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Departments of
*
Medicine and
Pathology, University of Washington, Seattle, WA 98195; and
Department of Immunology, Immunex Research and Development Corporation, Seattle, WA 98101
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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IL-4, a 20-kDa glycoprotein, has numerous biologic activities relevant
to the mediation of allergic inflammation (7). IL-4
stimulates proliferation of mast cells, anti-Ig-activated B cells,
and T cells. IL-4 also induces Ia and Fc
RII expression on monocytes
and lymphocytes, and class switching/secretion of IgE and IgG1 by B
cells. Sib-pair analysis of individuals from Amish families provides
evidence for linkage of the IL-4 gene in 5q31.1 with a gene controlling
total serum IgE concentrations (8). In bronchial biopsies
from both atopic and nonatopic asthmatics compared with nonasthmatic
controls, an increase in the number of cells expressing IL-4 and IL-5
mRNA and protein is observed (9). In these asthmatic lung
tissues, the majority of the IL-4 and IL-5 mRNA is expressed in
CD3+ T cells (CD4+ >
CD8+ T cells), with the remainder expressed in
mast cells and eosinophils (4). By immunocytochemistry,
most of the IL-4 and IL-5 protein colocalizes with mast cells and
eosinophils in the bronchial tissue from the atopic and nonatopic
asthmatics (4).
The biologic actions of IL-4 are mediated by its binding to its
receptor, IL-4R (molecular mass, 139 kDa in the mouse), expressed on
diverse cells. IL-4R is composed of IL-4R
and 
c subunits or
IL-4R and IL-13R subunits (10, 11, 12, 13, 14). Bronchial
biopsies from atopic asthmatics compared with atopic control subjects
exhibit increased expression of IL-4R mRNA and protein in the
epithelium, subepithelium, and endothelial cell layer
(15). IL-4R mRNA expression is also demonstrated in
CD3+ T cells and mast cells in lung tissue from
atopic patients with asthma (15). A naturally occurring
soluble form of IL-4R (sIL-4R; molecular mass, 39 kDa in the mouse) is
secreted and inhibits the biologic actions of IL-4. rIL-4R contains
only the extracellular portion of IL-4R and lacks the transmembrane and
intracellular domains. In vitro, sIL-4R blocks B cell binding of IL-4,
B cell proliferation, and IgE and IgGl secretion (16). In
vivo, sIL-4R inhibits IgE production by up to 85% in
anti-IgD-treated mice (17), suggesting that sIL-4R may
be useful in the treatment of IgE-mediated inflammatory diseases. A
previous study demonstrated that sIL-4R blocked IgE production and AHR
when administered at the time of sensitization (18). To
test the therapeutic potential of sIL-4R in asthma, we examined the
effect of exogenously administered sIL-4R in a murine model of
allergen-induced asthma (19, 20).
Insights into the mechanisms of airway inflammation and hyperreactivity
in asthma have come from investigations of the LAR in animal models.
Sensitization to a variety of allergens and subsequent airway challenge
with the allergen have been shown to produce typical features of the
LAR in a number of species including: mouse, rat, guinea pig, and
nonhuman primate. We have developed a protocol for administration of
OVA as a model allergen to induce late phase allergen-specific
pulmonary disease in mice (19, 20, 21). Our protocol includes
i.p. immunization of mice with OVA in alum adjuvant on days 1 and 14,
and single intranasal (i.n.) doses of OVA on days 14, 25, 26, and 27.
On day 28, OVA-treated mice display a disease similar to
allergen-induced human asthma including: 1) increased levels of total
and OVA-specific IgE in the blood, 2) increased release of leukotrienes
B4 and C4 in BAL fluid, 3)
a striking eosinophil influx into BAL fluid and lung tissue, 4) airway
mucus hypersecretion, 5) increased expression of Th2 and Tc2 cytokines
(IL-4, IL-5, and IL-13) and decreased expression of Th1 cytokines (IL-2
and IFN-) in bronchial lymph node tissue, and 6) AHR, as assessed by
a significantly greater decrease in airway conductance and dynamic
compliance in response to methacholine compared with control mice.
Employing this model, we found that sIL-4R treatment before OVA
challenge in OVA-immunized mice inhibits mucus hypersecretion and
eosinophil influx into the lungs, but not AHR, to methacholine
following allergen challenge.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Recombinant sIL-4R mouse (M) was prepared as previously described (17). For a negative control protein, a 5 mg sample of sIL-4R(M) (10 mg/ml) in PBS was inactivated by acidification to pH 3 in 1 N HCl and heating for 4 h at 100°C. After cooling in ice water to room temperature, the samples were neutralized to pH 6–7 with 2 M NaOH. Inactivation of the sIL-4R(M) sample was confirmed using an in vitro assay that measures inhibition of IL-4-induced B cell proliferation (16). Recombinant sIL-4R human (H), which does not exhibit cross-species binding with mouse IL-4, and mouse serum albumin (MSA; Sigma, St. Louis, MO) were used as additional negative controls.
Animals
Female BALB/c mice (6–8 wk of age; obtained from D&K, Seattle, WA, and The Jackson Laboratory, Bar Harbor, ME) were used. All animal study protocols were approved by the University of Washington Animal Care Committee.
Allergen induction of eosinophil infiltration
To induce eosinophil infiltration into murine airway tissue, mice were sensitized and later challenged with OVA (Pierce, Rockford, IL) as the allergen. Mice were immunized with OVA (100 µg) complexed with aluminum potassium sulfate (alum) in a 0.2 ml vol, administered by i.p. injection on days 0 and 14, as previously described (19). On days 14, 25, 26, and 27, mice were anesthetized with 0.2–0.3 ml of ketamine (6.5 mg/ml) and xylazine (0.44 mg/ml) diluted in normal saline. The OVA and sIL-4R/OVA groups all received 100 µg OVA in 0.05 ml normal saline by the i.n. route on day 14, and 50 µg of OVA in 0.05 ml normal saline by the i.n. route on days 25, 26, and 27. The control group received normal saline with alum by the i.p. route on days 0 and 14, and 0.05 ml of normal saline by the i.n. route on days 14, 25, 26, and 27.
Administration of sIL-4R
The sIL-4R/OVA group received a sIL-4R dosage of 0.1–100 µg/mouse/day. The sIL-4R dose was administered by either i.n. or i.p. injection 30 min before challenge with OVA on day 25. Prior data indicate that i.n. administration of small particles (i.e., 1% solution of carbon particles, 0.027–0.050 µm diameter) in mice results in the uniform distribution of the particles throughout the upper and lower lungs reaching to the alveoli (E. Y. C., unpublished observations). Previous pharmacokinetic studies indicated that the plasma t1/2 of sIL-4R in the mouse is 4.6 h after i.p. administration (22). Therefore, to increase sIL-4R levels above endogenous levels during the course of the allergen challenge, mice received additional sIL-4R dosages on days 26 and 27, by the i.n. or i.p. route, 30 min before OVA treatment.
Pulmonary function testing
On day 28, 
24 h following the last i.n. administration of
either normal saline or OVA, pulmonary mechanics in response to an i.v.
infusion of methacholine were evaluated in the mice by a
plethysmographic method (19), modified from previously
described methods (23, 24). After pentobarbital anesthesia
(70–90 mg/kg by the i.p. route), the jugular vein was cannulated, the
trachea was intubated and connected to a Harvard ventilator, and the
thorax was opened by a thoracotomy, as previously described
(19). The animal was placed in a supine position in one
compartment (0.25 ml dead space) of a two-chamber whole body
plethysmograph. The following minute ventilation maintained normal
arterial blood gases: tidal volume = 0.2 ml/20 g, frequency 120
breaths/min, and positive end expiratory pressure of 2.5–3 cm
H2O (25). Copper mesh in the
plethysmograph served as a heat sink for rapid gas compression during
each tidal breath. Because the plethysmograph is a closed system, the
change in box pressure (Pbox) of the
first chamber (measured by a sensitive transducer (±0.7 cm
H20)) represented the change in lung volume
(
vol = Pbox) of the mouse;
Pbox is equivalent to lung pleural
pressure. Ambient pressure swings and temperature increases in the
first chamber were offset as previously described (19).
Pressure movements at the opening of the tracheal tube
(Paw) were measured by another
transducer, referenced to Pbox to
determine transpulmonary pressure (Ptp
= Paw -
Pbox). After initial inflation to a
Paw of 30–35 cm
H2O, the lungs were inflated at least once 1–2
min before each measurement to prevent partial collapse. An
analogue-to-digital data acquisition system (Strawberry Tree) was
employed to sample Ptp and
Pbox at 5-ms intervals, with a
smoothing function employed to dampen background noise, as previously
described (19). The change in volume from
Pbox:point-to-point/5 ms was used to
calculate flow.
Data from seven consecutive breaths collected three times during the
first 10 min were used to calculate basal pulmonary function.
Methacholine was then infused by hand delivery into the jugular vein
over 10 s at a concentration of 120 µg/kg, after 10 min of
ventilation. Resistance (R), lung conductance
(GL = 1/R), and dynamic compliance
(Cdyn) determined for both the control
period and during the peak response to methacholine challenge.
R was calculated as the difference in
Ptp and airflow at midtidal volume on
inflation and deflation. Tracheal tube resistance (0.63 cm
H2O x ml-1 x s) was
subtracted from all airway resistance measurements.
Cdyn was calculated as the change in
tidal volume (Vt) divided by the difference between
Ptp at end inspiration and end
expiration when flow is zero (Cdyn =
Vt/Ptp). At the completion of
pulmonary function testing, each mouse was exsanguinated by cardiac
puncture; plasma samples were collected from all mice in each
experimental group and stored at -70°C until assay of sIL-4R.
Bronchoalveolar lavage
After tying off the left lung at the mainstem bronchus, the right lung was lavaged three times with 0.4 ml of normal saline. The three BAL fluid samples collected from each animal were pooled, and the number of cells in a 0.05-ml aliquot was determined using a hemocytometer. The remaining sample was centrifuged at 4°C for 10 min at 200 x g, and the supernatant was stored at -70°C until assay of sIL-4R levels. The cell pellet was resuspended in normal saline containing 10% BSA, and BAL fluid cell smears were made on glass slides. To stain eosinophils, dried slides were stained with Discombe’s diluting fluid (0.05% aqueous eosin and 5% (v/v) acetone in distilled water) for 5–8 min, rinsed with water for 0.5 min, and counterstained with 0.07% methylene blue for 2 min.
Lung histology
The trachea and left lung (upper and lower lobes) were collected
and fixed in Carnoy’s solution at 20°C for 15 h. After embedding
in paraffin, the tissues were cut into 5-µm sections. For each mouse,
10 airway sections randomly distributed throughout the left lung were
assessed for the severity of the cellular inflammatory response and
mucus occlusion by morphometric analysis (19), by
individuals blinded to the protocol design. The intensity of the
cellular infiltration around pulmonary blood vessels and airways was
assessed on a semiquantitative scale ranging from
0–4+.
Eosinophils were stained in the lung tissue with Discombe’s Solution, as described above. The number of eosinophils per unit airway area (2200 µm) (2) was determined by morphometric analysis, as previously described (19, 26). Airway mucus (i.e., mucin and sulfated mucosubstances) was identified by the following staining methods: methylene blue, hematoxylin/eosin, and Alcian blue, as previously described (19). Occlusion of the airway diameter by mucus was assessed on a semiquantitative scale ranging from 0–4+ (19). Each airway section was assigned a score for airway diameter occlusion by mucus based on the following criteria: 0, 0–10% occlusion; 1, 10–30% occlusion; 2, 30–60% occlusion; 3, 60–90% occlusion; 4, 90–100% occlusion (19).
Immunocytochemistry
Paraffin sections of lung tissue were deparaffinized, hydrated, and washed in PBS. To localize VCAM-1, the sections were incubated at room temperature for 30 min with rat anti-mouse VCAM-1 mAb (PharMingen, San Diego, CA) diluted 1/40 in PBS. Control sections were treated with PBS and rat IgG. After rinsing in PBS, the sections were incubated for 30 min with goat anti-rat IgG (Vector Laboratories, Burlingame, CA). After rinsing in PBS, the sections were incubated for 60 min with alkaline phosphatase (AP) solution of Vectastain ABC-AP standard kit (Vector Laboratories) in PBS. The sections were rinsed in PBS and incubated for 30 min with the AP solution of Vector Red-AP substrate kit in Tris-HCl buffer, pH 8.2, containing 1% levamisol (Vector Laboratories). The AP reaction products stain pink. The sections were rinsed in distilled water, counterstained with 0.4% methylene blue in 70% ethanol and 0.01% NaOH, dehydrated in a series of ethanol concentrations up to 100%, cleared in xylene, and mounted with Permount (Fisher Scientific, Pittsburgh, PA).
Assay of sIL-4R levels in plasma and BAL fluid
Levels of sIL-4R(M) in the plasma and BAL fluid were determined by a capture ELISA protocol based on two anti-IL-4R mAbs, M1 and M2, as previously described (27).
Specific Ab levels
Total and OVA-specific IgE levels in the blood were determined
by ELISA. For the total IgE ELISA, the plates were coated with 100 µl
of 1 µg/ml rat anti-mouse IgE (Serotec, Kidlington, Oxford, U.K.)
in PBS overnight at 4°C. The plates were washed six times with
PBS/Tween and blocked with 150 µl of 10% FBS in PBS for 1 h at
room temperature. The plates were washed again six times with PBS.
Samples were titrated in 10% FBS in PBS with dilutions beginning at
1/20 and standards started at 1 µg/ml; the final volume per well was
100 µl. After washing six times with PBS/Tween, the plates were
incubated with rat anti-mouse IgE heavy chain biotin Ab (100
µl/well; Serotec) diluted 1/5000 in 50% goat sera (Life
Technologies, Gaithersburg, MD) in PBS for 1 h at room
temperature. The plates were washed 10 times with PBS/Tween. After
addition of 100 µl of 1:1000 streptavidin:HRP in 50% goat sera in
PBS, the plates were incubated for 1 h at room temperature. The
plates were washed 10 times with PBS/Tween, developed with 1:1
substrate of 100 µl tetramethylbenzidine (Kirkegaard & Perry
Laboratories, Gaithersburg, MD), and read at 650 nm or stopped with 50
µl/well of 2 N H2SO4 and
read at 450 nm. Data were analyzed on the Soft program (Molecular
Devices, Sunnyvale, CA). The protocol for determination of OVA-specific
IgE was the same as for total IgE ELISA, except that the first step was
performed with 100 µg/ml of OVA in PBS.
Statistical analysis
The data are presented as the mean ± SE of the mean. A Student’s two-tailed t test was used to compare data for BAL fluid eosinophil counts and airway mucus between the different experimental groups. For the evaluation of pulmonary mechanics, a Fisher PLSD test was used to compare values for GL and Cdyn between experimental groups. Differences were considered statistically significant for p values <0.05.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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From previous pharmacokinetic studies (22), it was
known that the elimination t1/2 of
sIL-4R in the mouse is 4.6 h after i.p. administration. sIL-4R(M)
doses from 0.1–100 µg were administered i.n. or i.p. 30 min before
OVA challenge on days 25, 26, and 27. On day 28, plasma and BAL fluid
samples were collected 24 h following administration of the final
sIL-4R dosage and assayed for sIL-4R levels (Fig. 1
). The levels of sIL-4R(M) were similar
in blood from saline control and OVA-sensitized/challenged mice
(13.6 ± 1.5 ng/ml (n = 7) and 16.2 ± 1.3
ng/ml (n = 9), respectively); sIL-4(M) levels were <1
ng/ml in the BAL fluid from control and OVA-treated mice. After i.n. or
i.p. delivery of 0.1–10 µg sIL-4R(M) before OVA on days 25 to 27,
sIL-4R(M) blood levels were increased <2-fold on day 28. At 100 µg
of sIL-4R(M), i.n. delivery increased blood sIL-4R(M) levels by
2.7-fold (44 ± 3.8 ng/ml, n = 8,
p = 0.0001 vs OVA) and i.p. delivery by 3.7-fold
(60.5 ± 2, n = 2, p = 0.0001 vs
OVA). Whereas i.n. administration of sIL-4R(M) at 100 µg dose
increased BAL fluid levels of sIL-4R(M) 283-fold (255 ± 81.6
ng/ml, n = 16, p = 0.004 vs OVA), i.p.
delivery of 100 µg sIL-4R(M) did not significantly increase BAL fluid
levels of sIL-4R(M). Pretreatment with 100 µg doses of
heat-inactivated sIL-4R(M), sIL-4R(H), or MSA by i.n. administration or
100 µg of i.p. sIL-4R(H) did not affect sIL-4R(M) levels of
OVA-treated mice.
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Blood was obtained on day 28 in the OVA-sensitized/challenged mice. Treatment with sIL-4R(M) 30 min before OVA challenge on days 25, 26, and 27 did not significantly affect the circulating levels of either total IgE or OVA-specific IgE in the OVA-treated mice (data not shown).
Effect of sIL-4R(M) on recruitment of inflammatory cells into the lungs
Twenty-four hours following the final i.n. OVA challenge in
animals from each experimental group, BAL was performed on the right
lung, and left lung tissue was obtained to assess the effect of sIL-4R
on airway inflammation. By light microscopy, compared with saline
controls (Fig. 2, A and
B) a marked influx of eosinophils and mononuclear cells into
the lungs around blood vessels and airways was observed in
OVA-sensitized/challenged mice (Fig. 3,
A and B). Administration of 100 µg of sIL-4R(M)
i.n. before OVA challenge on days 25, 26, and 27 inhibited the cellular
infiltration surrounding airways and pulmonary blood vessels (Fig. 4, A–C). In contrast, i.n.
pretreatment with 100 µg of MSA (Fig. 3C),
heat-inactivated sIL-4R(M) (Fig. 5, A and B), and
sIL-4R(H) (Fig. 6, A–C),
failed to affect the cellular inflammatory response in OVA-treated
mice.
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, A and B). Pretreatment with i.n.
sIL-4R(M) reduced the number of eosinophils in the pulmonary tissue of
OVA-treated mice by 89% (p = 0.0001, i.n.
sIL-4R(M)/OVA vs OVA). MSA, heat-inactivated sIL-4R(M), and sIL-4R(H)
pretreatment did not alter this infiltration of the airways by
eosinophils (Fig. 7B). Administration of 100 µg of
sIL-4R(M) by the i.p. route also reduced the cellular infiltrate by
eosinophils and other inflammatory cells in the lungs (Fig. 7, A and B), but to a lesser extent than by i.n.
administration.
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A; saline group vs OVA group,
p = 0.0001). Pretreatment with i.n. delivery of 100
µg of sIL-4R(M) significantly reduced the total number of cells in
the BAL fluid of OVA-sensitized/challenged mice
(p = 0.046, i.n. sIL-4R(M)/OVA vs OVA group).
The mean number of eosinophils in BAL fluid collected from the saline
group was 1.3 x 103 cells (Fig. 8B). After OVA treatment, the number of eosinophils in the
BAL fluid from the OVA group increased 22.7-fold to 3 x
105 (saline group vs OVA group, p
= 0.0001). Pretreatment with i.p. and i.n. 100 µg dosing of sIL-4R(M)
reduced eosinophil infiltration into the BAL fluid by 73.3%
(p = 0.0009, i.p. sIL-4R(M)/OVA vs OVA) and by
80% (p = 0.0003, i.n. sIL-4R(M)(100 µg)/OVA
vs OVA), respectively (Fig. 8B).
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B) compared with
saline-treated controls (Fig. 9A). VCAM-1 expression was
also noted in inflammatory cells recruited to the lung interstitium of
OVA-sensitized/challenged mice (Fig. 9A). Pretreatment on
days 25, 26, and 27 with 100 µg of sIL-4R(M) reduced VCAM-1
expression in the endothelium of the lung blood vessels and in the
interstitium surrounding the airways of the OVA-treated mice (Fig. 9C).
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Mucus hypersecretion and occlusion of the airways was a prominent
histopathologic feature of the OVA-sensitized/challenged mice (Fig. 3
, A and B). Mucus hypersecretion and occlusion of
airway diameter were significantly inhibited by pretreatment with 100
µg of sIL-4R(M) in the OVA-treated mice (Figs. 4A,
B, and C, and 7C). Mucus release in
OVA-treated animals was unaffected by i.n. administration of 100 µg
of MSA (Figs. 3C and 7C), heat-inactivated
sIL-4R(M) (Figs. 5A and 7C), and sIL-4R(H) (Figs. 6A, B, and C, and 7C). In
contrast to i.n. delivery, i.p. treatment with 100 µg of sIL-4R(M)
did not significantly reduce mucus occlusion of airway diameter in
OVA-treated mice (Fig. 7C).
Effect of sIL-4R(M) on allergen-induced AHR to methacholine
Airway reactivity was evaluated on day 28, which was 24 h
following the third i.n. challenge with OVA. In the OVA group, AHR was
seen following challenge with methacholine, with a significant decrease
in both GL and
Cdyn compared with the saline group
(Table I; saline group vs OVA group,
p < 0.05 for both GL and
Cdyn). At a dose of 100 µg
administered i.p. or i.n. before OVA on days 25, 26, and 27, sIL-4R(M)
did not reduce bronchial hyperresponsiveness to methacholine in the
OVA-sensitized and challenged mice. Similarly, pretreatment with 100
µg i.n. of sIL-4R(H) failed to alter the methacholine-induced AHR
observed in OVA-treated mice (Table I).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Goblet cell hyperplasia and mucus obstruction of the airways are key features of chronic asthma (28, 29). IL-4 is important in regulation of airway mucus release. Transgenic mice overexpressing IL-4 have increased MUC5AC, but not MUC2 mucin gene expression (30). These transgenic mice have up to a 10-fold increase in Alcian blue and periodic acid-Schiff (PAS)-positive mucus glycoprotein materals in their BAL fluid compared with transgene-negative controls (30). In our murine model, a marked increase in airway mucus is observed on day 22; by days 24 to 25, airway epithelial cells are extensively replaced by mucus-producing goblet cells (31). Our data indicate that blockade of IL-4 beginning on day 25 by i.n. delivery of sIL-4R inhibits airway mucus hypersecretion after airway OVA challenge in mice previously sensitized to this allergen. These data suggest an ongoing role for IL-4 in the maintenance of airway mucus glycoprotein secretion in the allergic airways. The failure of i.p. delivery of sIL-4R(M) to significantly reduce airway obstruction by mucus indicates that increasing the airway levels of sIL-4R is critical for its inhibitory action on mucus secretion.
Like CD4+ and CD8+ T cells,
eosinophils are a prominent cell type in the LAR and in the chronic
inflammation of asthma. Eosinophilia is noted in sputum, lavage, and
biopsy samples, and eosinophils are often activated, as evidenced by
morphologic changes and the identification of increased concentrations
of eosinophil products, such as cationic protein and major basic
protein (32). IL-4 is a critical mediator of airway
eosinophilia. IL-4 increases human bronchial tissue expression of
VCAM-1 (CD106) (33), which binds to the integrin
heterodimer VLA-4
(4
1; CD49d/CD29).
VLA-4 is expressed on eosinophils and all other circulating leukocytes
except neutrophils, and is important for the selective influx of
eosinophils and lymphocytes into the airways in asthma (21, 34). Of interest, we observed expression of VCAM-1 in
inflammatory cells infiltrating the lung interstitium of OVA-treated
mice. Prior reports have noted VCAM-1 expression in vascular dendritic
cells present in human atherosclerotic plaques (35) and in
lymphocytes and monocytes invading the renal interstitium of diabetic
KKAy mice (36).
In murine asthma models using IL-4-deficient (IL-4-/-) mice, IL-4 has been found important for both eosinophil (37) and Th2 cell (38) recruitment to the lungs, and Th2 cell production of IL-4 is necessary for the development of airway eosinophilia (39). We found that pretreatment of OVA-sensitized/challenged mice with sIL-4R(M) at a dosage of 100 µg i.n. or i.p. significantly inhibited eosinophil infiltration into the lung interstitium and BAL fluid. Elevation of circulating levels of sIL-4R by either i.p. or i.n. delivery is sufficient to block movement of eosinophils into the lungs, indicating that eosinophil recruitment by IL-4 may be a systemic effect of this cytokine. Although a number of studies have established a key role for IL-4 in VCAM-1 expression (40, 41, 42, 42), a connection between IL-4-mediated eosinophil migration and regulation of VCAM-1 expression has not been clearly shown in asthma models. Our results demonstrate that VCAM-1 expression is reduced in allergen-challenged mice following IL-4 inhibition. These results support the hypothesis that eosinophil migration in asthma is dependent upon VCAM-1 expression, and provide an indirect mechanism whereby IL-4 may affect eosinophil accumulation in the lungs.
It has been proposed that eosinophils and their products contribute to the chronic inflammation and the development of AHR. Alternatively, they may not be causally involved in the development of AHR, but their presence may be merely an epiphenomenon of the Th2 response with elaboration of IL-5. In our model, airway reactivity was evaluated on day 28, which was 24 h following the third i.n. challenge with OVA. Mice in the OVA group demonstrated AHR when challenged with methacholine (i.e., GL and Cdyn were significantly reduced compared with the control group). Despite significantly increasing BAL fluid levels of sIL-4R(M), treatment with sIL-4R(M) at a dosage of 100 µg failed to alter in vivo AHR in response to methacholine.
Renz et al. (18) examined the effect of a rsIL-4R on in
vivo immediate hypersensitivity responses and AHR in mice sensitized to
the airways to OVA by once weekly ultrasonic nebulizations for 4 wk. In
their studies, mice received sIL-4R before initial OVA sensitization
and throughout the OVA treatment period. Treatment with 150 µg of
i.p. sIL-4R(M) reduced by >50% anti-OVA IgE and IgG1 Ab titers.
In a dose-dependent manner (15–150 µg), sIL-4R(M) inhibited
allergen-specific immediate cutaneous hypersensitivity responses in the
mice. Treatment with i.p. sIL-4R(M) also prevented expansion of
V8.1/8.2 T cells in OVA-treated animals. Renz et al.
(18) found that local administration to the lungs of
sIL-4R(M) by aerosolization not only decreased the IgE/IgG1 responses
to OVA, but also reduced total serum IgE levels. In vitro studies by
Renz et al. (43) have also demonstrated that murine sIL-4R
and dimeric sIL-4R fusion protein significantly reduce
allergen-specific polyclonal IgE production by lymphocytes obtained
from allergen-sensitized mice. This inhibitory effect was greatest
during the first 3 days of culture and was not observed after 6 days,
suggesting that sIL-4R blocks the early period of B cell maturation/IgE
production (43).
In our studies, sIL-4R(M) given only before allergen challenge on days 25, 26, and 27 and not during sensitization did not reduce either total or OVA-specific IgE levels. We have previously demonstrated that mice receiving two doses of OVA by the i.p. route on days 1 and 14 have similar levels of OVA-specific IgG1 and IgE on day 28, irrespective of the number of i.n. OVA doses administered (20). In view of this robust IgE response to OVA in our model and the in vitro studies by Renz et al. (43), it is not surprising that administration of sIL-4R after allergen sensitization would not affect circulating IgE levels.
As reported by Renz et al., nebulized sIL-4R(M) given before and throughout OVA sensitization normalized airway responsiveness (as measured in vitro by electrical field stimulation of excised tracheal smooth muscle preparations) to a greater extent than i.p. administration of this protein. In contrast, administration of 100 µg sIL-4R(M) by either i.p. or i.n. routes did not alter AHR in our murine model of asthma. Corry et al. (44) found that anti-IL-4 mAb given during the systemic immunization period in a murine asthma model blocked AHR, but when administered during the allergen challenge period, had no effect on the response of the sensitized animals to acetylcholine. The results with sIL-4R and anti-IL-4 mAb treatments suggest that IL-4 generated during the sensitization period of lymphocyte priming by allergen is important for allergen-induced AHR, but that IL-4 is not required for maintenance of AHR after allergen challenge in immunized mice.
The discordance we observed between airway eosinophils and AHR has been noted in several other animal models. The presence of eosinophils clearly does not ensure AHR. Transgenic mice with IL-4 constitutively expressed in the lung develop an eosinophilic inflammatory cell infiltrate including eosinophilia but do not exhibit AHR (45), and airway administration of IL-5 produces airway eosinophilia without AHR in guinea pigs (46). AHR without eosinophilia is also observed in murine asthma models. In previous studies, we found that inhibition of leukotriene synthesis prevents allergen-induced eosinophil recruitment without affecting AHR (19). Furthermore, a CD49d mAb given by i.p. administration at doses that saturated circulating leukocytes blocked Ag-induced eosinophil accumulation in BAL fluid, but did not prevent AHR, mucus hypersecretion, or IL-4 and IL-5 release (21). Thus, the development of AHR does not appear dependent on pulmonary infiltration by eosinophils.
It remains unclear to what extent IL-4 vs IL-13 regulates the allergic
diathesis. IL-13 also binds to the -chain of the IL-4R (47, 48), and the IL-13 gene is closely linked to the IL-4 gene
(49). However, IL-13 induces IL-4-independent IgE and IgG4
by human B cells (50). Because T cells lack functional
IL-13R, IL-13 (unlike IL-4) does not induce Th2 cell differentiation
(51). By in situ hybridization, an increased number of
cells express both IL-13 and IL-4 mRNA in endobronchial biopsies of
patients with mild atopic asthma compared with normal controls
(3). IL-13 and IL-4 protein levels are significantly
increased 18 h after allergen challenge in patients with allergic
asthma (52). A key role for IL-13 in the mediation of
airway inflammation and hyperreactivity has recently been demonstrated
in murine models of human asthma (53, 54). In
OVA-sensitized mice, blockade of IL-13 by a
sIL-13R2-IgGFc fusion protein that
specifically binds to and neutralizes IL-13 (55) inhibits
airway eosinophilia, mucus release, and AHR (53, 54). In
addition, intratracheal or i.n. administration of either IL-13 or IL-4
induces airway eosinophilia, and AHR in naive animals (53, 54), but not in IL-4R-deficient mice, suggesting that
development of the asthma phenotype in mice is dependent on signaling
by either IL-4 or IL-13 through IL-4R.
Our results indicate that IL-4 is an important mediator in the late phase inflammatory response that occurs in this allergen-induced model of asthma. These data have therapeutic implications, in that they suggest that local antagonism of IL-4 in the lung may be beneficial even in the presence of full sensitization to an allergen. Clinical studies are currently ongoing to examine the effects of sIL-4R treatment on airway inflammation and function in patients with persistent asthma. In a phase I/II study in 25 patients with moderate persistent asthma who required inhaled corticosteroids for control of symptoms, after removal of corticosteroids, a single 1500 µg nebulization of sIL-4R stabilized lung function and decreased exhaled NO, a marker of pulmonary inflammation during the first week after sIL-4R inhalation (56). Moderation of lung cellular inflammation and mucus hypersecretion by sIL-4R in a mouse model of asthma suggests that sIL-4R may be useful in the treatment of patients with asthma.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. William R. Henderson, Jr., Department of Medicine, 1959 NE Pacific Street, University of Washington, Seattle, WA 98195-7185. E-mail address:
3 Abbreviations used in this paper: AHR, airway hyperreactivity; alum, aluminum potassium sulfate; AP, alkaline phosphatase; BAL, bronchoalveolar lavage; Cdyn, dynamic compliance; GL, conductance; H, human; i.n., intranasal; LAR, late airway response; M, mouse; MSA, mouse serum albumin; s, soluble; Tc, T cytotoxic.
Received for publication June 22, 1999. Accepted for publication November 1, 1999.
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