Levels of
lung hydroxyproline. White bars, PBS-treated group; black bars, OVA-
sensitized/challenged group. ***P < 0.001 versus corresponding PBS
controls; †P < 0.05, ††P < 0.01, or †††P < 0.001 versus group indicated.
n = 6 per group (A–C); n = 8 per group (D).
Penh responses following i.n. OVA antigen challenge in KitW/W-v mice
that had been engrafted with FcRγ–/– versus FcRγ+/+ BMCMCs. (A)
Responses to aerosolized methacholine 24 hours after the eighth OVA
or PBS challenge. (B) Penh measured 1 hour before and 1, 3, 6, 9, 12,
18, and 24 hours after the ninth OVA or PBS challenge. Data are from
OVA-sensitized/challenged FcRγ+/+ BMCMCs→KitW/W-v (closed circles)
and FcRγ–/– BMCMCs→KitW/W-v mice (open circles) and PBS-treated
FcRγ+/+ BMCMCs→KitW/W-v (closed squares) and FcRγ–/– BMCMCs→
KitW/W-v (open squares) mice. *P < 0.05 versus corresponding PBS
controls; †P < 0.05 versus OVA-sensitized/challenged FcRγ–/–
BMCMCs→KitW/W-v mice; ‡P < 0.05 versus values at that time point in
the corresponding PBS control group; n = 6 per group.
1638?The?Journal?of?Clinical?Investigation http://www.jci.org Volume 116 Number 6 June 2006
BMCMCs→KitW/W-v or FcRγ–/– BMCMCs→KitW-sh/W-sh mice, although
significant compared with responses in the PBS-challenged mice,
were significantly lower, and in many cases at least 50% lower, than
those in the OVA-challenged FcRγ+/+ BMCMCs→KitW/W-v or FcRγ+/+
BMCMCs→KitW-sh/W-sh mice. These results indicate that FcRγ-inde-
pendent mechanisms of MC activation can contribute to the devel-
opment of these features of this chronic asthma model but that
optimal responses require MC expression of FcRγ.
FcRγ-independent mechanisms of MC activation can significantly
contribute to elevations of serum histamine and increased numbers
of airway goblet cells. Serum histamine levels were significantly
lower in OVA-treated FcRγ–/– versus FcRγ+/+ BMCMCs→KitW/W-v
mice, but the reduction was only approximately 19% (Figure 7C).
Given that virtually all of the elevation of serum histamine in this
model is MC dependent (Figure 3B), the most straightforward inter-
pretation of this finding is that in WBB6F1
mice, OVA-treatment either induced MC
histamine release by FcRγ-independent
mechanisms or (less likely, we think) that
such mechanisms permit MCs to promote
histamine release from other sources. Nota-
bly, in mice on the C57BL/6 background,
FcRγ-independent mechanisms appear to be
less important than FcRγ-dependent mecha-
nisms in regulating OVA-induced histamine
release (Supplemental Figure 10C).
The increased numbers of goblet cells
induced in OVA-treated versus PBS-treated
FcRγ–/– BMCMCs→KitW/W-v mice reached
levels that were nearly as high as, and were
statistically indistinguishable from, the
levels induced in the corresponding FcRγ+/+
BMCMCs→KitW/W-v mice (Figure 8A). In
contrast, the enhanced levels of Muc5ac
and Muc5b mRNA in OVA-challenged mice
were significantly reduced in FcRγ–/– versus
FcRγ+/+ BMCMCs→KitW/W-v mice (Figure 8,
B and C). FcRγ-independent mechanisms
appeared to contribute significantly both
to the increases in goblet cell numbers
and to the enhanced levels of Muc5ac and
Muc5b mRNA in OVA-challenged C57BL/6
mice as well (Supplemental Figure 11).
Our findings indicate that FcRγ-independent activation of MCs
can contribute significantly to the enhanced goblet cell numbers
(especially in mice on the WBB6F1 background) and to the mark-
edly increased mucin gene expression that develops in a largely
MC-dependent manner in this chronic asthma model.
We have developed a mouse model in which repetitive i.n. chal-
lenge of sensitized mice with antigen induces changes in the air-
ways and lungs that mimic many of the features of chronic asthma
in humans. Our studies in MC-engrafted KitW/W-v mice indicate
that MCs are not required for the generation of antigen-specific
IgE or IgG1 antibody responses in this model but are required for
the development of every other feature of the model that we ana-
lyzed. Moreover, in many instances, the optimal development of
Features of allergic inflammation in this
chronic asthma model 24 hours after the ninth
OVA or PBS challenge in FcRγ–/– BMCMCs→
KitW/W-v mice versus FcRγ+/+ BMCMCs→
KitW/W-v mice. (A and B) Numbers of lung MCs
(A) and eosinophils (B). (C) Serum histamine
concentration. (A–C) White bars, PBS-treated
group; black bars, OVA-sensitized/challenged
group. (D) Numbers of leukocytes in BAL fluid
from the right lungs of FcRγ+/+ BMCMCs→
KitW/W-v (black bars) and FcRγ–/– BMCMCs→
KitW/W-v (white bars) mice. **P < 0.01 or
***P < 0.001 versus corresponding PBS con-
trols; †P < 0.05, ††P < 0.01, or †††P < 0.001
versus group indicated (n = 6 per group).
Airway goblet cell numbers and mucin gene expression in this chronic asthma model 24
hours after the ninth OVA or PBS challenge in FcRγ–/– BMCMCs→KitW/W-v mice versus FcRγ+/+
BMCMCs→KitW/W-v mice. (A) Numbers of goblet cells along the airway epithelium. (B and C)
Lung mRNA levels of genes encoding mucin 5AC and mucin 5B. White bars, PBS-treated
group; black bars, OVA-sensitized/challenged group. **P < 0.01 or ***P < 0.001 versus cor-
responding PBS controls; †P < 0.05 versus group indicated (n = 6 per group).
? The?Journal?of?Clinical?Investigation http://www.jci.org Volume 116 Number 6 June 2006
individual features of the pathology and pathophysiology in this
model require that MCs express FcRγ, and MC expression of FcRγ
appears to be required for virtually all of the increase in lung MC
numbers observed in OVA-treated mice (Supplemental Table 2).
Notably, using the more recently described MC-engrafted
C57BL/6-KitW-sh/W-sh mouse model (49, 50), in which both the adop-
tively transferred FcRγ–/– or FcRγ+/+ BMCMCs and the recipient
genetically MC-deficient KitW-sh/W-sh mice are all on the C57BL/6
background, we obtained results very similar to those obtained
in experiments employing the transfer of C57BL/6-FcRγ–/– versus
C57BL/6-FcRγ+/+ BMCMCs into WBB6F1-KitW/W-v mice.
The simplest interpretation of these findings is that these MC-
FcRγ–dependent features of the response reflect consequences of
the activation of MCs by antigen and antigen-specific IgE and/
or IgG1 antibodies (11, 14, 18). However, some features of the
response, including the marked increase in lung MCs, may also
(or alternatively) reflect actions of IgE on MCs that are mediated
through FcRγ but can be observed in the absence of known specific
antigen (47, 48); these effects include the promotion of MC sur-
vival, proliferation, and cytokine production (44–48). Neverthe-
less, our findings clearly identify the activation of MCs via FcRγ-
dependent mechanisms as a key promoter of many of the features
of this chronic asthma model. Such findings support the view that
IgE-dependent MC activation represents an attractive therapeutic
target in asthma (14, 30, 51).
However, our study also shows that certain features of this
model are significantly influenced by effects of MCs that are inde-
pendent of MC-FcRγ. Such antigen- and MC-dependent but MC-
FcRγ–independent effects contribute importantly in this model
to the striking increases in the numbers of epithelial goblet cells
(especially in mice on the WBB6F1 background) and also contrib-
ute significantly to the elevated levels of mRNA for mucins 5AC
and 5B in the lungs (in both WBB6F1 and C57BL/6 mice). These
findings have interesting implications for our understanding of
the factors that contribute to enhanced mucin production in
human asthma. Among the 18 Muc genes that have been identified
in humans, Muc5ac and Muc5b gene products represent the major
gel-forming mucins in airway secretion (7, 43). And it has been
reported that increased levels of mucin 5AC and mucin 5B can be
detected in patients with asthma (6, 7). Increased mucin produc-
tion contributes significantly to airway obstruction and AHR in
asthma; accordingly, several potential therapies targeting mucus
hypersecretion are currently under development (7, 52). Our data
indicate that, at least in mice, MCs are major contributors to gob-
let cell hyperplasia and increased expression of Muc5ac and Muc5b
that are observed in this model of chronic asthma.
FcRγ-independent mechanisms of MC engagement also contrib-
ute significantly to elevations of levels of serum histamine (espe-
cially in mice on the WBB6F1 background), lung infiltration with
eosinophils and lymphocytes, and recruitment of leukocytes to
the airways in response to antigen challenge, as reflected in counts
of cells in BAL fluid. Moreover, some of the entirely MC-depen-
dent airway responsiveness to antigen challenge in WBB6F1 mice
(Figure 1B) and perhaps even some of the MC-dependent AHR to
methacholine (Figure 1A) in this model also appear to reflect con-
tributions by MC-dependent but MC-FcRγ–independent processes
(Figure 6, A and B). In contrast, in C57BL/6 mice, there was no
detectable contribution of MC-FcRγ–independent processes to the
MC-dependent enhancement of Penh responses to either metha-
choline or antigen (Supplemental Figure 9).
In summary, our findings in this mouse model show that MCs
can represent key drivers of many important inflammatory, struc-
tural, and functional changes of the lungs that are also observed
in chronic human asthma. These observations are thus in accord
with the many reports implicating MCs and their products in the
pathology of human asthma (13, 24). However, while some of the
features of our chronic asthma model appear to be strongly depen-
dent on MC expression of FcRγ, other effects are clearly antigen- and
MC-dependent but also significantly MC-FcRγ–independent. Our
findings show that this MC-FcRγ–independent pathway can elicit
important contributions of MCs to several critical features of chron-
ic asthma, including epithelial goblet cell hyperplasia and markedly
enhanced expression of genes encoding mucins 5AC and 5B.
On the other hand, the importance of such MC-dependent but
MC-FcRγ–independent pathways in this model may be influenced
by the genetic background of the mice, in that such pathways
appeared to be more important in the development of goblet cell
hyperplasia and in the elevations of serum histamine after OVA
challenge observed in WBB6F1 as opposed to C57BL/6 mice.
Adding even more complexity, a very large number of candidate
mechanisms might be proposed to account for the MC-dependent
but MC-FcRγ–independent pathways that appear to contribute to
the development of some of the features of this model. MCs can
be activated independently of FcRγ by multiple factors that may
be present in tissues affected by asthma in humans or by models
of asthma in mice, including products of complement activation
(C5a and C3a) as well as certain neuropeptides, neurotrophins,
chemokines, products of recruited leukocytes, and other types of
products that can induce or enhance MC activation (10, 39, 40,
53–55). Complicating further any analysis of the potential roles of
such factors in our model are observations indicating that many
(if not all) of these factors may be able to influence features of
asthma independently of any effects they may have on MCs.
For example, we previously reported that C5a and C3a can
promote contractile responses in intestinal smooth muscle inde-
pendently of any contribution of MCs (56). Notably, aerosolized
C5a can also induce elevations of Penh and increased numbers
of BAL leukocytes independently of MCs in mice on either the
WBB6F1 or C57BL/6 background (Supplemental Figures 13 and
14). Accordingly, we expect that extensive work (and probably
some good fortune) may be required to identify the important
MC-dependent but MC-FcRγ–independent pathways that can
contribute significantly to the development of some of the fea-
tures of this model of chronic asthma.
The extent to which our observations can be extended to human
asthma remains to be determined. However, the findings in this
mouse model raise the possibility that therapies that target the
IgE-dependent activation of MCs and other effector cells may not
be fully effective in the treatment of chronic asthma even if it were
possible fully to eliminate IgE-dependent effector cell activation
by such approaches. Indeed, our data strongly suggest that MCs
themselves and FcRγ-independent pathways of MC activation in
addition to IgE represent promising therapeutic targets in patients
with chronic asthma.
Mice. Genetically MC-deficient (WB/ReJ-W/+ × C57BL/6J-Wv/+)F1-KitW/W-v
(WBB6F1-KitW/W-v) mice, congenic normal WBB6F1-Kit+/+ (Kit+/+) mice, and
C57BL/6J mice were purchased from Jackson Laboratory. C57BL/6-KitW-sh/W-sh
mice were generously provided by Peter Besmer (Molecular Biology Program,
1640? The?Journal?of?Clinical?Investigation http://www.jci.org Volume 116 Number 6 June 2006
Memorial Sloan-Kettering Cancer Center, New York, New York, USA); these
mice were then backcrossed to C57BL/6J for more than 6 generations. FcRγ–/–
mice (57) on the C57BL/6 background (B6.129P2-Fcer1gtm1Rav N12) were
purchased from Taconic. Age-matched female mice were used for all experi-
ments. All animal care and experimentation reported herein were conducted
in compliance with the guidelines of the NIH and with the specific approval
of Institutional Animal Care and Use Committee of Stanford University.
MC engraftment. Selective engraftment of MCs in MC-deficient WBB6F1-
KitW/W-v or C57BL/6-KitW-sh/W-sh mice was performed as described (33), with
minor modifications. Bone marrow cells derived from 4-week-old female
WBB6F1-Kit+/+, C57BL/6J-FcRγ+/+ (FcRγ+/+), or C57BL/6J-FcRγ–/– (FcRγ–/–)
mice were cultured in WEHI-3–conditioned medium (ATCC number
TIB-68), as a source of IL-3, for 4–5 weeks to generate cell populations that
contained more than 95% immature MCs. Via the tail vein, 5 × 106 BMCMCs
were injected into each mouse, and the recipients (e.g., WBB6F1-Kit+/+
BMCMCs→KitW/W-v or C57BL/6-Kit+/+ BMCMCs→ KitW-sh/W-sh mice) were
used for experiments 18 weeks later. Engraftment of KitW/W-v mice with
BMCMCs did not repair their anemia (as examined by hematocrit), con-
firming the selectivity of the MC engraftment (11, 33).
Immunization and airway challenge with antigen. Mice were immunized by
3 i.p. injections of 50 μg OVA (Sigma-Aldrich) in 0.1 ml PBS on days 1,
4, and 7. Starting on day 12, mice were challenged i.n. with 20 μg OVA
in 30 μl PBS weekly for 9 weeks; control mice received i.p. injections and
i.n. challenges with PBS on the same schedule. As shown in Supplemental
Figures 15 and 16, repeated i.n. administrations of OVA also appeared to
sensitize the mice to this antigen, albeit to a significantly lesser extent than
seen in mice that also received i.p. injections of OVA. Accordingly, for the
experiments presented herein, we compared responses in mice that had
been sensitized and challenged with OVA to those in mice that had been
mock sensitized and challenged with PBS.
All experiments employed OVA preparations that contained low levels of
LPS (i.e., <100 pg of LPS was administered during the entire sequence of OVA
sensitization and challenge). Eisenbarth et al. (58) showed that low levels of
LPS were required for successful induction of Th2 responses upon i.n. sen-
sitization of BALB/c mice with OVA that lacked aluminum hydroxide but
that LPS signaling via TLR4 was not required for the development of a Th2
response after i.p. sensitization with OVA and aluminum hydroxide. LPS levels
(measured with a QCL-1000 kit; Cambrex) in the OVA/PBS preparations used
for i.p. sensitization versus i.n. challenge in our experiments were 170 versus
225 pg/ml, respectively, and therefore each i.p. versus i.n. challenge dose of
OVA/PBS contained approximately 17 versus approximately 6.8 pg of LPS.
Measurement of airway reactivity to methacholine. Twenty-four hours after
the eighth OVA or PBS challenge, responses to aerosolized methacholine
were measured using whole-body plethysmography (Buxco) (33). Respons-
es to antigen (OVA) or PBS were assessed by recording Penh over 10-minute
time periods at intervals before and after the ninth (final) OVA or PBS
challenge (in the absence of methacholine). In some studies, we also per-
formed invasive measurements of airway reactivity in anesthetized, trache-
ostomized, mechanically ventilated mice (59). Aerosolized methacholine
was administered in increasing concentrations (0, 6.25, 12.5, 25, 50, and
100 mg/ml), with individual doses separated by 4 minutes. RL and Cdyn
were continuously computed by fitting flow, volume, and pressure to an
equation of motion for each aerosol challenge period, which consisted of a
2-minute aerosol exposure and a 4-minute period after exposure.
BAL and histology. For BAL, mice were killed by CO2 inhalation 24 hours
after the last (ninth) OVA or PBS challenge. The left lung was ligated and
removed whereas the right lung lobes were lavaged once with ice-cold HBSS
(BAL) then fixed (10% formaldehyde) and paraffin-embedded. Sections of
5 μm were mounted on Superfrost Plus glass slides (Fisher Scientific) and
stained with H&E, toluidine blue, Congo red, or Masson trichrome stains.
Quantitative RT-PCR. Total RNA isolated from the left lungs using TRIzol
Reagent (Invitrogen Corp.) was reverse transcribed using the High Capac-
ity cDNA Archive Kit (Applied Biosystems) according to the manufactur-
er’s instructions. Quantitative real-time PCR was performed using the
ABI 7700 Real-Time PCR System (Applied Biosystems) with the following
cycling parameters: 10 minutes at 95°C, then 40 biphasic cycles of 15 sec-
onds at 95°C, and 1 minute at 60°C. Primers and probes (see Supplemen-
tal Table 1) designed using Primer Express Software version 1.1 (Applied
Biosystems) were obtained from Operon Technologies. Units are expressed
as copy number per pg of RNA equivalents.
Measurement of serum antibodies and histamine. Serum was collected 24
hours after the ninth OVA or PBS challenge. Total serum IgE and IgG1
concentrations were measured by ELISA (Bethyl Laboratories). OVA-spe-
cific IgE and IgG1 concentrations were measured by ELISA (60). The OVA-
specific IgE standard was a generous gift from E.W. Gelfand (National Jew-
ish Medical and Research Center, Denver, Colorado, USA). Pooled serum
from 4 randomly selected OVA-sensitized/challenged WBB6F1-Kit+/+ mice
was used as the internal standard, which was assigned the arbitrary value
of 50 experimental units/ml for OVA-specific IgG1. Serum histamine was
measured using an Enzyme Immunoassay Kit (Beckman Coulter) accord-
ing to the manufacturer’s instructions.
Hydroxyproline assay. Tissue hydroxyproline concentration was used to
measure the collagen content of the lung. Sections of the left lung 1-mm
thick were dried with acetone, approximately 10 mg of the dried samples
were hydrolyzed with 0.5 ml 6 N HCl at 110°C overnight, and hydroxypro-
line concentration was determined as in ref. 61.
Statistics. Unless otherwise specified, differences in airway response
between different groups were tested for statistical significance using
ANOVA. The unpaired Student’s t test (2 tailed) was used for all other
analyses. P < 0.05 was considered statistically significant. Unless otherwise
specified, all data are presented as mean ± SEM or mean + SEM.
We thank Zhen-sheng Wang and Alian Xu for technical assistance,
Peter Besmer for his gift of C57BL/6-KitW-sh/W-sh mice, Erwin W. Gel-
fand for the gift of an OVA-specific IgE standard, and members
of the Galli laboratory for discussions. This work was supported
by United States Public Health Service grants HL67674, AI23990,
and CA72074 (to S.J. Galli) and a Ruth L. Kirschstein National
Research Service Award (to M. Yu).
Received for publication May 20, 2005, and accepted in revised
form April 11, 2006.
Address correspondence to: Stephen J. Galli, Department of
Pathology, L-235, Stanford University School of Medicine, 300
Pasteur Drive, Stanford, California 94305-5324, USA. Phone: (650)
723-7975; Fax: (650) 725-6902; E-mail: email@example.com.
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