Induction, distribution and modulation of upper airway allergic inflammation in mice

Induction, distribution and modulation of upper airway

allergic inflammation in mice

I. HUSSAIN*, D. RANDOLPH*, S. L. BRODY², S.-K. SONG³, A. HSU*,A. M. KAHN*, D. D. CHAPLIN*,§ and D. L. HAMILOS*

*Division of Allergy and Immunology, ²Division of Pulmonary and Critical Care Medicine, Department of Internal

Medicine, ³Department of Chemistry at Washington University School of Medicine, and §Howard Hughes Medical

Institute, St Louis, USA

Summary

Background To further elucidate mechanisms of human allergic rhinosinusitis, we

studied the induction, distribution and modulation of allergen-induced upper airway

inflammation in a BALB/c mouse model.

Methods Allergic inflammation induced with ovalbumin (OVA) by intraperitoneal (IP)

injection in alum was compared to repeated intranasal instillation. The type and

distribution of inflammatory cells was compared in the respiratory and olfactory epithelial

compartments. Eosinophil distribution was assessed using Scarlet Red stain and a

polyclonal antibody recognizing eosinophil major basic protein (MBP). The role of

interleukin (IL)-5 in upper airway inflammation was tested by administration of polyclonal

anti-IL-5 antibody during the sensitization protocol.

Results Unsensitized control mice receiving saline failed to develop upper airway

eosinophil infiltration. IP OVA-sensitized mice developed marked upper airway mucosal

eosinophil infiltration after aerosol OVA challenge, whereas repeated intranasal instillation

of OVA produced qualitatively similar, but less intense eosinophil infiltration. Using either

sensitization protocol, eosinophil infiltration was seen in areas of the lower portion of the

nasal septum, the floor and the lower lateral walls of the mid-caudal region of the nasal

cavity. Immunofluorescence staining for MBP confirmed this distribution of eosinophils

but also demonstrated some eosinophils in the maxillary sinuses and in circumscribed

regions of the ethmoturbinates. All areas of eosinophil infiltration were lined by respiratory

epithelium. The selective infiltration of respiratory but not olfactory epithelium by

eosinophils was unassociated with a measurable induction of epithelial ICAM-1 or eotaxin

expression. OVA-induced upper airway eosinophil infiltration was found to be IL-5

dependent, since administration of a polyclonal anti-IL-5 antibody (TRFK-5) during OVA

sensitization resulted in a marked modulation (80% decrease) in eosinophil infiltration in

response to subsequent OVA challenge.

Conclusion The mouse upper airway, specifically in areas containing respiratory

epithelium, is a target for OVA-induced allergic inflammation. This selective infiltration

of respiratory, but not olfactory, epithelium is, in part, dependent upon IL-5. This model is

useful for further dissection of the inflammatory response with genetic manipulations and

targeted immunological approaches.

Keywords: allergic, inflammation, ovalbumin, eosinophil, mice, respiratory, olfactory

Clinical and Experimental Allergy, Vol. 31, pp. 1048±1059. Submitted 29 September

2000; revised 21 December 2000; accepted 22 January 2001.

Clinical and Experimental Allergy, 2001, Volume 31, pages 1048±1059

1048 q 2001 Blackwell Science Ltd

Correspondence: Dr Daniel L. Hamilos, Washington University School of

Medicine, Division of Allergy and Immunology, Box 8122, 660 South

Euclid Avenue, St Louis, MO 63110, USA

Introduction

Allergic rhinitis is a common chronic illness, affecting

approximately 10% of the population in the United States

of America [1]. Natural allergen exposure is associated

with chronic inflammation and mucosal oedema of the

respiratory epithelium in nasal and sinus passages giving

rise to symptoms of nasal congestion, rhinorrhea, nasal

blockage and reduced olfaction [2,3]. These symptoms

have a significant negative impact on quality of life, and

account for billions of dollars of health care expenditure

yearly on anti-allergic medications [4].

Although numerous studies have examined the induction

of allergic inflammation in the lower airways, there have

been relatively few such studies in the upper airway. While

the upper and lower airway mucosae are similar, there are

differences in innervation, connective tissue elements,

submucosal lymphatic structures and specialized epithelia

(such as the olfactory epithelium) that may affect the

quality or intensity of the inflammatory response. It is

likely that eosinophils play a major role in the allergic

response in both the upper and lower airways [5±7], and

studies in humans and rodents suggest a role for epithelial

induction of ICAM-1 and eotaxin in promoting eosinophil

infiltration [8±13]. Furthermore, the upper airway response

may differ from the lower airways in terms of its response

to locally applied allergen. Hence, there is a need for

experimental systems that can be easily modulated to

examine localization and dynamics of the allergic inflam-

mation in the upper airway after either systemic vs.

intranasal sensitization.

Similarly, the target epithelium for allergic inflamma-

tion is unclear. Human allergic rhinitis is associated with

a reduced olfactory function during seasonal allergen

exposure but not out of season [14±17]. This question is

difficult to address in humans but can be addressed in

mice, since the olfactory epithelium occupies a much

larger fraction of the upper airway area and is easily

identified by its anatomic localization and expression of

an epithelial cell-specific marker, olfactory marker protein

(OMP) [18].

In the study of the pathophysiology of asthma, several

groups have employed a model of intraperitoneal (IP)

sensitization followed by aerosol challenge with chicken

ovalbumin (OVA) to induce allergic inflammation in the

lower airways [19±26]. In this model, there is a

characteristic eosinophilic inflammatory response that is

dependent upon elaboration of Th2-type cytokines, parti-

cularly IL-5 [25±27]. Interleukin-5 is known to increase the

survival of eosinophils in tissues and to promote eotaxin-

induced airway eosinophil infiltration [28]. Because the

respiratory epithelial cells and inflammatory responses of

the upper airway share many characteristics with the lower

airway, we also wished to determine whether OVA-induced

upper airway allergic inflammation is IL-5 dependent.

Materials and methods

OVA sensitization and challenge protocols

Female BALB/c mice 8±12 weeks of age (Harlan Labora-

tories, Indianapolis, IN, USA) were housed in a pathogen±

free barrier facility and maintained on OVA-free diets. All

experimental animals used in this study were under a

protocol approved by the Animal Studies Committee of

Washington University School of Medicine.

Mice were sensitized with OVA given intraperitoneally

(IP) or intranasally (see Fig. 1). In the IP protocol, each

mouse received 8 mg of OVA (Chicken ovalbumin, Grade

V, Sigma, St Louis, MO, USA) adsorbed to 2 mg of alum

(Sigma) in 0.5 mL of sterile phosphate buffered saline

(PBS) on day 1, with a booster on day 6. Age-matched

control mice received only alum 2 mg in 0.5 cc of PBS on

the same days. OVA-sensitized mice received 1% OVA

(wt/vol) by aerosol on day 13 and 19 in a small plexiglas

chamber device attached to a clinical nebulizer (Ultra-Neb

99, DeVilbiss Health Care, Somerset, PA, USA). On each

challenge day, mice received aerosolized OVA for 20 min,

first in the morning and then in the afternoon. Mice were

killed 24±72 h after the last challenge. In the intranasal

sensitization protocol, mice received 10 mL of 1% OVA

intranasally to each nostril for 10 consecutive days. Sham-

sensitized mice received 10 mL of saline intranasally to

each nostril on the same days. Mice were killed 48 h after

the last intranasal dose.

In separate experiments, mice were treated with 100 mg of

rat antimurine IL-5 monoclonal antibody TRFK-5 (ATCC,

Manassas, VA, USA) administered IP on days 4, 6 and 8 during

the intranasal OVA sensitization protocol as previously

described [25]. Control mice received 100 mg of isotype-

matched rat IgG IP on the same days. Mice were killed 48 h

following 10 consecutive days of intranasal OVA sensitization.

Mouse sinus MRI

Upper airway anatomy was defined by high resolution magnetic

resonance imaging (MRI) scans. Mice were anaesthetized with

halothane/O2 mixture (5% induction, 1.5% maintenance). A

1.5-cm outer diameter circular surface coil was placed on the

mouse head between the rostral aspect of the snout and the eyes

for MR signal reception while a 12-cm inner diameter

Helmholtz coil was used as the transmitter. A multislice spin

echo imaging sequence was used for the data collection. The

imaging parameters used were: repetition time 2 s, echo time

18 msec, field of view 1.5 cm2, data matrix 256 � 256,

in-plane resolution 60 mm, and slice thickness 500 mm.

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Upper airway sectioning, staining and immunohistochem-

istry

Mice were anaesthetized with ketamine/xylazine then killed

by cardiac exsanguination. Blood was flushed from the

circulation by intracardiac injection of sterile Dulbecco's

PBS. Heads were separated from the body, and the scalp was

removed. Whole heads were fixed in 10% buffered formalin

for 24±48 h. After fixation, the heads were decalcified in

14% EDTA at 218C for 2 week. After decalcification, heads

were sectioned coronally into four regions for paraffin

embedding: A at the incisor teeth, C at the anterior margin of

orbit, B halfway between A and C, and D at the posterior

margin of the orbit [29±31]. Tissue sections were cut at

5 mm thickness for evaluation.

To identify epithelial cells, tissue sections were deparaffi-

nized and hydrated and then incubated with a rat anti-mouse

Pan-cytokeratin (BD PharMingen, SanDiego, CA, USA)

1 : 200 diluted in Saponin buffer (0.5% Saponin in PBS) with

3% horse serum overnight at 4 8C. Control slides were

incubated with isotype-matched rat antibody under similar

conditions. To identify respiratory epithelial cells, antibody

directed toward HFH-4 was used. HFH-4 is a transcription

factor with tissue-specific expression on epithelial cells

possessing motor but not sensory cilia [32]. Rabbit anti-rat

HFH-4 polyclonal antibody (diluted 1 : 500) was used on

paraformaldehyde-fixed samples in paraffin as previously

described [32]. To identify neuro-olfactory epithelial cells,

goat anti-serum against mouse olfactory marker protein (OMP,

dilution 1: 4000) (a generous gift of Dr Frank Margolis,

Baltimore, MD, USA) was used [18,33]. To identify epithelial

expression of ICAM-1, a hampster anti-mouse monoclonal

anti-ICAM-1 antibody was used at a dilution of 1 : 500 (3E2,

BD PharMingen). To identify epithelial expression of eotaxin,

a polyclonal goat anti-mouse eotaxin antibody was used (AF-

420-NA, R & D Systems, Minneapolis, MN, USA). A

polyclonal rabbit anti-mouse anti-serum for major basic

protein (a generous gift of Dr Gerald Gleich, Mayo Clinic,

Rochester, MN, USA) was used at a dilution of 1 : 500.

Secondary antibodies of the appropriate species labelled with

Fig. 1. OVA sensitization and challenge protocols. (a) IP sensitization protocol. (b) Intranasal sensitization protocol. (c) Intranasal

sensitization protocol combined with IP anti-IL-5 protocol. See Materials and methods for details of each protocol.

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alkaline phosphatase or horseradish peroxidase (Vector

Laboratories, Inc., Burlington, CA, USA) was used for

detection, and sections were counterstained with haema-

toxylin. For immunofluorescence, antibody binding was

visualized using a fluorescent CY3-labelled anti-rabbit IgG

secondary antibody. Control experiments were done with

isotype-specific IgG for each antibody.

Biebrich Scarlet staining for eosinophils

To detect basic protein at alkaline pH (pH � 9.5), Biebrich

Scarlet stain, previously shown to be highly specific for

eosinophils, was used [34,35]. tissue sections were

deparaffinized, rehydrated gradually to 30% ethanol and

washed in PBS for 5 min. Slides were immersed in

Biebrich Scarlet stain for 1 h at 218C. Biebrich Scarlet

stain was prepared by mixing 1.9 gram borax with 20 mg

of Biebrich Scarlet powder (Sigma) in 50 mL of water and

titrating the pH to 9.5. Slides were counterstained with

methyl green for 2 min, washed with 100% ethanol for one

min and dehydrated before applying coverslips.

Cell quantification

The number of eosinophils was quantified per unit of

epithelial basement membrane to a uniform depth of

submucosa encompassing an area of 1.26 mm2 using an

eyepiece reticule. The average of cells counted in at least 6

sections was used for data analysis. All slides were

examined by a single observer, blinded to the experimental

condition. The percentage reduction in numbers of upper

airway eosinophils caused by anti-IL-5 antibody treatment

was calculated using the formula:

% reduction � ��OVA-induced eosinophils�/�OVA

1 anti-IL-5Ab-induced eosinophils��4 ��OVA-induced eosinophils�2 �Saline-induced eosinophils��

Data analysis

Data from each experimental condition were averaged

and compared by non-parametric Mann±Whitney U-test

[36]. A P-value of 0.05 was regarded as statistically

significant.

Results

Mouse upper airway anatomy

To define the distribution of inflammation in response to

OVA, the anatomy of the mouse upper airway needed to be

defined. For histological analysis, the upper airway was

sectioned rostral to caudal into four blocks A, B, C and D as

described under Materials and methods. Because the upper

airway anatomy is quite different in these four areas, we

illustrate the anatomy of the mouse upper airway in coronal

sections taken rostral to caudal by MRI scanning. The

images corresponding to the caudal ends of each block are

illustrated in Fig. 2. Key structures are identified in Fig. 3

(see below).

Distribution of respiratory and olfactory epithelium in the

mouse upper airway

The mouse olfactory epithelium produces a specific

protein known as olfactory marker protein or OMP

[18,33]. The BALB/c mouse olfactory epithelium was

found to have a characteristic high-intensity MRI signal

(shown in Fig. 2) and a thick, multinucleated appearance

on H & E stained tissues. The distribution of olfactory

epithelium was confirmed by immunostaining with OMP

anti-serum. OMP-positive epithelium was found in the

superior region of the nasal cavity, including the superior

aspect of the septum and the lateral nasal walls (Fig. 3).

The superior half of the vomeronasal body, an olfactory

organ, was also defined by strong OMP expression

[18,33].

The distribution of cytokeratin-positive epithelium in the

mouse upper airway was determined by immunostaining

with a rat anti-mouse pancytokeratin antibody (data not

shown). Positive immunostaining was observed throughout

the entire upper airway but was most intense in the lower

half of the septum, the lateral nasal wall and the floor of the

nasal cavity. Olfactory epithelium also showed weak

immunostaining for cytokeratin.

The distribution of respiratory epithelium in the mouse

upper airway was also examined by immunostaining for the

transcription factor HFH-4 (Fig. 3). Using a polyclonal

anti-HFH-4 antibody, the distribution of HFH-4 immuno-

staining matched the distribution of high-intensity cyto-

keratin immunostaining, namely involving the floor of the

nasal cavity, the inferior half of the lateral wall and the

septum and maxillary sinuses. As expected, HFH-4

immunostaining was specific for respiratory epithelium

and exclusive of cells that expressed OMP. In subsequent

experiments, we found that OVA-induced upper airway

eosinophil infiltration was specific for areas lined by

respiratory epithelium.

Compartments of bone marrow were also identified in

the superior portion of the nasal septum and just superior

to the nasal cavity within the skull (refer to Fig. 6 below).

The eosinophil density in this compartment was used to

assess the magnitude of inflammatory response to

Upper airway allergic inflammation in mice 1051

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systemic sensitization with IP vs. intranasal OVA (see

below).

OVA-sensitized, OVA-challenged mice develop upper

airway eosinophil infiltration

Mice sensitized by IP OVA and challenged with aero-

solized OVA developed significant upper airway eosinophil

infiltration. In preliminary experiments, the extent of

eosinophil infiltration was determined at 24, 48, 72 and

96 h after the 19 day sensitization/challenge protocol. The

peak of infiltration was found to occur at 48±72 h after the

last OVA challenge. Hence, in subsequent experiments,

mice were euthanized 48 h after the last OVA challenge.

The appearance of upper airway eosinophil infiltration in

OVA-sensitized, OVA-challenged mice identified by

Biebrich Scarlet Red staining is illustrated in Fig. 4. The

saline-sensitized/saline-challenged mice had rare eosino-

phils in the nasal mucosa. In contrast, the OVA-sensitized/

OVA-challenged mice developed a marked increase in

mucosal eosinophils (32.5 ^ 11.8 vs. 5.0 ^ 0.6,

P � 0.0005) as summarized in Fig. 5. The eosinophil

infiltration was almost exclusively confined to areas of

respiratory epithelium and the underlying submucosa.

Eosinophil infiltration was consistently most intense in

blocks C and D within the lower one-half of the nasal

septum and lateral nasal wall and the floor of the nasal

cavity (n � 5 experiments). The submucosa in these areas

Fig. 2. Coronal sections through the mouse upper airway obtained by MRI. Serial scans were taken with an in-plane resolution of 60 mm

and a slice thickness 500 mm. The areas with high intensity in the MRI correspond to areas of olfactory epithelium (see below). See scale

bar for magnification. Block sections A, B, C and D described under Materials and methods correspond best with images 3, 6, 9 and 12,

respectively, on the MRI scan. The multiple ethmoturbinales are shown in images 10±12 (45).

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remained relatively thin, but there was a suggestion of

expansion in some areas with the most intense eosinophil

infiltration. In contrast, virtually no eosinophils were seen

in areas of olfactory epithelium.

Enhanced detection of OVA-induced upper airway eosino-

phil infiltration

Eosinophils were easily identified in areas of respiratory

epithelium by Scarlet Red staining. However, their identifica-

tion was less precise in the thicker olfactory epithelium. To

more precisely examine the entire mouse upper airway, we

used immunofluorescent staining with a primary MBP

antibody and a fluorescent CY3-conjugated secondary anti-

body and examined the distribution of eosinophils in OVA-

sensitized/OVA-challenged mice. Figure 6 shows a colour

enhanced reconstruction of the entire mouse upper airway

corresponding to block D stained for MBP. The background

colour was enhanced to demonstrate the anatomy. The normal

red-orange fluorescence of the CY3 has been colour enhanced

to yellow for better visualization of eosinophils. As shown, the

predominant distribution of eosinophils by MBP staining was

similar to that detected by Scarlet Red stain, namely involving

the lower one-half of the nasal septum and lateral nasal wall

and the floor of the nasal cavity. In addition, a few eosinophils

could be detected in the epithelium of the maxillary sinuses,

the inferior portions of the inferior turbinates and in the ventral

scroll and the base of the second ethmoturbinates. Eosinophils

were only seen in areas lined with respiratory epithelium.

Upper airway eosinophil infiltration requires OVA sensiti-

zation followed by aerosol OVA challenge

To determine whether upper airway eosinophil infiltration

required both systemic OVA sensitization and aerosol OVA

challenge, we compared eosinophil infiltration in IP saline-

sensitized/intranasal saline-challenged, IP OVA-sensitized/

intranasal saline-challenged, and IP OVA-sensitized/intranasal

OVA-challenged mice. The extent of eosinophil infiltration

was determined by MBP immunofluorescence. As shown in

Fig. 7, the IP OVA-sensitized/intranasal saline mice showed

Fig. 4. Upper airway eosinophilia in

OVA-sensitized/OVA-challenged mice.

(a) Section of the nasal septum in a saline-

sensitized/saline-challenged mouse

showing absence of eosinophils. (b)

Section of the nasal septum in an OVA-

sensitized/OVA-challenged mouse

showing numerous eosinophils in the

submucosa (arrows) with mild expansion

of the submucosal space.

Fig. 3. Composite view of the mouse upper airway from block C

immunostained for olfactory marker protein (OMP) and HFH-4

(see Materials and methods). The olfactory epithelium (blue-black

immunostain) was distributed in the upper half of the nasal cavity,

including the upper half of the septum and the lateral nasal wall.

The superior half of the vomeronasal body, an olfactory organ,

was also strongly OMP-positive. HFH-4 immunostaining (brown

immunostain) was found to be specific for respiratory epithelium

and to have a distribution precisely opposite the distribution of

OMP immunostaining.

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only rare upper airway eosinophils, similar to the IP saline-

sensitized/intranasal saline-challenged mice. In contrast, the IP

OVA-sensitized/intranasal OVA-challenged mice showed a

large increase in the numbers of eosinophils as expected.

Hence, IP OVA sensitization alone was insufficient to induce

upper airway eosinophil infiltration.

Intranasal vs. IP sensitization

To more closely approximate the process of intranasal

sensitization to allergens, BALB/c mice were sensitized by

intranasal instillation of 1% OVA (10 mL per nostril) daily for

10 consecutive days. This protocol was previously reported to

elicit a more selective IgE anti-OVA response, whereas the IP

OVA sensitization protocol elicited both an IgE and an IgG

anti-OVA response [25]. Control mice that received repeated

intranasal instillation of saline failed to develop upper airway

eosinophil infiltration. Mice administered repeated intranasal

OVA developed upper airway eosinophil infiltration in a

pattern similar to that observed after IP OVA sensitization and

aerosol OVA challenge. This infiltration was statistically

significant compared to intranasal saline alone (23.3 ^ 20.3

vs. 2.0 ^ 0.1, P � 0.02). In comparison with the IP

Fig. 6. Composite view of the mouse

upper airway corresponding

approximately to panel 11 in Fig. 2 (block

D) stained for eosinophil MBP in an

OVA-sensitized/OVA-challenged mouse.

MBP was visualized using a fluorescent

CY3-labelled antirabbit secondary

antibody. The background colour (red)

was enhanced to demonstrate the

anatomy. The normal reddish-orange

fluorescence of the CY3 was colour

enhanced to yellow to allow for better

visualization of eosinophils. Eosinophils

were seen in the lower one-half of the

nasal septum and lateral nasal wall and the

floor of the nasal cavity. In addition, a few

eosinophils were also seen in the

epithelium of the maxillary sinuses, the

inferior portions of the inferior turbinates

and in the ventral scroll and the base of the

second ethmoturbinates. All of these areas

are lined with respiratory epithelium.

Virtually no eosinophils were found in

areas of olfactory epithelium. Note that

eosinophils are also visible within the

bone marrow cavity of the upper septum.

Fig. 5. Comparison of the number of upper airway eosinophils in

saline-sensitized/saline-challenged mice vs. OVA-sensitized/

OVA-challenged mice. Eosinophils were identified by Scarlet

Red staining and counted with an eyepiece reticule along a

uniform length of basement membrane in the lower half of the

nasal septum and lateral nasal wall. The difference is statistically

significant (P � 0.0005). Three experiments were performed and

representative data are shown (n � 4 mice per condition).

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sensitization protocol, the repeated intranasal sensitization/

challenge protocol elicited a less intense (approximately 70%

intensity) eosinophil infiltration.

We compared the magnitude of sensitization with OVA

administered via the IP vs. the intranasal protocol. This was

done by measuring the incremental rise in eosinophil

numbers per unit area of the bone marrow after each type

of sensitization. Both protocols elicited an increase in bone

marrow eosinophils. As expected, the IP protocol elicited a

greater increase in bone marrow eosinophils (4.33 ^ 1.48

eosinophils per hpf) than the intranasal protocol

(1.55 ^ 0.69 eosinophils per hpf) (P � 0.03). This differ-

ence may explain the relatively greater intensity of the upper

airway eosinophil response observed using the IP protocol.

No significant induction of epithelial ICAM-1 or eotaxin

immunostaining after OVA sensitization and challenge.

We sought to determine whether the selective infiltration of

eosinophils into areas lined by respiratory epithelium was due to

induction of ICAM-1 in respiratory but not olfactory

epithelium. Induction of epithelial ICAM-1 has been reported

to occur in human allergic rhinitis and may facilitate the

transepithelial migration of eosinophils [8,37]. Tissues from

control and OVA sensitized/challenged mice (both IP and

intranasal at 24, 48 and 72 h) were immunostained for ICAM-1.

Control mice demonstrated constitutive ICAM-1 immunostain-

ing in the nasal-associated lymphoid tissue (NALT) along the

lateral aspect of the floor of the nasal cavity and the overlying

respiratory epithelium (Fig. 8). Of note, eosinophils were not

found in the NALT regions either by Biebrich or immuno-

fluorescence staining. In contrast, no other areas of epithelium

were stained. No measureable induction of epithelial immu-

nostaining for ICAM-1 was observed in either respiratory or

olfactory epithelium after OVA sensitization and challenge.

In a similar manner, we immunostained the same tissues

with a polyclonal antibody recognizing eotaxin. Eotaxin is

known to be expressed in human and mouse airway epithelium

and to be induced in human allergic rhinitis [11,38]. Control

mice demonstrated mild focal eotaxin immunostaining in an

area of respiratory and olfactory epithelium along the mid-

portion of the nasal septum and throughout the lower airway

bronchioles as previously reported. OVA-sensitized/chal-

lenged mice showed similar immunostaining (data not

shown). Hence, we found no significant induction of epithelial

immunostaining for eotaxin in either respiratory or olfactory

epithelium after OVA sensitization and challenge.

Inhibition of upper airway eosinophilia by an anti-IL-5

antibody

The lower airway eosinophilia induced by OVA sensitization

and aerosol challenge has previously been shown to be

Fig. 7. Comparison of upper airway eosinophil infiltration,

stained for eosinophil MBP, under various conditions. Antibody

binding was visualized using a fluorescent CY3-labelled anti-

rabbit secondary antibody. (a) Section from a saline-sensitized,

saline-challenged mouse showing no eosinophils. (b) Section from

an OVA-sensitized, saline challenged mouse showing very few

eosinophils. (c) Section from an OVA-sensitized, aerosol OVA-

challenged mouse showing numerous eosinophils.

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dependent upon the production of IL-5 during the sensitization

procedure [25]. To determine whether upper airway eosino-

phil infiltration was dependent upon IL-5, mice were treated

with an anti-IL-5 antibody (TRFK-5) or a control rat IgG

during the 10 day intranasal OVA challenge protocol. Mice

were evaluated for eosinophil infiltration 48 h after the last

intranasal OVA dose (Fig. 9). Mice receiving intranasal OVA

plus control rat IgG antibody showed no significant reduction

in upper airway eosinophils. In contrast, mice receiving

intranasal OVA plus anti-IL-5 antibody showed roughly an

80% reduction in upper airway eosinophils (P � 0.005).

Discussion

The results of this study demonstrate that BALB/c mice

sensitized systemically or intranasally and then challenged

with OVA develop a specific and localized upper airway

infiltration with eosinophils. Eosinophil infiltration was most

intense in the lower portion of the nasal septum, the floor and

the lower lateral walls of the mid-caudal region of the nasal

cavity. These areas are lined by respiratory epithelium. These

observations are similar to those reported in the lower airway

and suggest an epithelial cell-specific response [25,27].

Virtually no eosinophil infiltration was observed in areas

lined by olfactory epithelium. Immunofluorescence staining

with MBP antibody confirmed the distribution of eosinophils

but also demonstrated some eosinophils in the maxillary

sinuses and in circumscribed regions of the superiorly located

ethmoturbinates in areas lined by respiratory epithelium. The

Fig. 8. Constitutive expression of ICAM-1 on nasal-associated lymphoid tissue (NALT) and the epithelium overlying the NALT in

control mice. Panel a (insert): A section of mouse upper airway from Block C stained with control rat IgG showing no specific

immunostaining in the epithelium. The NALT is visible as an accumulation of lymphocytes beneath the epithelium along the inferior

aspect of the lateral nasal wall. Panel b: Similar section of the mouse upper airway immunostained with a polyclonal anti-ICAM-1

antibody showing focal staining in the respiratory epithelium overlying the NALT (arrows) but minimal staining of respiratory epithelium

elsewhere (arrowheads). Sections were counterstained with haematoxylin to better demonstrate the lymphoid cells of the NALT.

Fig. 9. OVA-induced upper airway eosinophilia is abrogated by

administration of rat anti-IL-5 antibody (TRFK-5). BALB/c mice

were sensitized by repeated intranasal instillation of OVA for 10

successive days and simultaneously given IP injections of TRFK-

5 or control rat IgG (100 mg) on days 4, 6 and 8. In comparison

with the OVA-sensitized mice, the anti-IL-5 treated mice showed

an 80% reduction in upper airway eosinophil infiltration

(P � 0.0005). Mice given IP injections of control rat IgG

developed eosinophil infiltration equal to that of OVA-sensitized/

OVA-challenged mice. (Saline control and intranasal OVA-treated

mice, N � 3 mice per condition. Anti-IL-5 and rat IgG-treated

mice, N � 5 mice per condition).

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demonstration of eosinophils in the ethmoturbinates, but not in

more inferiorly located areas lined by olfactory epithelium,

argues further for an anatomically specific distribution of

eosinophil infiltration. This may reflect differences in the

ability of the respiratory and the olfactory epithelia to mobilize

an allergic inflammatory response.

The epithelium participates in eosinophil recruitment

through production of adhesion molecules and chemokines

[38]. In human allergic rhinitis, an increase in epithelial

expression of ICAM-1 has been observed during natural

allergen exposure [8] and after experimental allergen

challenge [37]. There is little information on ICAM-1

expression in the mouse upper airway, but Asakura et al.

found that topically applied monoclonal anti-ICAM-1 anti-

body caused a significant attenuation of the eosinophil

infiltration into nasal mucosa 24 h after nasal challenge with

OVA in sensitized rats [9]. Furthermore, in a mouse model of

Ova-induced asthma, treatment with anti-ICAM-1 antibody

blocked Ova-inhalation-induced infiltration of eosinophils by

greater than 70% [10]. Similarly, an increase in epithelial

expression of eotaxin has been found in nasal mucosa after

local allergen challenge in patients with allergic rhinitis [11]

and after natural allergen exposure [39]. Intranasal application

of eotaxin has also been shown to increase eosinophils in nasal

secretions in patients with allergic rhinitis [13]. Given these

data, we hypothesized that intranasal OVA challenge may be

associated with an increase in epithelial expression of either

ICAM-1 or eotaxin in OVA-sensitized mice. However, we

were unable to confirm that either of these proteins was

induced in OVA sensitized and challenged mice. Our failure to

detect the induction of either ICAM-1 or eotaxin may reflect a

lack of their induction, insensitivity of the immunostaining

procedure or perhaps the failure to detect an early response.

Certain epitopes of key immunological interest could have

been lost owing to the tissue fixation and/or decalcification

processes employed. Indeed, we found it difficult to detect

lymphocyte surface markers or cytokines in these tissues.

However, this is unlikely to be the case, since we found regions

of constitutive expression of both ICAM-1 and eotaxin in

control mice. Using a similar OVA allergen challenge model,

Rothenberg et al. found that eotaxin mRNA expression

occurred rapidly in the lungs, peaking at 3±6 h [40]. In eotaxin

null mice, the influx of eosinophils into the lungs after allergen

challenge was reduced by 70% at 18 h but was normal 48 h

after allergen challenge [40]. Hence, in our experiments, it is

possible that we could have missed a critical early induction of

epithelial eotaxin expression even though we examined mice

as early as 24 h after allergen challenge. Obviously, more

studies are needed to examine the mechanism of eosinophil

infiltration into the upper airway, including strategies to

selectively inhibit the function of either ICAM-1 or eotaxin in

OVA sensitized and challenged mice.

The development of upper airway eosinophilia required

both systemic OVA sensitization and aerosol or intranasal

OVA challenge. Hence, direct application of OVA to the

upper airway was required to induce eosinophil infiltration.

Intraperitoneal sensitization was found to elicit a greater

increase in bone marrow eosinophils than intranasal

sensitization. This difference in sensitization may explain

the less intense eosinophil infiltration of the upper airway

seen using the intranasal protocol.

Repeated intranasal exposure to OVA has previously been

shown to elicit a weaker sensitization than IP injection with

alum but greater selectivity for eliciting an IgE anti-OVA

response [25]. As a result, the intranasal sensitization protocol

was initially used by Hamelmann et al. to demonstrate IL-5

dependence of lower airway eosinophilia. However, in a

subsequent study, this same group [26] showed that OVA

sensitization by the IP route was also abrogated by anti-IL-5

antibody. Hence, OVA sensitization by either protocol

appears to give rise to a similar IL-5 dependent response.

OVA-induced upper airway eosinophilia was found to be

IL-5-dependent, since administration of IL-5 antibody during

OVA sensitization resulted in an 80% reduction in the

eosinophil response to subsequent OVA challenge. This

finding is consistent with previously published reports. For

example, Okano et al. [41] found that mice sensitized by

repeated intranasal instillation of Schistosoma mansoni egg

antigen (SEA) produced SEA-specific IgE antibody, devel-

oped nasal eosinophilia and had infiltration of the nasal

secretions with lymphocytes that could be demonstrated to

produce IL-5 in vitro. In another study, Asakura et al. [9]

investigated the effect of anti-IL-5 antibody pretreatment on

allergen-induced late-phase nasal eosinophil infiltration in

mice. Consistent with our results, they reported that the nasal

eosinophil response was significantly abrogated by anti-IL-5

mAb pretreatment. In contrast, the numbers of mast cells in

nasal secretions was unaffected by anti-IL-5 mAb. Hence,

similar to mouse models of allergic lower airways disease

[25±27], the mouse upper airway, specifically in areas lined

by respiratory epithelium, is a target for allergic inflamma-

tion, and this process is largely IL-5-dependent.

Most recently, van de Rijn et al. developed a mouse model

of allergic rhinitis in 129/SvEv mice with Aspergillus

fumigatus (Af) antigen [12]. Consistent with our results, they

showed that sensitized mice developed nasal mucosal

eosinophil infiltration after nasal Af challenge. A similar

response was induced in IgE-deficient mice thus suggesting

that IgE is not required for eosinophil infiltration. They also

reported that the upper airway response was characterized by a

paucity of mononuclear cells and a predominance of

eosinophils, whereas comparable studies in the lower airway

have shown a greater admixture of eosinophils and mono-

nuclear cells [12]. Similarly, we detected very few lympho-

cytes (or neutrophils) in the OVA-sensitized, OVA-challenged

nasal tissues. Nonetheless, our results using the anti-IL-5

Upper airway allergic inflammation in mice 1057

q 2001 Blackwell Science Ltd, Clinical and Experimental Allergy, 31, 1048±1059

antibody (TRFK-5) provide evidence that allergic upper

airway eosinophil infiltration, like that of the lower airway, is

dependent on the elaboration of IL-5. We hope to extend these

observations to a more detailed examination of the participa-

tion of Th2 and Th1 lymphocytes in nasal eosinophil

infiltration using a strategy of passive transfer of OVA-

specific T lymphocytes as recently reported by Randolph and

coworkers [22].

An attractive additional feature of the mouse upper

airway allergen model is the ability to simultaneously

examine mucosal inflammation and bone marrow effects of

allergen sensitization and/or nasal allergen challenge.

Hence, we were able to show that OVA sensitization alone

induced a significant increase in bone marrow eosinophils

but no increase in nasal mucosal eosinophils, and we were

able to show that the IP protocol elicited a stronger

sensitization than the intranasal protocol. By simulta-

neously examining the bone marrow and upper airway

compartments, it may be possible to study in a coordinated

fashion mechanisms of induction of eosinophil bone

marrow progenitor cells and recruitment of these cells into

the upper airway as has been described in the dog [42±44].

To our knowledge, this study represents the first

demonstration of mouse sinus anatomy using MRI images.

Relatively high resolution of sinus mucosal thickness was

achieved with our protocol. In preliminary experiments, we

did not observe any significant change in nasal or sinus

mucosal thickness in response to allergic inflammation,

However, we postulate that MRI imaging may prove useful

in future studies in which more dramatic levels of

inflammation may be seen, such as chronic allergic

inflammation or acute infection. Serial MRI imaging may

offer the advantage of tracking dynamic mucosal changes,

such as the response to treatment, without killing the animal.

Acknowledgements

This work was funded in part by the NIH SCOR grant #

1P50HL54619 (Dr Chaplin) and by the Janssen Pharmaceutica

Research Award in Allergic Rhinitis (Dr Hussain). Dr Chaplin

is an Investigator of the Howard Hughes Medical Institute. The

authors thank Ms. Lisa Fitzgerald for manuscript preparation;

Michael Holtzman for use of Core Laboratory facilities;

Deepak Sampath, Tim Birkland, Brian Faddis, Richard Chole,

Theresa Tolley and Jill Roby for assistance in tissue

preparation and Dwight Look for review of the manuscript.

References

1 Mackay IS, Durham SR. ABC of allergies. Perennial rhinitis.

BMJ 1998; 316 (7135):917±20.

2 Juniper EF. Rhinitis management: the patient's perspective.

Clin Exp Allergy 1998; 28 (Suppl. 6):34±8.

3 Demoly P, Crampette L, Mondain M et al. Assessment of

inflammation in noninfectious chronic maxillary sinusitis. J

Allergy Clin Immunol 1994; 94 (1):95±108.

4 Meltzer EO. The prevalence and medical and economic impact

of allergic rhinitis in the United States. J Allergy Clin

Immunol 1997; 99 (6, part 2):S805±28.

5 Durham SR, Ying S, Varney VA. et al. Cytokine messenger

RNA expression for IL-3, IL-4, IL-5, and granulocyte/

macrophage-colony-stimulating factor in the nasal mucosa

after local allergen provocation: relationship to tissue

eosinophilia. J Immunol 1992; 148 (8):2390±4.

6 Bentley AM, Meng Q, Robinson DS, Hamid Q, Kay AB,

Durham SR. Increases in activated T lymphocytes, eosino-

phils, and cytokine mRNA expression for interleukin-5 and

granulocyte/macrophage colony-stimulating factor in bron-

chial biopsies after allergen inhalation challenge in atopic

asthmatics. Am J Respir Cell Mol Biol 1993; 8 (1):35±42.

7 Robinson DS, Hamid Q, Ying S. et al. Predominant TH2-like

bronchoalveolar T-lymphocyte population in atopic asthma. N

Engl J Med 1992; 326 (5):298±304.

8 Ciprandi G, Pronzato C, Ricca V, Bagnasco M, Canonica GW.

Evidence of intercellular adhesion molecule-1 expression on

nasal epithelial cells in acute rhinoconjunctivitis caused by

pollen exposure. J Allergy Clin Immunol 1994; 94 (4):738±46.

9 Asakura K, Saito H, Watanabe M, Ogasawara H, Matsui T,

Kataura A. Effects of anti-IL-5 monoclonal antibody on the

murine model of nasal allergy. Int Arch Allergy Immunol

1998; 116 (1):49±52.

10 Chin JE, Winterrowd GE, Hatfield CA. et al. Involvement of

intercellular adhesion molecule-1 in the antigen-induced

infiltration of eosinophils and lymphocytes into the airways

in a murine model of pulmonary inflammation. Am J Respir

Cell Mol Biol 1998; 18 (2):158±67.

11 Minshall EM, Cameron L, Lavigne F. et al. Eotaxin mRNA

and protein expression in chronic sinusitis and allergen-

induced nasal responses in seasonal allergic rhinitis. Am J

Respir Cell Mol Biol 1997; 17 (6):683±90.

12 van de Rijn M, Mehlhop PD, Judkins A, Rothenberg ME,

Luster AD, Oettgen HC. A murine model of allergic rhinitis:

studies on the role of IgE in pathogenesis and analysis of the

eosinophil influx elicited by allergen and eotaxin. J Allergy

Clin Immunol 1998; 102 (1):65±74.

13 Hanazawa T, Antuni JD, Kharitonov SA, Barnes PJ. Intranasal

administration of eotaxin increases nasal eosinophils and nitric

oxide in patients with allergic rhinitis. J Allergy Clin Immunol

2000; 105 (1, part 1):58±64.

14 Cowart BJ, Flynn-Rodden K, McGeady SJ, Lowry LD.

Hyposmia in allergic rhinitis. J Allergy Clin Immunol 1993;

91 (3):747±51.

15 Apter AJ, Mott AE, Frank ME, Clive JM. Allergic rhinitis and

olfactory loss. Ann Allergy Asthma Immunol 1995; 75

(4):311±6.

16 Klimek L, Eggers G. Olfactory dysfunction in allergic rhinitis

is related to nasal eosinophilic inflammation. J Allergy Clin

Immunol 1997; 100 (2):158±64.

17 Moll B, Klimek L, Eggers G, Mann W. Comparison of

1058 I. Hussain et al.

q 2001 Blackwell Science Ltd, Clinical and Experimental Allergy, 31, 1048±1059

olfactory function in patients with seasonal and perennial

allergic rhinitis. Allergy 1998; 53 (3):297±301.

18 Keller A, Margolis FL. Isolation and characterization of rat

olfactory marker protein. J Biol Chem 1976; 251 (20):6232±7.

19 Renz H, Smith HR, Henson JE, Ray BS, Irvin CG, Gelfand

EW. Aerosolized antigen exposure without adjuvant causes

increased IgE production and increased airway responsive-

ness in the mouse. J Allergy Clin Immunol 1992; 89

(6):1127±38.

20 Renz H, Bradley K, Larsen GL, McCall C, Gelfand EW.

Comparison of the allergenicity of ovalbumin and ovalbumin

peptide 323±339. Differential expansion of V beta-expressing

T cell populations. J Immunol 1993; 151 (12):7206±13.

21 Randolph DA, Stephens R, Carruthers CJ, Chaplin DD.

Cooperation between Th1 and Th2 cells in a murine model

of eosinophilic airway inflammation. J Clin Invest 1999; 104

(8):1021±9.

22 Randolph DA, Carruthers CJ, Szabo SJ, Murphy KM, Chaplin

DD. Modulation of airway inflammation by passive transfer of

allergen- specific Th1 and Th2 cells in a mouse model of

asthma. J Immunol 1999; 162 (4):2375±83.

23 Cohn L, Homer RJ, Marinov A, Rankin J, Bottomly K.

Induction of airway mucus production By T helper 2 (Th2)

cells: a critical role for interleukin 4 in cell recruitment but not

mucus production. J Exp Med 1997; 186 (10):1737±47.

24 Kim TS, DeKruyff RH, Rupper R, Maecker HT, Levy S,

Umetsu DT. An ovalbumin-IL-12 fusion protein is more

effective than ovalbumin plus free recombinant IL-12 in

inducing a T helper cell type 1-dominated immune response

and inhibiting antigen-specific IgE production. J Immunol

1997; 158 (9):4137±44.

25 Hamelmann E, Oshiba A, Loader J. et al. Antiinterleukin-5

antibody prevents airway hyperresponsiveness in a murine

model of airway sensitization. Am J Respir Crit Care Med

1997; 155 (3):819±25.

26 Hamelmann E, Cieslewicz G, Schwarze J. et al. Anti-

interleukin 5 but not anti-IgE prevents airway inflammation

and airway hyperresponsiveness. Am J Respir Crit Care Med

1999; 160 (3):934±41.

27 Hamelmann E, Takeda K, Schwarze J, Vella AT, Irvin CG,

Gelfand EW. Development of eosinophilic airway inflamma-

tion and airway hyperresponsiveness requires interleukin-5 but

not immunoglobulin E or B lymphocytes. Am J Respir Cell

Mol Biol 1999; 21 (4):480±9.

28 Collins PD, Marleau S, Griffiths-Johnson DA, Jose PJ,

Williams TJ. Cooperation between interleukin-5 and the

chemokine eotaxin to induce eosinophil accumulation in vivo.

J Exp Med 1995; 182 (4):1169±74.

29 Hardisty JF, Garman RH, Harkema JR, Lomax LG, Morgan

KT. Histopathology of nasal olfactory mucosa from selected

inhalation toxicity studies conducted with volatile chemicals.

Toxicol Pathol 1999; 27 (6):618±27.

30 Ishida M, Amesara R, Ukai K, Sakakura Y. Antigen (DNP-As)

-induced allergic rhinitis model in guinea pigs. Ann Allergy

1994; 72 (3):240±4.

31 Ressler KJ, Sullivan SL, Buck LB. A zonal organization of

odorant receptor gene expression in the olfactory epithelium.

Cell 1993; 73 (3):597±609.

32 Blatt EN, Yan XH, Wuerffel MK, Hamilos DL, Brody SL.

Forkhead transcription factor HFH-4 expression is temporally

related to ciliogenesis. Am J Respir Cell Mol Biol 1999; 21

(2):168±76.

33 Keller A, Margolis FL. Immunological studies of the rat

olfactory marker protein. J Neurochem 1975; 24 (6):1101±6.

34 Mlynek ML. Comparative investigations on the specificity of

Adams' reaction and the Biebrich scarlet stain for the

demonstration of eosinophilic granules. Klin Wochenschr

1985; 63 (14):646±7.

35 Schaefer HE, Fischer R. Specific staining of eosinophilic

granulocytes with Biebrich scarlet. Klin Wochenschr 1968; 46

(7):396±7.

36 Daly LE, Bourke GJ, McGilvray JW. Interpretation and uses

of medical statistics. 4th edn. Oxford. Boston. Blackwell

Scientific Publications., 1991.

37 Ciprandi G, Ricca V, Passalacqua G, Fasolo A, Canonica GW.

Intranasal fluticasone propionate reduces ICAM-1 on nasal

epithelial cells both during early and late phase after allergen

challenge. Clin Exp Allergy 1998; 28 (3):293±9.

38 Rothenberg ME, Eosinophilia. N Engl J Med 1998; 338

(22):1592±600.

39 Pullerits T, Linden A, Praks L, Cardell LO, Lotvall J.

Upregulation of nasal mucosal eotaxin in patients with allergic

rhinitis during grass pollen season: effect of a local

glucocorticoid. Clin Exp Allergy 2000; 30 (10):1469±75.

40 Rothenberg ME, MacLean JA, Pearlman E, Luster AD, Leder

P. Targeted disruption of the chemokine eotaxin partially

reduces antigen-induced tissue eosinophilia. J Exp Med 1997;

185 (4):785±90.

41 Okano M, Nishizaki K, Abe M. et al. Strain-dependent

induction of allergic rhinitis without adjuvant in mice. Allergy

1999; 54 (6):593±601.

42 Inman MD, Denburg JA, Ellis R, Dahlback M, O'Byrne PM.

Allergen-induced increase in bone marrow progenitors in

airway hyperresponsive dogs: regulation by a serum hemopoie-

tic factor. Am J Respir Cell Mol Biol 1996; 15 (3):305±11.

43 Inman MD, Ellis R, Wattie J, Denburg JA, O'Byrne PM.

Allergen-induced increase in airway responsiveness, airway

eosinophilia, and bone-marrow eosinophil progenitors in mice.

Am J Respir Cell Mol Biol 1999; 21 (4):473±9.

44 Woolley MJ, Denburg JA, Ellis R, Dahlback M, O'Byrne PM.

Allergen-induced changes in bone marrow progenitors and

airway responsiveness in dogs and the effect of inhaled

budesonide on these parameters. Am J Respir Cell Mol Biol

1994; 11 (5):600±6.

45 Mery S, Gross EA, Joyner DR, Godo M, Morgan KT. Nasal

diagrams: a tool for recording the distribution of nasal lesions

in rats and mice. Toxicol Pathol 1994; 22 (4):353±72.

Upper airway allergic inflammation in mice 1059

q 2001 Blackwell Science Ltd, Clinical and Experimental Allergy, 31, 1048±1059