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* Division of Allergy, La Jolla Institute for Allergy and Immunology, San Diego, CA 92121;
Pharmaceutical Research and Development, Johnson & Johnson, La Jolla, San Diego, CA 92130; and
Department of Dermatology, University of California, Davis, School of Medicine, Sacramento, CA 95817
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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RI
-chain-deficient (FcRI-/-) mice to
investigate the role of IgE-dependent mast cell activation in these
models. When mice were either 1) immunized once with OVA in alum i.p.
and then challenged with OVA intranasally, or 2) repeatedly immunized
with OVA in the absence of adjuvant and subsequently challenged with
nebulized OVA, FcR-/- mice had significantly fewer
eosinophils and lower IL-4 levels in their bronchoalveolar lavage fluid
compared with wild-type mice. When mice were given anti-IL-5
antibody before OVA challenge in protocol 1, eosinophilic infiltration
into the airways was significantly suppressed in both genotypes, but
only FcRI-/- mice showed significantly reduced
airway hyperresponsiveness (AHR). In addition, when mice immunized and
challenged with OVA also received a late OVA provocation at a higher
concentration and were then exposed to methacholine, only wild-type
mice developed a substantial increase in AHR. Since FcRI is
expressed mainly on mast cells in mouse airways, we conclude that
IgE-dependent activation of this cell type plays an important role in
the development of allergic airway inflammation and AHR in mice. The
models used may be of value for testing inhibitors of IgE or mast cells
for development of therapeutic agents for human
asthma. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Mast cells have long been regarded as an important cell type involved in allergic inflammation. These cells are ordinarily distributed throughout the connective tissue, where they are often situated adjacent to blood and lymphatic vessels and beneath epithelial surfaces that are exposed to environmental Ags (5). Activation of these cells results in the release of spasmogenic and vasoactive mediators as well as chemical mediators that promote infiltration of other leukocytes, including eosinophils, to sites of inflammation (6). Mast cells can signal naive and memory T cells to preferentially differentiate into Th2 cells (7) and induce IgE synthesis in B cells (8). It has also been reported that activation of mast cells in the airways of mice by anti-IgE Ab results in enhanced airway responsiveness to cholinergic stimulation by methacholine (9).
However, there are conflicting reports in the literature with regard to the role of mast cells in murine models of asthma, primarily based on studies in mast cell-deficient mice. While some reports have demonstrated that mast cell deficiency results in attenuated eosinophilic airway inflammation (10, 11), others have shown that this deficiency does not affect allergic airway inflammation and AHR (12, 13, 14, 15). Recent studies have suggested that the extent to which mast cells contribute to airway inflammation and AHR in mice is highly dependent on the experimental model used to generate the airway response (16, 17).
The role of IgE in mast cell and basophil activation is critical in
human allergic diseases (9). Recently, anti-IgE
therapy currently in clinical trials has provided additional evidence
for the role of IgE in both allergic rhinitis and asthma
(18, 19, 20, 21). Ag-specific IgE Abs bind mast cells via FcRI.
These cell surface-bound IgE molecules can subsequently become
cross-linked upon binding to the Ag, resulting in mast cell activation
and the release of a plethora of pro-inflammatory molecules. However,
allergic airway inflammation and AHR can be elicited in mice in the
absence of IgE (22, 23) or all classes of Abs
(24). The existing literature suggests that a role for IgE
or other classes of Abs in murine models of asthma is dependent on
experimental variables. As an example, it has been proposed that IgE
plays an important role in the development of airway inflammation and
AHR only under experimental conditions in which eosinophilic
inflammation is relatively low (25).
IgE-mediated mast cell activation is critically dependent on FcRI.
It appears that mice deficient in FcRI may be well suited for
delineation of the roles of mast cells and IgE in allergic airway
inflammation. Since in mouse airways FcRI is expressed only on mast
cells, the phenotypic alterations detected in these mice could be
connected to mast cells. Studies with these mice would obviously shed
light on the role of IgE, since FcRI is the major receptor for IgE.
In addition, while the mast cell-deficient mice commonly used to study
the role of mast cells in various disease models have defects other
than the lack of mast cells, the defect of FcRI-deficient mice is
restricted to IgE-mediated mast cell/basophil responses. Also, IgE can
exert its effects through both FcRI and FcRII, and thus the
phenotype of IgE-deficient mice may reflect the functions of IgE
mediated through either of these two receptors. The phenotype of
FcRI-deficient mice, on the other hand, should be related only to
those mediated through FcRI.
Thus, studies in FcRI-deficient mice should complement earlier
studies in mast cell-deficient mice and IgE-deficient mice and provide
additional insights into the role of IgE-mediated mast cell activation
in murine models of asthma. We now report that 1) FcRI-deficient
mice exhibit reduced airway eosinophilic inflammation and AHR when
sensitized to the Ag systemically and challenged subsequently with the
Ag through the airways; and 2) the sensitivity/threshold of this
response are differentially regulated in
FcRI-/- and wild-type mice.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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RI-/- mice
FcRI-/- mice were produced by
targeted disruption of the gene encoding the FcRI-chain. A 2.4-kb
DNA fragment (knockout construct) containing part of exon 1 and
complete exons 2 and 3 of the mouse FcRI gene (26) was
obtained by PCR of embryonic stem cell genomic DNA. The mouse
FcRI-chain gene-specific primers used in PCR are
5'-CCCCTCGAGCCTAGCACTGCTGTTCATGT-3' and
5'-CCCGCGGCCGCGCTTCATGGATTGTTCAGAGC-3'. The mouse FcRI gene
was disrupted by inserting the neomycin-resistant gene cassette of 1.2
kb into the BamHI site of exon 3. Mouse D3 embryonic stem
cells from 129/SV mice were maintained in an undifferentiated state of
growth on a feeder layer of mitomycin C-treated embryonic fibroblasts
in DMEM supplemented with 100 U/ml leukemia inhibitory factor (Life
Technologies, Gaithersburg, MD) and 15% FBS. Embryonic stem cells were
transfected with the knockout construct DNA by electroporation, and
transfected cells were selected in medium containing 300 µg/ml G418.
Homologous recombination was detected by PCR as previously described
(27).
Chimeric mice were generated from blastocysts injected with the
selected embryonic stem cells. Mice carrying the disrupted FcRI gene
were obtained by breeding chimeric mice with C57BL/6J mice and
screening for agouti offspring. The disrupted FcRI gene in agouti
mice was detected by Southern blot analysis of tail DNA (see
Results). Mice heterozygous for the disrupted gene were
interbred to homozygosity. The mice were then backcrossed for nine
generations with BALB/cBYJ mice (The Jackson Laboratory, Bar Harbor,
ME). The heterozygous animals were intercrossed to obtain homozygous
FcRI-/- and wild-type animals with the
same genetic background. Confirmation of the disruption of the
FcRI-chain gene was accomplished by PCR and Southern blot
analysis. Experiments were performed on age- and sex-matched 8- to
12-wk-old mice. The animals were maintained in pathogen-free conditions
and on chicken egg albumin-free diets.
Reagents
Acetyl-
-methacholine chloride (methacholine (MCh)) was
purchased from Sigma-Aldrich (St. Louis, MO). Rat anti-IL-5 mAb was
purified from the TRFK-5 hybridoma cell line provided by Dr. R. Coffman
(DNAX-Research Institute of Molecular and Cellular Biology, Palo Alto,
CA) (28). Monoclonal anti-DNP IgE was purified from
ascites from hybridoma 26.82 as previously described
(29).
Flow cytometric analysis
Bone marrow-derived mouse mast cells (BMMC) from naive
FcRI-/- and wild-type mice were generated
by maintaining femoral bone marrow cells in vitro for 4–5 wk according
to a published protocol (30). The cells were incubated
first with 10 µg/ml anti-DNP IgE for 40 min and then with
FITC-conjugated anti-mouse IgE (BD PharMingen, San Diego, CA) for
another 30 min. Flow cytometric analysis of the stained cells was
performed with FACScan (BD Biosciences, Mountain View, CA) equipped
with CellQuest software.
Hexosaminidase release assay
BMMC were plated at 2 x 106/ml in 25-ml tissue culture flasks and sensitized with 0.5 µg/ml anti-DNP IgE overnight at 37°C in a CO2 incubator. The cells were washed once and resuspended at 10 x 106/ml. Then, 0.5-ml aliquots of the cell suspension were placed in Eppendorf tubes. The cells were then incubated with 0, 3, 10, 30, 100, and 300 ng/ml multivalent Ag DNP-BSA for 45 min. Afterward, the cells were centrifuged, and the supernatants were collected. The cell pellets were lysed with 0.1% Triton. All samples were kept at -20°C until tested for enzyme activity.
For quantitating -hexosaminidase, 30 µl supernatants or
Triton-lysed cell pellets were placed in wells of 96-well flat-bottom
plates (Fisher Scientific, Los Angeles, CA) containing 20 µl of the
substrate, 3 mM
p-nitrophenyl-N-acetyl--D-glucosaminide
(Sigma-Aldrich), in 0.1 M citrate buffer, pH 4.5. The assay was
terminated 20 min later by the addition of 0.1 M sodium carbonate
buffer, pH 10.0. The color developed was measured
spectrophotometrically at 405 nm. The enzyme release was expressed as
the percentage of the total -hexosaminidase content of the cells
after subtraction of unstimulated release (31).
Passive cutaneous anaphylaxis reaction
The ears and shaved dorsal skin of naive
FcRI-/- and wild-type mice were injected
with either saline or dilutions of anti-DNP IgE hybridoma ascites
intradermally. Three hours later, 200 µl DNP-BSA at 1 mg/ml was
injected i.v., and 30 min later the mice were euthanized. Ear and skin
biopsies were placed directly in 10% zinc formalin. The
paraffin-embedded 5-µm sections were cut and hydrated, then stained
with toluidine blue. The number of mast cells was counted in 20
100-mm2 areas using an ocular grid. The
morphology of the mast cells was graded as normal (<10% of
cytoplasmic granules exhibiting fusion, staining alterations, or
extrusion from the cell), moderately degranulated (10–50% of granules
altered as described above), and extensively degranulated (>50%
granules altered as described above) (32).
Sensitization protocol
Mice were immunized with OVA (chicken egg albumin grade V from Sigma-Aldrich) i.p. according to the following three protocols: 1) 10 µg OVA and 2 mg/ml aluminum hydroxide gel (alum) as adjuvant in 0.5 ml saline once, 2) 10 µg OVA in 0.5 ml pyrogen-free saline without adjuvant 11 times on alternate days, and 3) OVA-alum as described above, followed by a booster OVA-alum injection on day 14.
Airway Ag challenge
Mice immunized according to protocol 1 were challenged 14 days later intranasally with 10 µg OVA in 15 µl saline on 4 alternate days. Mice immunized according to protocol 2 were challenged with 1% aerosolized OVA in PBS 30 min each day for 6 consecutive days 14 days after the last immunization. Control mice were immunized in an identical fashion, but challenged with aerosolized PBS. The aerosol was generated in a Plexiglas chamber connected to a nebulizer (Ultra Neb Nebulizer, 120VAC, with a maximum aerosol output of 6 ml/min and an average particle size of <4 µm with an airflow of up to 30 lpm). Mice immunized according to protocol 3 were challenged with aerosolized OVA on 7 consecutive days 10 days after the last immunization. Seven days after the last Ag challenge, mice were exposed to OVA aerosol generated from a 5% OVA solution, while control mice received aerosolized PBS.
Anti-IL-5 Ab treatment
Mice immunized once with OVA in alum (sensitization protocol 1) were given 14 days later either 10 µg rat anti-IL-5 Ab or, as a control, purified normal rat IgG (Zymed, San Francisco, CA) i.p. in pyrogen-free saline. Two hours later the mice received 10 µg OVA intranasally. The anti-IL-5 treatment and intranasal Ag challenge were performed on 4 alternate days.
Determination of AHR
The airway response was measured in unrestrained animals using whole-body plethysmography (Buxco, Troy, NY), as described previously (33). Readings were obtained at baseline and after exposure to aerosolized saline or MCh (5–50 mg/ml). Data were collected for 5 min after 3 min of inhalation, and average values were expressed as the enhanced pause (Penh): Penh = [(Te - Tr)/Tr] x (PEP/PIP), where Te is the expiratory time (seconds), Tr is the relaxation time (time of the pressure decay to 36% of total box pressure during expiration), PEP is the peak expiratory pressure (milliliters per second), and PIP is the peak inspiratory pressure (milliliters per second). Results are expressed as the percent increase in Penh following challenge with each concentration of MCh, where baseline Penh is expressed as 100%, or as Penh values. The airway response of mice receiving aerosolized Ag was measured 4 h after the last challenge; it was measured 24 h after the last challenge in mice that received the Ag intranasally.
Collection and analysis of bronchoalveolar lavage fluid (BALF)
BALF was collected by cannulating the upper part of the trachea and lavaging three times with 1 ml pyrogen-free PBS (85–90% of the 1-ml input volume was retrieved). The lavage fluid collected was centrifuged at 400 x g for 5 min at 4°C, and the cells were separated from the fluid. The fluid was then centrifuged at 1000 x g for 2 min at 4°C to remove cellular debris and then stored at -20°C until it was evaluated. The cells were resuspended in PBS containing 1% FBS, and the total number of viable cell was determined by trypan blue exclusion using a hemocytometer. Differential cell counts were determined with cytospin preparations, followed by Wright-Giemsa staining (Fisher Scientific, Los Angeles, CA).
Cytokine and Ig quantifications by ELISA
IL-4. Ninety-six-well plates (Maxisorp, Nunc, Roskilde, Denmark) were coated overnight with the capture Ab 11B11 (National Cancer Institute, Biological Response Modifier Program, Bethesda, MD) at 4°C and then blocked with the assay diluent (1% BSA in PBS containing 1% FBS and 0.05% Tween 20) for at least 15 min. BALF samples and rIL-4 standard (a gift from Dr. J. Ohara, Denver, CO) were placed in appropriate wells, and the plates were incubated for 2 h at room temperature. The bound cytokine was detected with biotinylated rat anti-mouse IL-4 (BD PharMingen) with an incubation period of 2 h at room temperature, followed by avidin-HRP (Bio-Rad, Richmond, CA) at 1/1000 dilution for 30 min at room temperature. The plates were developed by addition of the substrate o-phenylenediamine dihydrochloride (Sigma-Aldrich). The reaction was stopped by adding 25 µl of 4 N sulfuric acid before reading the plates in an ELISA plate reader (Spectramax 250; Molecular Devices, Sunnyvale, CA) at 490 nm. The experimental values were read off a standard curve generated using the plate reader software.
Total IgE.
The plates were coated with affinity-purified goat anti-mouse IgE Ab
(29) overnight at 4°C and then blocked with 1% BSA in
PBS for 2 h at room temperature. The samples and appropriate
dilutions of a standard anti-DNP IgE preparation (29)
in the assay diluent (1% BSA in PBS containing 0.05% Tween 20) were
incubated overnight at 4°C. The bound IgE was detected after 1-h
incubation with purified rat anti-mouse-IgE-HRP (-chain
specific; Southern Biotechnology Associates, Birmingham, AL) at room
temperature and subsequent addition of substrate as described
above.
Histological studies
After BAL the lungs were perfused with 10 ml PBS via the right heart ventricle. The exsanguinated left lung was then removed from the chest cavity and fixed in 10% zinc formalin (10 ml). The lobes were sagittally sectioned, paraffin embedded, cut in 5-µm sections, and stained with Wright-Giemsa to identify eosinophils in perivascular and peripheral areas of lung tissue, which were counted using a 10 x 10-mm grid (x10) plus x40 magnification (x400 final magnification).
Statistical analysis
Values are expressed as the mean ± SEM. The data were compared using Student’s two-tailed t test with Excel 5.0; p < 0.05 was considered significant.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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RI-/- mice
FcRI in rodents has a tetrameric structure consisting of
noncovalently associated subunits, 
2
(34). The subunit is responsible for the IgE-binding
function of the receptor, while the and subunits are both
primarily intracellular and critically involved in transducing signals
induced by cross-linking of the receptor. All three subunits are
required for cell surface expression of FcRI in mice
(35). FcRI-/- mice were
generated by gene targeting according to standard protocol, with the
FcRI gene being disrupted within exon 3. Confirmation of
homologous recombination was achieved by Southern blot analysis.
Briefly, mouse genomic DNA was digested with EcoRI,
BglII plus HindIII, or BglII plus
EcoRV, and the genomic blots were hybridized with a 0.7-kb
DNA probe located at the 3'-flanking region of the knockout construct
(Fig. 1
). As shown in Fig. 1, a 4-kb
EcoRI DNA fragment was detected for the wild-type FcRI
gene, and this DNA fragment was shortened to 2.5 kb in the disrupted
gene due to the presence of an EcoRI site in the
neomycin-resistant gene cassette. The
HindIII-BglII DNA fragment (4 kb) and the
EcoRV-BglII DNA fragment (2.5 kb) of the
wild-type gene were increased to 5.7 and 3.7 kb, respectively, in the
disrupted gene due to insertion of the neomycin-resistant gene
cassette.
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RI-/- and
wild-type mice have comparable numbers of mast cells when stained with
toluidine blue, consistent with previous reports (36, 37).
The number of mast cells per 100-mm2 dermis was
7.9 ± 1.6 for wild-type mice and 9.2 ± 2.1 for
FcRI-/- mice. Since FcRI is primarily
expressed by mast cells in mice (35), we confirmed the
functional deficiency of this receptor by studying mast cell responses
in these mice. We found that there was no detectable IgE binding by
BMMC from FcRI-/- mice by flow cytometry,
while such binding was readily detectable with BMMC from wild-type mice
(Fig. 2A). In addition, the
absence of functionality of FcRI on BMMC from
FcRI-/- mice was demonstrated in vitro by
a lack of IgE-dependent degranulation.
FcRI-/- and wild-type BMMC were first
incubated with DNP-specific IgE and then with DNP-BSA. No
-hexosaminidase release was detected in BMMC from
FcRI-/- mice, while BMMC from wild-type
mice released the enzyme in a dose-dependent manner (Fig. 2B). The results are not due to a defect in the synthesis of
the enzyme, since total BMMC lysates from both genotypes contained
comparable amounts of the enzyme (data not shown). Finally,
FcRI-/- mice showed a defective
IgE-dependent mast cell response in vivo. When sensitized with
DNP-specific IgE s.c. and then challenged with the Ag DNP-BSA
systemically, FcRI-/- mice did not
exhibit an appreciable cutaneous reaction, in contrast to wild-type
mice, which showed extensive dermal mast cell degranulation (Fig. 2C). Very few mast cells in the skin sections of control
mice from both genotypes degranulated. Furthermore, the total numbers
of mast cells in control vs Ag-challenged mice were comparable for both
genotypes, suggesting that most, if not all, degranulated mast cells
were detected by the method we used. The data were obtained from three
mice per group for each genotype.
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RI-/- mice exhibit reduced airway responses
upon immunization to an Ag with adjuvant and then challenged with the
Ag intranasally
When mice were immunized twice i.p. with OVA in alum and then
challenged with nebulized OVA once a day for 6 consecutive days, both
FcRI-/- and wild-type mice showed marked
airway responses measured 4 h after the last Ag challenge, as
gauged by the development of AHR and by the number of eosinophils as
well as the levels of IgE and IL-4 in BAL fluid. However, there was no
significant difference between the two genotypes (data not shown). In
mice, mast cells are distributed primarily peritracheally and not in
lung parenchyma (38). Therefore, the extent of the
contribution of mast cells in the airway response is probably dependent
on where and how the Ag is administered. Indeed, when we challenged
OVA-immunized mice with OVA intranasally 24 h after the last Ag
challenge, we found a significant difference in certain airway
responses between FcRI-/- mice and
wild-type mice in three independently performed experiments. Both the
number of eosinophils infiltrating the lungs (Fig. 3A), as well as the IL-4
levels in BALF were significantly lower in
FcRI-/- mice than in wild-type mice (Fig. 3B), but IgE levels in BALF (Fig. 3C) and AHR
(Fig. 3D) were very similar. In addition,
FcRI-/- mice developed a significantly
lower extent of eosinophil infiltration in perivascular areas in the
lungs compared with wild-type mice (numbers of eosinophils in the lungs
of FcRI-/- mice: Ag challenged,
38.43 ± 12.8/100 mm2; controls, 1.0 ±
0.66/100 mm2; wild-type mice: Ag challenged,
74.07 ± 12.39/100 mm2; controls, 2.33
± 2.3/100 mm2).
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RI-/- mice consistently showed lower
numbers of eosinophils and lower levels of IL-4 in BAL fluid (data not
shown).
FcRI-/- mice exhibit significantly attenuated
airway responses when immunized with an Ag in the absence of adjuvant
and challenged with the Ag through the airways
Williams et al. (16) reported recently that mast
cell-deficient mice exhibit reduced airway responses compared with
wild-type mice only when the mice are sensitized with OVA in the
absence of alum and then challenged with OVA through the airways. We
reasoned that since FcRI is expressed primarily by mast cells, such
a protocol would more probably reveal phenotypic defects in
FcRI-/- mice. We sensitized both
genotypes with OVA 11 times in the absence of adjuvant every other day
and then, 14 days later, challenged the mice with nebulized OVA once a
day for 6 consecutive days. The BALF from
FcRI-/- mice collected 4 h after the
last Ag challenge contained a significantly lower number of eosinophils
than wild-type mice (Fig. 4A).
Further, BALF from FcRI -/- mice
contained a significantly lower amount of IL-4 compared with that from
wild-type mice (Fig. 4B). The two genotypes, however, showed
no difference in their IgE levels in BALF and AHR (Fig. 4, C
and D). The data were generated from three independently
performed experiments.
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RI-/-
mice
The causal relationship between eosinophils and AHR remains
controversial. Treatment of mice with anti-IL-5 Ab, which results
in a nearly complete suppression of the airway eosinophil response,
does not always result in a reduction in AHR (see
Discussion). We believe that the effect of reduction in
eosinophil infiltration may have a more pronounced effect on AHR when
the mast cell response is blunted, such as in
FcRI-/- mice. To test this hypothesis,
mice were immunized with OVA in alum and then challenged with OVA
intranasally four times, while they also received rat anti-IL-5 mAb
or normal rat IgG. The responses were measured 24 h after the last
Ag challenge. Consistent with our other experiments, described above,
BALF from rat IgG-treated FcRI-/- mice
contained significantly lower number of eosinophils than that from
comparably treated wild-type mice (Fig. 5A). As expected, eosinophil
numbers decreased dramatically in the anti-IL-5 Ab-treated groups
for both genotypes (Fig. 5A). Also consistent with the
results described above, FcRI-/- mice
were not defective in the development of AHR and, in fact, showed
higher AHR than wild-type mice in the absence of anti-IL-5
treatment (Fig. 5B, rat IgG treated groups). However, the
anti-IL-5 Ab treatment caused a substantial reduction in AHR in
FcRI-/- mice only (Fig. 5B),
suggesting a greater dependency of the bronchial response on eosinophil
infiltration in these mice. The data were generated from three
independently performed experiments.
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RI-/- mice develop significantly lower AHR in
response to Ag provocation
Previously we showed that when mice were immunized with Ag
systemically and subsequently challenged by the same Ag through the
airways, Ag-specific IgE remained detectable in the airways for >2 wk
after the last airway challenge (39). Thus, the airway
mast cells should remain sensitized by Ag-specific IgE even after the
airway inflammation has subsided and should respond when the animals
are provoked with the same Ag through the airways at a later time
point. We reasoned that under these conditions the airway response is
likely to be more dependent on mast cells, and thus a reduced airway
response is more likely to be noted in
FcRI-/- mice. Therefore, mice were
immunized twice with OVA, challenged 10 days later with nebulized OVA
(1%) for 7 consecutive days, and 7 days after the last Ag challenge
were exposed to OVA aerosol generated from a 5% OVA solution.
Immediately afterward, AHR to MCh challenge was measured repeatedly
over time. As shown in Fig. 6, a
significant increase in Penh was already detectable in wild-type mice
in the first measurement (10 min) after the 5% OVA provocation, and
the Penh value reached a peak 1 h after provocation. In
significant contrast, FcRI-/- mice did
not show such an increase in Penh during the entire period of
observation. Their response was, in fact, similar to that of mice that
were sensitized but not challenged by the Ag, which showed only a slow
increase in Penh in response to the MCh challenge following the Ag
provocation. All groups returned to a similar low Penh 2 h after
the 5% OVA provocation. The data were generated from two independently
performed experiments.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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RI-/- mice
develop significantly lower allergic airway inflammatory response and
AHR compared with wild-type mice when immunized with OVA systemically
and then challenged by the same Ag through the airways. Firstly, when
mice were immunized with OVA in alum and challenged with the Ag through
the intranasal route or were immunized with OVA in the absence of
adjuvant and challenged with nebulized Ag,
FcR-/- mice developed a significantly
lower degree of eosinophilic airway inflammation compared with
wild-type mice. Secondly, anti-IL-5 treatment before airway OVA
challenge caused a significant decline in AHR in
FcRI-/- mice, but not in wild-type mice.
Thirdly, in an OVA provocation protocol, the wild-type mice with intact
mast cell function responded with a higher AHR, while
FcRI-/- mice failed to respond to this Ag
provocation, essentially demonstrating a background level AHR. Because
in mice FcRI is the major receptor for IgE on mast cells and
basophils (35), our findings indirectly imply a role for
IgE-mediated mast cell/basophil activation in the allergic airway
response in murine models of asthma.
It has been reported that germinal centers are formed in mice in the
parenchyma of inflamed lungs following airway Ag challenge. These
contain IgG- and IgE-producing plasma cells, which have been shown to
produce the corresponding isotypes when isolated from the lungs and
cultured in vitro (40). The locally produced IgE as well
as circulating IgE are likely to sensitize mast cells in the airways.
Because mast cell activation has been shown to be a source of induction
of eosinophil infiltration as well as AHR (9, 41), it is
conceivable that mast cells play an important role in the airway
response in murine models of asthma. Indeed, there are studies
supporting the role of mast cells in these models (10, 11). However, there are other reports in the literature that
question mast cell participation in this response
(12, 13, 14, 15). Recently, strong evidence has been presented
that ties the role of mast cells in murine models of asthma to the
experimental protocols used. Thus, Kobayashi et al. (17)
noted that mast cell-deficient mice developed reduced AHR compared with
normal congenic mice only under certain experimental conditions. They
proposed that AHR could be induced by different mechanisms, and only
certain protocols would enlist mechanisms that involve IgE-mediated
mast cell activation. Likewise, Williams and Galli (16)
noted that while mast cell-deficient mice developed airway eosinophilia
and AHR comparable to normal congenic mice, only the former developed
significantly reduced responses when the mice were immunized with OVA
in the absence of adjuvant. Therefore, our finding that
FcRI-/- mice exhibit reduced airway
responses only under certain experimental conditions is in accord with
these previous reports. It has to be noted, however, that Kobayashi et
al. (17) described a contribution of mast cell activation
to AHR, but not to eosinophil recruitment, whereas we found a
FcRI-dependent contribution to eosinophil recruitment, but not to
AHR. Regardless of these differences, our findings together with these
recent reports strongly support the importance of IgE-dependent mast
cell activation in airway inflammation in murine models of asthma.
The reduced airway inflammatory response (eosinophil infiltration and
IL-4 production) in FcRI-/- mice was
observed only when mice were immunized with OVA in alum and then
challenged with OVA administered intranasally and not when equally
immunized mice were challenged with nebulized OVA. The results suggest
that IgE-dependent mast cell activation contributes to the airway
inflammation through a local effect in the airways and not in the phase
of systemic sensitization to the Ag. In the airways, mast cell
activation may lead to enhanced eosinophil infiltration through
secreted mediators that promote the diapedesis of eosinophils by acting
on these cells directly or on the blood vasculature. With regard to the
reduced IL-4 production in FcRI-/- mice,
the results are consistent with the fact that this cytokine is produced
by mast cells. Alternatively, mast cells are known to regulate T cell
functions (42), and thus the diminished mast cell
activation in FcRI-/- mice could result
in a down-regulated T cell response in the airways and consequently
reduced Th2 cytokine production.
Our finding that anti-IL-5 Ab treatment caused a significant
reduction in AHR only in FcRI-/- mice and
not in wild-type mice sheds light on the relationship between
infiltration of eosinophils and induction of AHR. Presently, the
participation of eosinophils in the development of AHR is not clear.
Studies by Nagai et al. (14) and Corry et al.
(43) showed that the anti-IL-5 Ab treatment inhibited
airway eosinophilia, but not AHR. Kobayashi et al. (17)
also described markedly inhibited airway eosinophilia without a
concomitant reduction in AHR in mice treated with anti-IL-5 Ab.
However, there are studies showing that IL-5-deficient mice, which do
not develop eosinophilia, also fail to develop AHR, supporting a causal
relationship between eosinophilic airway inflammation and AHR
(44). Hogan et al. (45) proposed that the
contribution of eosinophils to the development of AHR in mice might
sometimes be masked by coexisting pathways that operate independently
of eosinophils. We propose that IgE-dependent mast cell activation may
represent such a pathway, and that in its absence, as in the case of
FcR-/- mice, a positive correlation
between eosinophilia and AHR can be more easily demonstrated (Fig. 5
).
Our results also indicate that there is a synergy between mast cells
and eosinophils in the induction of AHR in murine models of asthma.
This suggests that mast cells and eosinophils secrete different
mediators that may activate bronchial smooth muscle through distinct
pathways, and these pathways cooperate synergistically in inducing
heightened bronchial responsiveness.
We were able to show that IgE-dependent mast cell activation can
clearly contribute to the development of AHR in experiments in which
mice were given a late airway Ag provocation with 5% OVA, and the MCh
challenge was administered immediately thereafter. Previously Martin et
al. (9) showed with mast cell-deficient mice and normal
congenic mice that IgE-mediated activation of mast cells enhances
pulmonary responsiveness to cholinergic stimulation. The most likely
explanation for our finding is that the observed increase in Penh in
OVA-provoked wild-type mice represents a mast cell-dependent reaction
induced by the Ag, since in both treatment groups of
FcRI-/- mice and in wild-type mice that
did not receive OVA provocation (PBS control), the Penh value did not
achieve the same level as in the test mice (Fig. 6). The results
support the idea that IgE-dependent mast cell activation can result in
AHR, probably through certain mediators that alone are not potent
enough to cause a detectable bronchial response under our experimental
conditions. Mice develop bronchoconstriction in response to MCh
challenge because of the stimulation of smooth muscles via
muscarinergic M3 receptors. This mechanism is effective in connection
with mast cell degranulation after the Ag provocation. The localization
of mast cells in lungs in mice is mainly in the tracheal and main
bronchial submucosal areas (38). Thus, the release of mast
cell mediators triggered by Ag provocation and inhaled MCh takes place
at the effector sites. We think that in our model
FcRI-/- mice only show the smooth muscle
contraction through MCh provocation, whereas the wild-type mice are
highly sensitized and respond with a mast cell-dependent reaction after
Ag provocation plus cholinergic stimulation of smooth muscles.
In humans FcRI is expressed on cell types other than mast cells and
basophils, suggesting that this receptor may have broader functions
than previously envisioned. In particular, the expression of FcRI on
monocytes and Langerhans cells that are classically considered APC
suggests a possible role of this receptor in IgE-dependent Ag
processing and presentation. In fact, peripheral monocytes and
dendritic cells have been shown to take up Ags through FcRI in an
IgE-dependent fashion and to present these Ags to T cells (46, 47). The role of FcRI in IgE-dependent Ag presentation by
mast cells to T cells has also been demonstrated (48).
However, in this study we did not observe any significant difference in
IgE production between FcRI-/- mice and
wild-type mice. These results are consistent with the lack of
expression of this receptor in classical APC in mice and also suggest
that mast cell FcRI does not play an important role in Ag
presentation under our experimental conditions.
Our results show that FcRI is important in the development of
allergic airway inflammation. Hence, this receptor could be a useful
target for the treatment of human asthma. Our study demonstrates that
the role of FcRI in allergic airway inflammation is strongly
dependent on how mice are exposed to Ag. By using other experimental
conditions, an even more pronounced amplification of the FcRI
participation in the allergic response could be achieved. Our
experimental approaches provide important information for both
mechanistic investigations as well as further testing of mast cell and
IgE inhibitors. Furthermore, this report points out that mechanisms of
asthma are multivariant and complicated, and therefore the treatment of
this disease may require combinations of drugs targeting different
components of the allergic response pathways. In addition,
FcRI-/- mice provide one important tool
for testing treatments that are targeted at cell types other than mast
cells and molecules other than IgE and FcRI.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Fu-Tong Liu, Department of Dermatology, University of California, Davis, 4860 Y Street, Suite 3400, Sacramento, CA 95817. E-mail address: fliu{at}ucdavis.edu
3 Abbreviations used in this paper: AHR, airway hyperresponsiveness; BAL, bronchoalveolar lavage; BALF, bronchoalveolar lavage fluid; BMMC, bone marrow-derived mouse mast cell; MCh, methacholine; Penh, enhanced pause.
Received for publication March 4, 2002. Accepted for publication June 5, 2002.
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in A).
Southern blot patterns obtained from tail DNA of wild-type,
heterozygous, and homozygous Fc
) and
Fc
) mice were immunized with OVA in alum
and 14 days later were challenged with OVA or PBS intranasally on 4
alternate days. BALF was obtained 24 h after the last challenge
and analyzed for the number of eosinophils (A) and
levels of IL-4 (B) and total IgE (C). The
number of eosinophils and IL-4 levels in BALF of OVA-challenged
Fc
