Abstract
purpose. To determine the impact of interleukin-1 (IL-1) inhibition using IL-1 receptor
antagonist (IL-1Ra) in a mouse model of allergic eye disease.
methods. A/J mice sensitized and challenged with cat dander in the eye were
treated with topical IL-1Ra or vehicle alone. Control mice were treated
with IL-1Ra or vehicle but sensitized and challenged with
phosphate-buffered saline alone. Immediately after the final allergen
challenge, the mice were observed for behavioral changes and assessed
for lid injection and chemosis. The animals were then killed, eyes and
attached lids were removed for either RNA extraction or histology, and
draining lymph nodes were removed for either RNA extraction or in vitro
stimulation assays. Differences in chemokine message between
experimental and control groups were determined by RNase protection
assays.
results. Treatment with IL-1Ra in allergen-challenged animals
significantly reduced allergen-induced changes in photosensitivity
(60%, P = 0.0002), chemosis (50%, P = 0.0151), and injection (86.7%, P = 0.0068) compared with vehicle-treated controls.
Interleukin-1Ra reduced the number of degranulated mast cells and
caused a significant reduction in the number of eosinophils
infiltrating the conjunctival matrix (P < 0.001)
after allergen challenge. Examination of chemokine mRNA taken from the
conjunctiva and draining lymph nodes by RNase protection assay showed a
profound decrease in the production of a number of C–C
chemokines.
conclusions. These findings suggest that IL-1Ra is suppressing allergic eye disease
by a down-modulation of the recruitment of eosinophils and other
inflammatory cells essential for the immunopathogenesis of ocular
atopy.
Allergic conjunctivitis (AC), atopic inflammation of the
mucous membrane that lines the eyelid and outer eyeball, affects over
40 million patients per year in the United States.
1 Patients with AC experience itching and burning sensations in the eye
in response to allergens that are innocuous for normal individuals and
show clinical signs of chemosis, tearing, conjunctival hyperemia, and
lid edema.
1 The diagnosis of AC is a clinical one mainly
based on the history and ophthalmologic findings. Conjunctival
scrapings, although not commonly used, are of help because eosinophils
are not ordinarily found in the conjunctival scrapings form nonallergic
individuals, making the presence of even one eosinophil considerable
evidence in favor of a diagnosis of AC.
1
We have developed an experimental murine model for cat-dander–induced
AC, which provides the clinical, cellular, and humoral parameters of
allergic disease. Sensitized mice are challenged via eyedrops with cat
dander extract containing defined amounts of the major cat allergen, a
35-kDa protein known as
Felis domesticus allergen 1
(
Fel d1).
2 As the predominant human IgE binding
component in cat dander,
Fel d1 is known to elicit the
symptoms of perennial AC.
2 Sensitization and challenge
with cat dander results in significant increases in photosensitivity,
itching, chemosis, and conjunctival injection compared with
phosphate-buffered saline (PBS)–challenged control animals. These
early clinical symptoms correlate with mast cell degranulation observed
within 1 hour after challenge, and significant infiltration of
inflammatory cells, including eosinophils, 24 hours later. This model
enables us to carefully study disease pathogenesis and to evaluate new
therapies, for the treatment of AC.
Interleukin 1 (IL-1), a 17.5-kDa cytokine synthesized primarily by
activated macrophages, plays a central role in the initiation and
coordination of host defenses in response to a range of physiological
insults, including trauma, infection, and inflammation. The IL-1 family
consists of two agonists, IL-1α and IL-1β, and a structurally
related specific receptor antagonist, IL-1Ra. Interleukin-1Ra binds
with high avidity to IL-1 receptors but fails to trigger intracellular
responses, thus acting as a competitive inhibitor.
3 Recombinant human IL-1Ra has been used both in vitro and in vivo to
block a range of IL-1–induced biological activities, thereby
validating its use in determining the precise role of IL-1 in
short-term animal models of disease.
In the study reported in this article, we used recombinant IL-1
receptor antagonist to study the effects of IL-1 inhibition in a mouse
model of AC. Interleukin-1 has been found to regulate chemokines and
adhesion factors, the upregulation of which has been closely linked to
the development of allergic disease.
3 Therefore, we
hypothesized that treatment with IL-1Ra could suppress ocular allergy.
Our results suggest that IL-1Ra can effectively treat allergic eye
disease by decreasing the expression of chemokines essential for the
recruitment of allergy-inducing inflammatory cells, including
eosinophils.
Six-week-old A/J mice were obtained from Jackson Laboratory (Bar
Harbor, ME). To prevent any potential aerosolization of allergen from
affecting PBS-challenged control animals, all mice were kept in cages
with filter-topped lids. The studies reported here conformed to the
principles for laboratory animal research outlined by the Animal
Welfare Act and the National Institute of Health guidelines for the
experimental use of animals and complied with the ARVO Statement for
the Use of Animals in Ophthalmic and Vision Research. We used 10 mice
for each experimental group at each time point studied, unless
specified otherwise.
We sensitized the mice to cat dander by local administration via
eye drops of prepared cat dander extract containing quantitated amounts
of Fel d1 (ALK; 250 AU/eye) drop-wise in the eye
(2.5 μl/eye) with the use of a p10 micropipetteman (Eppendorf). Mice
were then challenged with the same amount of allergen via eyedrops once
per week for the next 4 weeks. Control mice were challenged with a
similar volume of PBS (sham challenge).
Two percent IL-1Ra (20 mg/ml) in 0.2% sodium hyaluronate was
administered to both cat dander–challenged and sham-challenged (PBS)
control mice three times daily for the duration of the experiment (28
days), starting 30 minutes before the initial sensitization. The dosage
and administration of IL-1Ra treatment used in these experiments
conformed to previous protocols using topical IL-1Ra in models of
inflammatory eye disease.
4 To control for any potential
response to sodium hyaluronate alone, two additional groups (either cat
dander–challenged or sham-challenged) received a similar volume of
vehicle (0.2% sodium hyaluronate).
In all the challenge protocols, the identity of treatment in each
of the cages was masked, and mice were observed by two independent
investigators every 5 minutes for the first half hour after the final
antigen challenge for changes in behavior (i.e., increased
photosensitivity as evidenced by squinting or excessive face washing,
suggesting a response to itching eyes). The animals were also assessed
by two ophthalmologists (DM and ID), using a slit lamp for assessment
of injection and chemosis. Representative photographs of mouse eyes
were used to assess disease severity, and all symptoms were recorded
as ± during each 1-minute timed assessment. The percentage of
animals per cage experiencing a particular symptom was then recorded.
The animals were then killed at 1, 24, 48, or 72 hours after the final
antigen challenge. In all cases, the mice were anesthetized with
ketamine/xylasine (200 mg/kg ketamine + 10 mg/kg xylasine) and
exsanguinated via cardiac puncture for antibody analysis of their sera.
To assess the cellular infiltrate in the conjunctiva and
surrounding tissue, the eyes were removed with the attached lids and
intact conjunctiva, immediately fixed in 4% paraformaldehyde, and
embedded in Historesin (Leica Instruments GmbH, Heidelberg, Germany).
Serial sections were cut from each eye and stained with Giemsa for mast
cells and hematoxylin and eosin (H&E) or Congo red (both from Sigma)
for the identification of eosinophils. All slides were masked, and cell
counts were made by two independent investigators. For cellular
quantitation, five sections were examined from each mouse per treatment
group (n = 10 mice per group), and cells were counted
in five 400× nonoverlapping fields per eyelid section, for a total of
250 400× fields per treatment group. Eosinophils were identified by
red eosin staining of their cytoplasmic granules and their distinctive
bilobed nuclei. Mast cells were identified by their oval contour, mean
diameter of approximately 12 to 13 μm, and cytoplasm filled with
distinctive pink-to-purple granules.
To determine relative chemokine mRNA levels, we used the Riboquant
Multiprobe RNase Protection Assay system (PharMingen, San Diego, CA).
Briefly, draining (cervical) lymph nodes and eyeballs containing
conjunctival samples were removed from the mice immediately after they
were killed. The conjunctiva was dissected from its attachments to the
eyelids. The tissues were placed in 2 ml of RNAStat 60, homogenized,
and immediately frozen on dry ice. The samples were stored at −80°C
until used. For use, separate RNA samples from five mice per
experimental group were defrosted on ice, and the RNA was extracted
with a phenol chloroform procedure using DEPC-treated materials.
Aliquots of the samples were run on a 1% agarose gel to determine
concentration and ascertain any potential degradation. The samples were
then used as per manufacturer’s instructions with the mCK-5 probe
positive for lymphotaxin (Ltn), Regulated on activation normal T cell
expressed (RANTES), eotaxin, macrophage inflammatory peptide-1α
(MIP-1α), MIP-1β, MIP-2, interleukin-gamma–induced protein 10
(IP-10), monocyte chemotactic protein-1 (MCP-1), T cell activation
gene-3 (TCA-3), and the housekeeping genes L32 and GAPDH. To
semiquantify differences in amounts of RNA, the film was scanned after
development, and the bands were subjected to densitometry using Digital
Science ID software (Kodak). The numerical values were normalized
according to the densitometry of the housekeeping genes GAPDH and L32.
Data are summarized as mean ± SE. The statistical analysis
of the results was performed by ANOVA using Fisher’s least significant
differences test for multiple comparisons. P < 0.05
was considered significant.
To control for any potential effects of either IL-1Ra treatment or
vehicle, a second set of mice were sham-challenged with PBS. Previous
studies in our laboratory did not find any discernible difference
between PBS-challenged and naive control animals. Antigen treatment of
sensitized mice caused significant increases in clinical signs of AC,
including an increase in photosensitivity (86.6%,
P =
0.0001) indicated by marked squinting, itching as evidenced by vigorous
face-washing and scratching about the eyes
5 (66.6%,
P = 0.001), and chemosis (50%,
P =
0.001) and injection (100%,
P = 0.0001) of the
conjunctiva compared with sham-challenged control animals. The allergic
symptoms began within 5 minutes after antigen challenge and peaked 20
minutes after the final challenge
(Fig. 1) . Control mice sham-challenged with PBS displayed none of these
symptoms and appeared physiologically normal. Treatment with IL-1Ra in
allergen-challenged animals significantly reduced allergen-induced
changes in photosensitivity (60%,
P = 0.0002),
chemosis (50%,
P = 0.0151), and injection (86.7%,
P = 0.0068), reducing symptoms to levels
indistinguishable from the sham-challenged control groups.
Reduction in Allergen-Induced Eosinophil Infiltration
and Mast Cell Degranulation after IL-1Ra Treatment
To assess the cellular infiltration in the conjunctiva and
surrounding tissue, the intact eyes and lids were sectioned and stained
with either hematoxylin and eosin or Congo red for the assessment of
eosinophils, or with Giemsa for the assessment of intact mast cells.
The groups were masked, and slides were counted by two independent
investigators. There was a significant increase in the numbers of
degranulated mast cells in the matrix surrounding the conjunctiva after
allergen challenge compared with sham-challenged controls (301%,
P = 0.03;
Fig. 2 A). Treatment with IL-1Ra caused a modest, statistically insignificant
decrease in mast cell degranulation in cat dander–challenged animals
(
P = 0.1846;
Fig. 2A ).
Sham-challenged control mice had few eosinophils in their eyelids and
conjunctiva. At 24 hours after the final allergen challenge, there were
significant increases in the total number of eosinophils infiltrating
into the matrix surrounding the conjunctiva, and especially in the
fornical region, compared with sham-challenged controls
(Fig. 2B) (598%,
P = 0.0001). Cell counts on conjunctiva from
animals treated with IL-1Ra showed a reduction in the numbers of
eosinophils to levels similar to that observed in the sham-challenged
control animals (
P = 0.998), suggesting an ablation of
antigen-induced eosinophil influx.
The comparatively small amount of tissue available from the mouse
conjunctiva resulted in relatively low total concentrations of RNA and
therefore weaker overall signals than what could be observed from other
tissues, including the draining lymph nodes
(Fig. 3) . However, we were able to observe a reduction in eotaxin and RANTES
mRNA in the conjunctiva after IL-1Ra treatment in both the
allergen-challenged and sham-challenged control groups (
Fig. 4 A). The levels of gene expression for eotaxin and RANTES in cat dander–
and IL-1Ra–treated conjunctival samples were comparable to levels seen
in sham-challenged control animals.
Because there are a large number of articles showing that
chemokine expression in the draining lymph node of an inflamed organ
affects the expression of immunity and inflammation in the organ
itself
6 ; we also examined the level of chemokine
expression in the draining (cervical) lymph nodes. Cat dander challenge
caused significant increases in the gene expression of the chemokines
RANTES, eotaxin, MIP-1α, and IP-10 in the draining lymph nodes
(Figs. 3 and 4B) compared with sham-challenged PBS controls. Interleukin-1Ra
treatment substantially reduced the expression of these chemokines in
both the cat dander–challenged mice and in the sham-challenged (PBS)
mice. We used densitometric analysis of the autoradiogram with Digital
Science ID software to approximate differences in chemokine mRNA.
RANTES production was decreased 15%
(Fig. 4) in samples taken from
draining lymph nodes of mice that had been treated with IL-1Ra compared
with control animals, although the actual differences may have been
greater because the bands in the cat-dander + vehicle group appeared to
have reached saturation. Differences in eotaxin and MIP-1a expression
between allergen-challenged animals treated with IL-1Ra and controls
were even more striking, with a 41% and a 47% decrease, respectively,
in allergen-challenged IL-1Ra–treated animals compared with controls.
Levels of IP-10 after IL-1Ra treatment showed the biggest differences,
with a 50.7% decrease compared with vehicle-treated animals.
Increased levels of IL-1 have been found in late phase reactions
in the skin after allergen skin challenge in sensitive
humans,
3 suggesting an important role for IL-1 in allergic
disease. Moreover, a critical element of all immunoinflammatory
responses, including allergic disease, is the recruitment of leukocytes
by chemokines and upregulation of adhesion factors. Interleukin-1
increases chemokine production, increases adhesion factors, increases
macrophage infiltration and activity, and increases lymphocyte
proliferation,
3 all of which play a role in the
immunopathogenesis of allergic disease.
The administration of IL-1Ra used in the present study proved effective
in significantly reducing the clinical stigmata of allergy in response
to cat dander. In contrast, treatment with vehicle alone did not
significantly alter responses in either sham-challenged or cat
dander–challenged mice. The reduction in clinical symptoms after
IL-1Ra treatment coincided with the ablation of the allergen-induced
eosinophilia, because the number of eosinophils in allergen-challenged
IL-1Ra–treated animals was similar to that seen in the sham-challenged
control animals. A number of studies in recent years have shown
the infiltration of eosinophils and the subsequent release of
eosinophil-derived granular proteins and lipid metabolites to be
central in the pathogenesis of allergic disease.
7 Initially, we thought the decrease in eosinophil infiltration into the
conjunctiva after IL-1Ra treatment was due to a modification in
cytokine levels. A number of articles have stressed the importance of
T
H2 cytokines, especially IL-4 and IL-5, in the
induction of eosinophilia.
7 However, although we did see
an allergen-induced increase in the concentrations of IL-4 and IL-5 in
lymphocyte cultures from draining lymph nodes after allergen challenge,
blockade of IL-1 did not significantly alter IL-4 and IL-5 cytokine
production in this model (data not shown).
The traffic of eosinophils to the sites of allergic reactions is
presumed to be regulated at several levels, including the expression of
chemokines.
8 Increased levels of RANTES, eotaxin, and
MIP-1α have been detected in the nasal secretions of atopic patients
exposed to allergen challenge and have been reported to be increased in
nasal washings of ragweed-sensitive subjects during the pollen allergen
season.
9 In this model of AC, several chemokines including
RANTES and eotaxin are upregulated in the conjunctiva of sensitized
mice after allergen challenge. Treatment with IL-1Ra substantially
decreases the concentrations of these chemokines, both of which have
been found to be important in eosinophil chemotaxis.
8
Additionally, examination of chemokine profiles in the draining lymph
nodes shows a substantial reduction in the allergen-induced chemokines
RANTES, MIP-1α, eotaxin, and IP-10. All of these chemokines are
thought to play an important role in the allergic inflammatory process,
because they are chemotactic for activated T cells, eosinophils,
basophils, and monocyte/macrophages.
8 We do not contend
that chemokine patterns in the draining lymph nodes directly influence
cell traffic into the conjunctiva. However, the importance of lymph
node chemokine data is underscored by the central role lymph nodes play
in bringing together mature antigen presenting cells and naive T cells
whose priming in the nodes leads to clonal proliferation of effector T
cells.
6 Accordingly, suppression of RANTES and MIP-1α
gene expression in the lymph node as a result of IL-1Ra treatment may
affect immune responses by the down-modulation of the activation and
eventual recruitment of T cells to the eye, which could, in turn,
influence the infiltration of histamine-releasing cells such as
eosinophils into the conjunctiva. Interestingly, the reduction in
RANTES and eotaxin production was even more striking in the
IL-1Ra–treated, sham- challenged groups in both the conjunctiva and
the draining lymph nodes. This finding suggests that IL-1Ra was able to
down-modulate both the endogenous and the antigen-induced gene
expression of chemokines.
Interleukin-1 has been shown to regulate the expression of eotaxin in
epithelial and endothelial cells in vitro,
10 again
suggesting a strong association between IL-1 production, eosinophil
infiltration, and allergic disease. Unlike the more general effects of
the other chemokines examined, eotaxin exclusively attracts eosinophils
when applied in vivo, and its expression is enhanced in animal models
of allergic inflammation and in tissue cells at sites of eosinophil
accumulation. Because of this preferential and potent action on
eosinophils and its occurrence in different species, eotaxin is
considered perhaps the most relevant chemokine in the pathophysiology
of allergic conditions and asthma.
8 The major cellular
sources of eotaxin are thought to be the epithelia and activated
infiltrating leukocytes like eosinophils. The localization of eotaxin
production is important not only to our understanding of the basic
mechanisms of tissue eosinophilia and tissue damage but also to the
design of drug delivery, which can now target the specific cells that
generate the signal responsible for the selective recruitment of
eosinophils.
8 IL-1Ra treatment yielded a substantial
decrease in eotaxin levels in conjunctiva and draining lymph nodes
compared with allergen-challenged animals that received vehicle alone.
Conjunctiva tends to be a relatively fragile tissue to work with,
particularly in small rodent species. It is for this reason that we
were not able to extract chemokine proteins themselves from our murine
samples. However, because we observed such a striking decrease in
clinical symptoms as well as an ablation of eosinophil infiltration
into the conjunctival matrix after IL-1Ra treatment, we propose that
the differences in mRNA assayed were biologically relevant and that
they explain, at least in part, the observed striking suppression of
clinical and histologic parameters of allergic eye disease.
The highly variable efficacy and myriad side effects of topical and
systemic nonspecific antiinflammatory pharmaceuticals including
corticosteroids are well known to clinicians who use these agents to
arrest allergic disease. The observations implicating eosinophils in
the pathogenesis of allergy make it likely that understanding the
mechanisms of eosinophil recruitment into the conjunctiva will offer
new therapeutic approaches for the treatment of this disease. Because
chemokine production appears to be essential for the recruitment of
eosinophils in the allergic response, strategies that block chemokine
production may be effective in treatment of allergic disease. Using an
in vivo model of allergic eye disease, we have shown that antagonism of
IL-1 activity by the topical administration of IL-1Ra offers an
effective means of suppressing eosinophilia and the resultant allergic
response in the eye. Our data strongly suggest that specific molecular
targeting strategies, such as the use of IL-1Ra to suppress IL-1
activity, may eventually offer a novel approach to the management of
AC.