Conjunctival mast cell as a mediator of eosinophilic response in ocular allergy

Article (PDF Available)inMolecular vision 14(182-84):1525-32 · February 2008with40 Reads
Source: PubMed
Abstract
To determine the contribution of conjunctival mast cells to the allergen-specific inflammatory responses in eyes with allergic conjunctivitis and to test the hypothesis that mast cells act as mediators of the early phase response. The participation of mast cells in allergen-induced inflammatory cell recruitment was studied in an experimental murine model of allergic conjunctivitis. Experimental allergic conjunctivitis was induced by a single or multiple sensitizing injections of an allergen. The conjunctiva of allergen-sensitized, mast cell-deficient (Kit(w)/Kit(w-v)) mice were reconstituted with conjunctival mast cells isolated from naïve wild type mice by subconjunctival transfer. Kit(w)/Kit(w-v) mice and conjunctival mast cell reconstituted Kit(w)/Kit(w-v) mice were evaluated for early phase reactions and late phase inflammatory responses. The early phase response was minimal in Kit(w)/Kit(w-v) mice after both a single injection and multiple sensitization injections of the allergen. The early phase responses were fully restored following adoptive transfer of isolated conjunctival mast cells from naïve wild type mice. Eosinophilic inflammatory responses were significantly depressed in Kit(w)/Kit(w-v) mice without the impairment of allergen-specific priming. Reconstitution of the conjunctiva of Kit(w)/Kit(w-v) mice with mast cells from wild type mice fully restored the allergen-specific eosinophilic responses but not the neutrophilic responses. Our data indicate that conjunctival mast cells are essential for eosinophilic inflammation but not for neutrophilia in allergic conjunctivitis that is mediated by mast cell activation.

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Conjunctival mast cell as a mediator of eosinophilic response in
ocular allergy
Dai Miyazaki,1 Takeshi Tominaga,1 Keiko Yakura,1 Chuan-Hui Kuo,1 Naoki Komatsu,1 Yoshitsugu Inoue,1
Santa J. Ono2
1Division of Ophthalmology and Visual Science, Faculty of Medicine, Tottori University, Yonago, Japan; 2Emory University School
of Medicine and Emory Eye Center, Dobbs Ocular Immunology Laboratories, Atlanta, Georgia
Purpose: To determine the contribution of conjunctival mast cells to the allergen-specific inflammatory responses in eyes
with allergic conjunctivitis and to test the hypothesis that mast cells act as mediators of the early phase response.
Methods: The participation of mast cells in allergen-induced inflammatory cell recruitment was studied in an experimental
murine model of allergic conjunctivitis. Experimental allergic conjunctivitis was induced by a single or multiple sensitizing
injections of an allergen. The conjunctiva of allergen-sensitized, mast cell-deficient (Kitw/Kitw-v) mice were reconstituted
with conjunctival mast cells isolated from naïve wild type mice by subconjunctival transfer. Kitw/Kitw-v mice and
conjunctival mast cell reconstituted Kitw/Kitw-v mice were evaluated for early phase reactions and late phase inflammatory
responses.
Results: The early phase response was minimal in Kitw/Kitw-v mice after both a single injection and multiple sensitization
injections of the allergen. The early phase responses were fully restored following adoptive transfer of isolated conjunctival
mast cells from naïve wild type mice. Eosinophilic inflammatory responses were significantly depressed in Kitw/Kitw-v
mice without the impairment of allergen-specific priming. Reconstitution of the conjunctiva of Kitw/Kitw-v mice with mast
cells from wild type mice fully restored the allergen-specific eosinophilic responses but not the neutrophilic responses.
Conclusions: Our data indicate that conjunctival mast cells are essential for eosinophilic inflammation but not for
neutrophilia in allergic conjunctivitis that is mediated by mast cell activation.
Allergic diseases affect approximately one-third of the
population and constitute one of the major health care
problems in the Western world [1]. Ocular allergy is an
example of an IgE-mediated immediate hypersensitivity
reaction. These reactions are initiated by the cross-linking of
IgE by an allergen, and their effects are mediated by the
degranulation of mast cells. Immediate hypersensitivity
reactions are characterized by early phase and late phase
responses. The early phase response develops immediately
after the exposure to the allergen, and clinical symptoms and
signs such as itching, chemosis, and congestion are
manifested very quickly. This is followed by the late phase
response after 8-24 h, which is characterized by conjunctival
eosinophilia and neutrophilia.
Eosinophilic inflammation is not only a hallmark of
allergic conjunctivitis but also a major cause for tissue injury
and remodeling. Therefore, to understand how this response
develops or is exacerbated is important in developing an
effective strategy to combat ocular allergy.
The role played by T cells in the eosinophilic
inflammation of severe allergic conjunctivitis has been
Correspondence to: Dr. Dai Miyazaki, MD, Division of
Ophthalmology and Visual Science, Faculty of Medicine, Tottori
University, 36-1 Nishicho, Yonago, Tottori, Japan, 683-8504;
Phone: 81-859-38-6617; FAX: 81-859-38-6619; email:
dm@grape.med.tottori-u.ac.jp
gaining interest. For example, we have reported that the potent
inhibitor of T cell activation, tacrolimus, will reduce the
symptoms and signs of a severe form of allergic conjunctivitis
involving a corneal ulcer [2]. In experimental settings, an
eosinophil infiltration can occur independent of the presence
of mast cells [3,4]. Thus, the question arises as to how mast
cells contribute to ocular inflammatory cell recruitment. Are
conjunctival mast cells simply serving to induce the clinical
symptoms during the early phase and play a subordinate part
in the final late phase inflammatory process?
To answer this question and to evaluate the contribution
of mast cells to the late phase inflammatory process, we have
developed a mast cell reconstituted model. Bone marrow-
derived mast cells (BMMCs) are commonly used to restore
mast cell populations. However, these cells are not
appropriate for the analyses of ocular responses because
BMMCs have a distinct lineage from mast cells in connective
tissues such as those residing in the conjunctiva [5]. Indeed,
important maturation markers of connective tissue type mast
cells such as CCR3, a receptor of eotaxin-1, are not expressed
by BMMCs [5]. For inflammatory responses, mast cells are
recruited to the effector site from the bone marrow, and they
mature in situ presumably by differentiating into an
inflammatory phenotype by chemokine receptors. Thus,
conjunctival allergic responses are mixed phenomena induced
by tissue-residing mast cells and newly recruited mast cells.
Molecular Vision 2008; 14:1525-1532 <http://www.molvis.org/molvis/v14/a182>
Received 4 July 2008 | Accepted 15 August 2008 | Published 22 August 2008
© 2008 Molecular Vision
1525
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We have developed an adoptive transfer model in which
the conjunctiva of mast cell-deficient mice is reconstituted
with conjunctival mast cells from wild type mice. After the
induction of allergen-specific allergic conjunctivitis, we
carefully assessed the contribution of mast cells to the early
phase and late phase responses.
METHODS
Animals: Mast cell-deficient mice were of the Kitw/Kitw-v
strain. c-Kit is a protein that is essential for the development
and activation of mast cell lineage cells. Kitw/Kitw-v mice carry
point mutations in the transmembrane and kinase domains and
therefore lack the ability to activate the signaling cascade
initiated by its ligand, stem cell factor (SCF). The mast cell
deficiency is greater than 99% in the systemic circulation,
airway, intestine, and skin of these mice [6]. The mast cell-
deficient WBB6F1-Kitw/Kitw-v (Kitw/Kitw-v) mice and congenic
wild-type (WBB6F1+/+) mice were purchased from Shimizu
Laboratories Supplies (Kyoto, Japan). The wild type mice
were age-matched and gender-matched and reared under
identical conditions as the mast cell-deficient Kitw/Kitw-v mice.
The procedures used conformed to all of the regulations for
laboratory animal research outlined by the Animal Welfare
Act, the Department of Health, Education, and Welfare (NIH)
guidelines, and the ARVO statement for the experimental use
of animals.
Immunization and induction of experimental allergic
conjunctivitis: Kitw/Kitw-v and wild type mice were allergen-
sensitized using protocols developed in our laboratory [7-9].
We used two established immunization protocols; one used a
single exposure and the other using multiple exposures [7-9].
In both models, the inflammatory cell recruitment in the late
phase is dependent on mast cell degranulation.
For the single exposure protocol [8], anesthetized mice
were injected with a suspension of 50 μg of ragweed pollen
(ICN, Aurora, OH) and 1 mg of aluminum hydroxide (Sigma,
St. Louis, MO) into the left hind footpad. On day 22,
conjunctivitis was induced by topical application of 1.5 mg of
ragweed suspended in 10 μl of phosphate buffered saline
(PBS). Control mice were mock-sensitized with aluminum
hydroxide and challenged identically with the ragweed
suspension.
The clinical responses were graded using a modified
version of our published method 20 min after the allergen
challenge [7-9]. Conjunctival edema, lid edema, tear/
discharge, and conjunctival redness were graded from 0 to 4
by an observer who was masked to the treatment protocol of
the mice (Table 1). The cumulative clinical score was
calculated as the sum of the scores of each of the four
parameters with a range from 0 to 16.
For the repeated exposure protocol, groups of mice (10
mice per group) were initially injected intraperitoneally with
1 mg of aluminum hydroxide that was conjugated with cat
dander extract (200 BAU/mouse; ALK Laboratories,
Horsholm, Denmark) on days 1, 14, and 24. Control mice were
injected with the vehicle of the dander extract on the same
days. Concomitantly, aluminum hydroxide (25 μg/eye)-
conjugated cat dander extract (10,000 BAU/ml) or vehicle
was applied topically to the eye on days 1, 2, 3, 7, and 14.
Thereafter, mice were exposed to the allergen for sensitization
once a week by topical instillation of cat dander extract
(10,000 BAU/ml) or vehicle onto the eye. Eight weeks after
the initial sensitization, affinity-purified Fel d 1 (0.5 mg/ml),
cat dander extract (10,000 BAU/ml; Greer Laboratories,
Lenoir, NC), or vehicle was applied to the eyes (10 μl /eye)
for two consecutive days for the final priming. The early phase
and late phase responses were elicited by instilling cat hair
allergen (Fel d 1) to all mice 24 h after the last allergen
sensitization.
For histological evaluation, mice were sacrificed 2 h after
the challenge to investigate the early phase response and 24 h
after the challenge to examine the late phase inflammatory
response. The tissues from the eyes were fixed in 4%
paraformaldehyde and embedded in HistoResin (Leica
Molecular Vision 2008; 14:1525-1532 <http://www.molvis.org/molvis/v14/a182> © 2008 Molecular Vision
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TABLE 1. GRADING CRITERIA OF CLINICAL SCORES.
Criteria 012 3 4
conjunctival edema none focal edema edema confined
within one quadrant
edema extending to 3
quadrants
massive edema.
lid edema none slightly narrowed palpebral
fissure (3/4 normal width)
with congestionsevere edema
(1/3 of normal width)
narrowed palpebral
fissure with edema
(2/3 of normal width)
narrowed fissure with
severe edema (1/3 of
normal width)
massive edema
(cornea barely visible).
tear/discharge minimal level of
tear meniscus
increased tear level with
concave meniscus
increased tear level
with convex meniscus
highly increased tear
level with mucous
secretion
excessive tearing with
copious discharge
conjunctival redness none barely detectable venous
dilatation
three or four dilated
vessels at corneal
limbus
five or six dilated
vessels at limbus
marked ciliary
injection
The clinical responses were graded at 20 min after allergen challenge.
Instruments GmbH, Heidelberg, Germany). Serial sagittal
sections (3 μm thick) were cut and stained with toluidine blue,
Giemsa, or hematoxylin and eosin. Three serial sections of the
conjunctival tissue were examined from each eye to determine
the number of inflammatory cells recruited into the
conjunctiva. A masked observer counted the number of cells
under a 400X microscopic field.
Measurement of allergen-specific IgE and IgG1 antibodies:
Sera were collected by cardiac puncture 24 h after the allergen
challenge. The Fel d 1-specific IgE and IgG1 levels were
measured by ELISA [9,10]. The sera were pipetted onto a Fel
d 1-coated microtiter plate (Maxisorp; Nalge Nunc
International KK, Tokyo, Japan), incubated with biotin-
conjugated anti-IgE (BD Biosciences, Franklin Lakes, NJ) or
anti-IgG1 (SouthernBiotech, Birmingham, AL) antibodies,
and developed for peroxidase-based substrate detection.
Isolation and FACS analysis of mast cells from the
conjunctiva: The protocol to isolate crude conjunctival mast
cells was developed and optimized based on an enzymatic
digestion method. Briefly, the conjunctiva of eight-week-old
to 12-week-old SWR/J mice were cut into fragments and
incubated in RPMI1640 medium supplemented with 10%
fetal bovine serum, 1.5 mg/ml collagenase (Nitta Gelatin,
Osaka, Japan), 0.5 mg/ml hyaluronidase (Sigma), and 0.5 mg/
ml DNase I (Sigma) for 2 h at 37 °C. The dispersed cells were
filtered through a 40 μm cell strainer, layered on an isotonic
Percoll density medium (density=1.041, Amersham
Pharmacia Biotech, Piscataway, NJ), and centrifuged at 800x
g for 20 min [11]. The pellet was cultured in RPMI1640 with
murine recombinant IL-3 (10 ng/ml; Peprotech, Rocky Hill,
NJ), recombinant SCF (10 ng/ml; Peprotech), and 5% serum
for two days. Colony-forming, non-adherent mast cells were
harvested by gentle shaking and decanting of the flasks. The
cells were then layered onto a Percoll density medium
(density=1.041) and centrifuged at 800x g for 20 min.
To precisely evaluate the roles of mast cells, a further
purification protocol was developed for the functional
analyses. Based on preliminary FACS analyses, contaminated
populations included dendritic cells, macrophages, and
plasma cells, which were characterized by the expression of
CD4, CD8a, CD11b, B220, and PDCA-1. These markers were
selected and confirmed for appropriateness for depletion
markers based on the co-expression of mature mast cell
markers such as CCR3 and CXCR3 (data not shown). To
deplete these cells, we used a magnet–based negative
depletion method to obtain maximum recovery. Briefly, cells
were blocked by anti-CD32 (Clone 93; eBiosciences, San
Diego, CA) and labeled with rat anti-CD4, rat anti-CD8a, rat
anti-CD11b, rat anti-B220 (all from eBiosciences), and rat
anti-PDCA-1 (Miltenyi Biotec Inc., Auburn, CA). The labeled
fractions were depleted using anti-rat immunoglobulin κ chain
antibody-conjugated microbeads (IMag; BD Biosciences).
For FACS analyses, cultured mast cells were blocked by
anti-CD32 and stained for immunofluorescence analysis. The
analysis was performed with the FACSCalibur flow
cytometer (Becton Dickinson, Franklin Lakes, NJ).
Phycoerythrin (PE)-conjugated anti-CXCR3 antibody (R&D
Systems, Minneapolis, MN), biotin-conjugated anti-c-Kit
antibody (2B8; eBiosciences), PE-conjugated anti-c-Kit
antibody (eBiosciences), FITC-conjugated-anti-FcεRI α
subunit (MAR-1, eBiosciences), and Peridinin-chlorophyll-
protein (PerCP)-conjugated streptavidin (BD Biosciences)
were used for staining the mast cells. FITC-conjugated IgG
(eBiosciences), PE-conjugated IgG (eBiosciences), PerCP-
conjugated IgG (BD Biosciences), and biotin-conjugated IgG
(eBiosciences) were used for isotype control staining.
Adoptive transfer of isolated conjunctival mast cells: Purified
mast cells (4×105 cells/eye) from wild type mice were injected
subconjunctivally into allergen-sensitized, mast cell-deficient
Kitw/Kitw-v mice. Immediate hypersensitivity was induced by
allergen exposure two weeks after the transfer. Connective
tissue type mast cells were identified in the recipient mice and
control wild type mice by measuring the conjunctival
transcripts of mMCP-5, mMCP-6, and FcεRI α subunit by
quantitative reverse transcription polymerase chain reaction
(RT–PCR) and Giemsa staining.
Statistical analyses: Data are presented as the means
±standard error of the means (SEMs). Statistical analyses
were performed by ANOVA.
RESULTS
Mast cells required for early phase response of allergic
conjunctivitis: We first examined whether the tissues on the
ocular surface of Kitw/Kitw-v mice had any mature mast cells
that could be identified by the presence of metachromatic
granules. An earlier investigation showed that mast cells were
not present in different mucosal tissues of these mice [6], and
in confirmation, we found that mast cells were absent in the
conjunctiva, eyelids, and choroid of naïve Kitw/Kitw-v mice.
To evaluate the contribution of mast cells to the ocular
symptoms during the early phase allergic response, we
challenged sensitized mice with the allergen following the
single exposure protocol. Mice were assessed for clinical
signs including conjunctival edema, conjunctival redness,
tearing, and lid edema. The degree of inflammation during the
early phase is summarized as clinical scores in Figure 1A. The
clinical scores for both sensitized and mock-sensitized Kitw/
Kitw-v mice were significantly lower following the allergen
challenge than that of wild type mice (sensitized wild type:
8.7±0.3, sensitized Kitw/Kitw-v: 1.8±0.7, p<0.05). The clinical
scores for Kitw/Kitw-v mice appeared not specific for the
allergen because no significant difference was observed
between the sensitized and mock-sensitized mice. This is
consistent with the concept that conjunctival mast cells
mediate the clinical signs after the allergen challenge.
To examine the contribution of mast cells to the clinical
signs in allergic inflammation in more detail, we restored
Molecular Vision 2008; 14:1525-1532 <http://www.molvis.org/molvis/v14/a182> © 2008 Molecular Vision
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Figure 1. Effect of adoptive transfer of
mast cells isolated from wild type mice
to mast cell-deficient mice on the
clinical responses in the acute phase.
A: Recovery of defective acute phase
clinical responses in Kitw/Kitw-v mice
after adoptive subconjunctival transfer
of conjunctival mast cells from wild
type cells is shown. The clinical scores
for both sensitized and mock-sensitized
Kitw/Kitw-v mice were significantly
lower following allergen challenge than
that of wild type mice. B: Allergen-
induced-mast cell degranulation in Kitw/
Kitw-v mice after adoptive transfer of
conjunctival mast cells from wild type
mice is demonstrated in the chart. Mast
cell degranulation in the conjunctiva,
undetectable in Kitw/Kitw-v mice, was
observed in both sensitized and mock-
sensitized adoptive transfer mice. C:
Expression of mast cell-restricted
proteases, mMCP-5 and mMCP-6, in
the conjunctiva of Kitw/Kitw-v mice after
adoptive transfer is shown. Mice
injected with the conjunctival mast cells
had levels of mMCP-5 and mMCP-6
that were comparable to those of wild
type mice. D: Expression of CXCR3 as
a maturation marker on conjunctiva-
derived FcεRI+ c-Kit+ mast cells by
FACS analysis is demonstrated. Red
indicates the CXCR3 stained
conjunctiva-derived mast cells. Black
shows the isotype control. RW:
sensitized with ragweed pollen; PBS:
mock sensitized; n=10 mice/group.
Molecular Vision 2008; 14:1525-1532 <http://www.molvis.org/molvis/v14/a182> © 2008 Molecular Vision
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conjunctival mast cells to the mast cell-deficient Kitw/Kitw-v
mice. Conjunctival mast cells, isolated by lineage marker
depletion after density-gradient separation, were tested for
allergen-induced degranulation and mast cell-restricted
proteases. The conjunctiva-derived mast cells expressed high
levels of the connective tissue type mast cell proteases,
mMCP-5, mMCP-6, and mMCP-7. When these cells were
sensitized with anti-DNP-specific IgE and exposed to DNP-
albumin, they appropriately degranulated in an allergen-
specific manner (data not shown).
Peripheral tissues, including the conjunctiva, are
continuously replenished by the migration of bone marrow-
derived mast cells. To exclude any effect of newly immigrated
cells from the bone marrow, the conjunctiva of mast cell-
deficient mice were reconstituted with the conjunctiva-
derived mast cells from the wild type mice.
The conjunctival mast cells that were expressing CXCR3
as a maturation marker of conjunctival mast cells (Figure 1D)
[5] and isolated from naive wild type mice were adoptively
transferred into the allergen-sensitized Kitw/Kitw-v mice by
subconjunctival injection. Conjunctival expression of the
mast cell restricted transcripts, mMCP-5 and mMCP-6, was
used to assess the success of the transfer. As expected,
mMCP-5 and mMCP-6 were not expressed at appreciable
levels in the conjunctiva of Kitw/Kitw-v mice (Figure 1C). In
contrast, mice injected with the conjunctival mast cells had
levels of mMCP-5 and mMCP-6 that were comparable to
those of wild type mice (Figure 1C). This confirmed that the
transferred mast cells maintained the properties of the
connective tissue type as conjunctival mast cells.
To examine their homing ability, real time PCR was
conducted to examine levels of mast cell-specific proteases in
the draining lymph nodes of the adoptive transfer mice.
Neither mMCP-5 nor mMCP-6 was detected in the adoptive
transfer mice (below detection limits). This indicated that the
transferred mast cells remained in the conjunctiva and did not
migrate elsewhere.
When the adoptive, transferred Kitw/Kitw-v mice were
challenged with an allergen, clinical signs of allergic
inflammation were restored (Figure 1A). Mast cell
degranulation in the conjunctiva, undetectable in Kitw/Kitw-v
mice, was observed in both sensitized and mock-sensitized
adoptive transfer mice. The sensitized mice had significantly
more allergen-specific mast cell degranulation than the mock-
sensitized mice (53% versus 17%, p<0.05; Figure 1B). The
restored response was indistinguishable from that of the wild
type mouse response, supporting the validity of our adoptive
transfer model using Kitw/Kitw-v mice with conjunctival mast
cells.
We have shown that the degree of mast cell degranulation
is linearly correlated with the clinical scores [9]. Thus,
conjunctival mast cells are necessary and sufficient for
activation of the acute phase clinical responses.
In clinical settings, allergic symptoms are provoked after
repeated exposure to an allergen. We have shown that
repeated exposure to an allergen aggravated the clinical signs
and altered mast cell restricted proteases [12]. Therefore, we
tested whether repeated allergic exposure could stimulate
mast cell recruitment or maturation to correct the conjunctival
mast cell deficiency in Kitw/Kitw-v mice. Our results showed
that even after repeated allergen exposures, conjunctival mast
cells were not detected in sensitized Kitw/Kitw-v mice. The
clinical scores were also significantly lower in the sensitized
mast cell-deficient mice than in sensitized wild type mice
following multiple allergen exposures, (1.8 versus 10.5,
p<0.05; Figure 2). The clinical signs were completely
suppressed, and the tear/discharge score was depressed by
78%.
To determine whether mast cell deficiency affected
antigen-specific B cell and T cell responses during allergic
conjunctivitis, levels of allergen-specific IgE and IgG1 were
measured after the final allergen challenge. The titers of IgE
and IgG reflect the function of both B cells and T cells because
an increased production of IgE and IgG1 requires numerous B
cell and T cell processes including antigen presentation,
antigen-specific T cell expansion, class switch recombination
of B cells, and specific antibody production.
Allergen-specific IgE and IgG1 were appropriately
induced following the allergen challenge in sensitized wild
type mice but not in mock-sensitized wild type mice (Figure
3). Levels of allergen-specific IgE and IgG1 were not
significantly different between Kitw/Kitw-v mice and wild type
mice, indicating that the inductive arm of Kitw/Kitw-v mice was
still functioning normally during the sensitization phase.
Requirement of mast cells for late phase response of allergic
conjunctivitis: We evaluated the dependency of the late phase
Figure 2. Abolishment of early phase clinical symptoms in mast cell-
deficient mice. The clinical scores were also significantly lower in
the sensitized mast cell-deficient mice than in sensitized wild type
mice following multiple allergen exposures. An asterisk indicates
that p<0.005. Fel d 1: sensitized with Fel d 1; PBS: mock-sensitized;
n=10 mice/group.
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inflammatory response on mast cells by examining
recruitment of inflammatory cells 24 h after the allergen
challenge. To exclude the possible effects of preconditioning
by repeated exposures, we first used the single exposure
protocol for sensitization. Allergen-specific, late phase
responses in wild type mice were characterized mainly by
conjunctival infiltration of eosinophils. Foci of inflammatory
cells were accompanied or surrounded by mast cells, implying
some role for the mast cells in this process (Figure 4A). No
increase in conjunctival infiltration of eosinophils and
neutrophils was observed in sensitized Kitw/Kitw-v mice 24 h
after the allergen challenge (Figure 4B). However, when
conjunctival mast cells were transferred to sensitized Kitw/
Kitw-v mice, the allergen-specific eosinophil recruitment in the
transferred mice was completely restored (Figure 4B). These
observations clearly demonstrated that the late phase
eosinophil infiltration is dependent on mast cell activity.
Interestingly, we did not observe an increase in neutrophil
recruitment in any of the sensitized animals compared to their
mock-sensitized counterparts (Figure 4B).
We next sensitized mice using the repeated allergen
exposure model [9] and then evaluated mast cell contributions
to the local activation or preconditioning of bystander cells or
mast cells. In wild type mice, an allergen challenge
significantly increased the number of conjunctival eosinophils
in the sensitized animals but not in the mock-sensitized
animals (Figure 5). However, the number of conjunctival
eosinophils was not significantly increased in sensitized Kitw/
Kitw-v mice (Figure 5B). The conjunctival eosinophil count
following allergen exposure was significantly lower in Kitw/
Kitw-v mice than in wild type mice (19.1 versus 9.0, p<0.005),
although there was no significant difference in the number of
eosinophils between the mock-sensitized wild type and Kitw/
Kitw-v mice. All changes in the neutrophil count following the
allergen challenge were not significant (Figure 5B).
Taken together, the data from the adoptive transfer model
have shown that conjunctival mast cells are essential for
eosinophil recruitment in the early phase and late phase
Figure 3. Induction of Fel d1-specific IgE and IgG1 in Kitw/Kitw-v
mice. Kitw/Kitw-v mice are not impaired for Fel d 1-specific IgE or
IgG1. Fel d 1: sensitized with Fel d 1; PBS: mock-sensitized; n=10
mice/group.
allergic responses in the eye but not essential for neutrophil
recruitment.
DISCUSSION
Our results demonstrated important aspects of mast cell
involvement in ocular allergy (i.e., the activation of locally
residing conjunctival mast cells) after an allergen exposure
induced the recruitment of eosinophils. This is in marked
contrast to the conclusions of Ueta et al. [4] who reported that
mast cells were not required for eosinophil recruitment in
allergic conjunctivitis using antigen-sensitized, mast cell-
deficient mice. However, their conclusion considered only
one aspect of allergic conjunctivitis. It is known that allergic
conjunctivitis is triggered by the mast cell degranulation-
mediated cascade, and this needs to be clearly shown in the
model being evaluated for mast cell dependence. However,
eosinophilia is a good indicator of allergic conjunctivitis.
Indeed, the mast cell-mediated activation, which was not
presented in their report, primes Th2-type T cells-dependent
eosiophil recruitment. For example, the transfer of Th2-type
T cells can induce massive eosinophilic inflammation. Also,
in the corneal transplantation model, inflammatory responses
Figure 4. Impairment of late phase inflammatory responses in Kitw/
Kitw-v mice and subsequent response recovery in Kitw/Kitw-v mouse
recipients of conjunctival mast cells. A: Kitw/Kitw-v mice,
subconjunctivally injected with wild type conjunctival mast cells,
were challenged with an allergen, and the conjunctiva were
processed for Giemsa staining after 24 h. The left image shows
conjunctiva of mock-sensitized Kitw/Kitw-v adoptive transfer mice.
The right image shows conjunctiva of allergen-sensitized Kitw/Kitw-
v adoptive transfer mice. Each asterisk in the images denotes the
presence of eosinophils, and an arrow indicates transferred mast
cells. B: Recovery of defective eosinophil recruitment in Kitw/Kitw-
v mice by subconjunctival adoptive transfer of conjunctival mast cells
from wild type mice is shown in the chart. The sharp (hash mark)
means that p<0.05. RW: sensitized with ragweed pollen; PBS: mock
sensitized; n=10 mice/group.
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can be eosinophilic in certain donor and recipient
combinations [13]. Thus, one can create experimental settings
where the involvement of mast cells is least required.
Evaluation of eosinophilic inflammation in allergic
conjunctivitis needs to be evaluated by the activation of mast
cells.
We do not argue that eosinophilia can occur with a
minimal activation of mast cells. This situation can be created
by repetitive and strong immunizations. Ueta et al. [4] used
an intradermal injection of ragweed allergen followed by
multiple boosting by two intraperitoneal injections and four
repetitive allergen exposures. This would usually result in a
vigorous T cell-mediated response and clonal expansion that
should culminate in a massive eosinophil recruitment after an
antigen challenge [3,14,15]. In addition, they claimed that the
attenuated protocol (one intradermal injection followed by
one intraperitoneal injection) still provoked allergen-induced
eosinophil recruitment in mast cell-deficient mice. However,
this model also appeared to be independent of IgE-mediated
mast cell degranulation because no allergen-specific IgE was
detected in the sera of their experimental mice.
Figure 5. Impairment of late phase inflammatory responses in Kitw/
Kitw-v mice. A: Kitw/Kitw-v mice were challenged with an allergen,
and the conjunctiva were processed for Giemsa staining after 24 h.
The asterisk in the image denotes mast cells, and an arrow denotes
the presence of eosinophils. 400X magnification. B: Recruitment of
eosinophils and neutrophils in the conjunctiva of Kitw/Kitw-v mice is
show in the chart. The sharp (hash mark) denotes that p<0.005 and
the double sharp denotes that p<0.05. Fel d1: sensitized with Fel d1;
PBS: mock sensitized; n=10 mice/group.
The contribution of mast cells to the eosinophilic
responses can be explained by the properties of the cytokines
and chemokines released upon activation. For example,
conjunctival mast cells activated by FcεRI produce IL-2, IL-3,
IL-4, IL-5, IL-6, IL-10, IL-12, GM-CSF, MIP-1α, and TNF-
α (unpublished observations). IL-3 and IL-5 are particularly
likely to contribute to eosinophilic inflammation as they play
critical roles in eosinophil development, survival, and
recruitment [16]. Other mast cell-derived mediators that might
contribute to eosinophil activity in ocular hypersensitivity
include the leukotrienes, B4 and D4, and tryptases [17,18].
These observations are consistent with the idea that T cell
priming is required for mast cell-related eosinophilia and
support our findings that mast cells mediated the eosinophil
recruitment in the late phase inflammation.
It has been suggested that the role of conjunctival mast
cells was to induce neutrophilic inflammation. For example,
when mast cell degranulation is induced in the conjunctiva by
compound 48/80, the resulting conjunctivitis is characterized
by infiltration of neutrophils, macrophages, and CD4+ T
lymphocytes but surprisingly few eosinophils [19]. This is in
marked contrast to our observations, which showed that
neutrophilic inflammation does not require conjunctival mast
cells. This might be due to the relative low levels of
neutrophilic cytokines derived from mature mast cells. The
mononuclear cells and macrophages are the major sources of
neutrophilic cytokines such as TNF-α and MIP-1α. We
suggest that the neutrophilic inflammation induced by
compound 48/80 may be explained by such mediators derived
from mononuclear cells and macrophages, which are
indirectly stimulated by mast cell-derived chemokines or
cytokines.
In conclusion, our new mast cell reconstituted model is a
better method to investigate what role conjunctiva-residing
mast cells play in experimental allergic conjunctivitis. This
allowed us to evaluate the direct contribution of mast cells to
eosinophilic inflammation. Our data support the idea that
regulation of mast cell activation and maturation in addition
to current therapy to suppress mast cell degranulation might
be promising therapeutic strategies. Direct targeting of c-Kit,
mast cell-derived chemokines, or chemokine receptors might
also yield good therapeutic results [9,20-22].
ACKNOWLEDGMENTS
This study was supported in part by Grant-in-Aid from the
Ministry of Education, Science, Sports, and Culture of Japan.
The authors thank Dr. Duco Hamasaki for helpful comments
and editing.
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article through that date. Details of any changes may be found in the online version of the article.
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    • "IL-8 is produced by eosinophils [28] and is involved in eosinophil migration and survival, which are two relevant aspects in chronic allergic diseases [29]. We also investigated the compound's action on cytokine and chemokine secretion in the human mast cell line HMC-1 as these can greatly influence eosinophil activity in inflamed ocular tissues [2]. Mapracorat and dexamethasone (0.001–10 μM) both reduced IL-8 release induced by ionomycin in eosinophils cells in a concentration-related manner (Figure 5). "
    [Show abstract] [Hide abstract] ABSTRACT: Glucocorticoids can either suppress gene transcription (transrepression) or activate it (transactivation). This latter process may contribute to certain side effects caused by these agents. Mapracorat (also known as BOL-303242-X or ZK 245186) is a novel selective glucocorticoid receptor agonist that maintains a beneficial anti-inflammatory activity but seems to be less effective in transactivation, resulting in a lower potential for side effects; it has been proposed for the topical treatment of inflammatory skin disorders. This study assessed the anti-allergic activity of mapracorat at the ocular level and whether eosinophils and mast cells are targets of its action. With in vitro studies apoptosis was evaluated in human eosinophils by flow cytometry and western blot of caspase-3 fragments. Eosinophil migration toward platelet-activating factor was evaluated by transwell assays. Interleukin (IL)-6, IL-8, tumor necrosis factor-α (TNF-α), and the chemokine (C-C motif) ligand 5 (CCL5)/regulated upon activation normal T cell expressed, and presumably secreted (RANTES) were measured using a high-throughput multiplex luminex technology. Annexin I and the chemochine receptor C-X-C chemokine receptor 4 (CXCR4) were detected by flow cytometry. With in vivo studies, allergic conjunctivitis was induced in guinea pigs sensitized to ovalbumin by an ocular allergen challenge and evaluated by a clinical score. Conjunctival eosinophils were determined by microscopy or eosinophil peroxidase assay. In cultured human eosinophils, mapracorat showed the same potency as dexamethasone but displayed higher efficacy in increasing spontaneous apoptosis and in counteracting cytokine-sustained eosinophil survival. These effects were prevented by the glucocorticoid receptor antagonist mifepristone. Mapracorat inhibited eosinophil migration and IL-8 release from eosinophils or the release of IL-6, IL-8, CCL5/RANTES, and TNF-α from a human mast cell line with equal potency as dexamethasone, whereas it was clearly less potent than this glucocorticoid in inducing annexin I and CXCR4 expression on the human eosinophil surface; this was taken as a possible sign of glucocorticoid-dependent transactivation. In the guinea pig, mapracorat or dexamethasone eye drops induced an analogous reduction in clinical symptoms of allergic conjunctivitis and conjunctival eosinophil accumulation. Mapracorat appears to be a promising candidate for the topical treatment of allergic eye disorders. It maintains an anti-allergic profile similar to that of dexamethasone but seems to have fewer transactivation effects in comparison to this classical glucocorticoid. Some of its cellular targets may contribute to eosinophil apoptosis and/or to preventing their recruitment and activation and to inhibiting the release of cytokines and chemokines.
    Full-text · Article · Dec 2011
    • "The early phase response develops immediately after exposure to the allergen with clinical symptoms and signs such as itching, chemosis and congestion. This is followed by the late phase response after 8-24 hours which is characterized by conjunctival cellular infiltrations particularly eosinophilia and neutophilia (Miyazaki et al., 2008). The pathophysiology of allergic conjunctivitis is not a simple process, and a wide range of cytokines, chemokines, proteases and growth factors are involved by complex interrelated interactions (Leonardi et al., 2008). "
    Full-text · Chapter · Nov 2011 · Molecular vision
    • "The activation of mast cells leads to the release of powerful vasoactive amines that are responsible for the vasodilatation and increased permeability of blood vessels. In addition, the activated mast cells can recruit eosinophils to participate in the allergic reaction [29]. Conjunctival epithelium and corneal epithelium are also involved in the pathogenesis of AC. "
    [Show abstract] [Hide abstract] ABSTRACT: Allergic conjunctivitis (AC) has been reported to induce the instability of the tear film. The tear protein and the lipid layer play important roles in maintaining the tear film. The aim of this study was to quantify the alteration of the major tear protein components and a lipid related protein secretory type IIa phospholipase A2 (sPLA2-IIa) in tears of seasonal allergic conjunctivitis (SAC) and perennial allergic conjunctivitis (PAC) patients. Twenty-one SAC and PAC patients and thirteen normal controls completed a symptom questionnaire and underwent regular ocular examination. SAC and PAC patients were diagnosed based on the clinical presentation and elevated serum IgE levels. Schirmer test paper was used to collect tear samples from SAC and PAC patients and normal controls. Soybean trypsin inhibitor (SBTI) was used as an internal standard to analyze tear samples in 15% SDS-PAGE gel. Total tear protein and its major components from the SAC and PAC patients and normal controls were quantified by band densitometry. The major tear protein bands were determined by MALDI-TOF/TOF spectrum analysis. Western blot was used to detect the content of sPLA2-IIa in tears of allergic conjunctivitis patients and normal controls. Schirmer test scores were more than 10 mm in all the SAC and PAC patients and control subjects. The tear film breakup time of SAC and PAC patients was much shorter than that of the normal controls. We obtained 15 bands of tear protein by one dimensional SDS-PAGE, in which 14 bands were determined by mass-spectrum analysis. The band densitometry analysis revealed that the total tear protein concentration was much higher in SAC and PAC patients than in normal controls (p<0.05). The quantity of tear protein band 4 (serum albumin precursor), band 6 (Ig gamma-2), band 9 (leukocyte elastase inhibitor) were also significantly higher in AC patients (p<0.05). Content of sPLA2-IIa, as shown by western blot, was much higher in AC patients than in controls. The total tear protein concentration and some of the major tear protein components was increased in tears of SAC and PAC patients. In addition, the content of sPLA2-IIa in tears of SAC and PAC patients was elevated. The tear protein changes in SAC and PAC patients may contribute to instability of tear film.
    Full-text · Article · Oct 2010
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