Full text of DOI:10.1038/labinvest.3780442

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LABORATORY INVESTIGATION
Vol. 82, No. 4, p. 495, 2002
Copyright © 2002 by The United States and Canadian Academy of Pathology, Inc.
Printed in U.S.A.
Eotaxin Expression by Epithelial Cells and Plasma
Cells in Chronic Asthma
Rakesh K. Kumar, Paul S. Thomas, Da-Qiang Seetoo, Cristan Herbert,
Andrew N. J. McKenzie, Paul S. Foster, and Andrew R. Lloyd
Inflammation Research Unit (RKK, PST, D-QS, CH, ARL), Department of Pathology, University of New South Wales,
Sydney; Prince of Wales Hospital Clinical School (PST), University of New South Wales, Sydney, Australia; Medical
Research Council Laboratory of Molecular Biology (ANJM), Cambridge, United Kingdom; and Department of
Biochemistry and Molecular Biology (PSF), John Curtin School of Medical Research, Australian National University,
Canberra, Australia
SUMMARY: Chemoattractants such as eotaxin are believed to play an important role in the recruitment of eosinophils into the
airways in asthma. We investigated expression of eotaxin in the airway wall in a model of chronic human asthma, in which
systemically sensitized mice were exposed to low mass concentrations of aerosolized antigen for 6 weeks. In these animals, the
number of intraepithelial eosinophils in the airways was significantly increased 3 hours after exposure and declined by 24 hours.
In parallel, immunoreactivity for eotaxin was strikingly up-regulated in airway epithelial cells and in inflammatory cells in the lamina
propria. The latter were identified as plasma cells by double immunofluorescent labeling. Increased expression of eotaxin by
epithelial cells and plasma cells was also demonstrated in a case of fatal human asthma. In contrast, sensitized mice that received
a single exposure to a high mass concentration of aerosolized antigen exhibited delayed eosinophil recruitment, which did not
correlate with eotaxin expression. Furthermore, in sensitized chronically exposed interleukin-13–deficient mice there was virtually
no recruitment of eosinophils into the airways, although eotaxin expression was greater than or equal to that in wild-type mice.
These results indicate that there are striking differences between acute and chronic exposure models in the time course of eotaxin
expression and eosinophil recruitment. Although high eotaxin levels alone are not sufficient to cause recruitment of eosinophils
into the airways, recurrent exposure may generate or up-regulate additional signals required for eosinophil chemotaxis. (Lab
Invest 2002, 82:495–504).
sthma is an inflammatory disease of the airways,
characterized by acute exacerbations superim-
posed upon a background of chronic inflammation
and airway wall remodeling (Bousquet et al, 2000). In
acute asthma, numerous eosinophils are recruited into
the lamina propria and epithelium of the airways
sence of eosinophil recruitment (Leckie et al, 2000).
Nevertheless, eosinophils clearly do play a major role
in acute asthma, and an increased number of eosino-
phils in sputum is predictive of an exacerbation of the
illness (Jatakanon et al, 2000). Inhibition of recruitment
of eosinophils is therefore attractive as a potential
therapeutic strategy.
A
(
Montefort et al, 1994; Shaver et al, 1997). The pres-
ence of intraepithelial eosinophils is regarded as a
distinctive histopathological feature of the disease
Leukocyte recruitment to the site of inflammation
typically involves both the increased local generation
of chemoattractant mediators as well as enhanced
expression or increased affinity of adhesion ligands
and their counter-receptors on leukocytes and endo-
thelial cells (Bochner, 2000). In the case of eosinophils,
a range of relatively selective chemoattractants for
these cells has been identified, including eicosatetrae-
noic acid derivatives (Guilbert et al, 1999) and mem-
bers of the CC chemokine family, notably eotaxin,
RANTES, and various molecules of the monocyte
chemoattractant protein (MCP) group (Kita and Gleich,
(
Bousquet et al, 1990), although the specific role of
these inflammatory cells in the pathogenesis of the
functional abnormalities associated with an acute at-
tack remains poorly understood. Eosinophils release a
variety of potentially toxic granule products (Kroegel et
al, 1994; Weller, 1991), which have long been re-
garded as important in causing epithelial injury leading
to airway hyperreactivity (AHR) (Coyle et al, 1994;
Frigas et al, 1981). However, recent studies using a
humanized antibody to interleukin-5 (IL-5), which in-
hibits accumulation of eosinophils, have provided per-
suasive evidence that AHR can develop in the ab-
1996; Rothenberg et al, 1999; Teran, 2000). Potentially
selective adhesion molecules that may play a critical
role in eosinophil accumulation have also been de-
scribed, in particular very late antigen 4 (VLA-4) and
vascular cell adhesion molecule-1 (VCAM-1) (Boch-
ner, 2000; Henricks et al, 1997).
Received December 12, 2001.
This work was supported by a grant from Asthma New South Wales.
Address reprint requests to: R. K. Kumar, Department of Pathology, Uni-
versity of New South Wales, Sydney, Australia 2052. E-mail:
R.Kumar@unsw.edu.au
In human asthma, increased expression of various
eosinophil-selective chemoattractants has been re-
Laboratory Investigation • April 2002 • Volume 82 • Number 4 495

Kumar et al
ported (Alam et al, 1996; Lamkhioued et al, 1997;
Tillie-Leblond et al, 2000; Ying et al, 1999) and in-
creased expression of VCAM-1 (Fukuda et al, 1996)
has been demonstrated. However, the inherent diffi-
culty of human studies has made it difficult to assess
the relative importance of different mediators and
mechanisms during the evolution of an allergic inflam-
matory response. Despite this, the specificity of
eotaxin as a chemoattractant for eosinophils has led
to widespread acceptance of the notion that this
chemokine is likely to play an important role in the
accumulation of eosinophils in tissues (Garcia-Zepeda
et al, 1996; Ying et al, 1997).
their ability to produce this cytokine. Our results
suggest that there are striking differences between
acute and chronic exposure models in the time course
of eotaxin expression and eosinophil recruitment, that
plasma cells express high levels of eotaxin in both the
chronic murine model and in fatal human asthma, and
that eotaxin expression is not by itself sufficient to
cause recruitment of eosinophils.
Results
Recruitment of Eosinophils in Chronic and Acute
Exposure Models
Animal experimental studies lend considerable cre-
dence to the notion that eotaxin plays a pivotal role in
eosinophil recruitment in allergic inflammation
Sensitized BALB/c mice that had been repeatedly
exposed to low levels of aerosolized antigen for 6
weeks exhibited recruitment of numerous eosinophils
into the tracheal epithelium at 3 hours following the
final exposure (Fig. 1A). Numbers of intraepithelial
eosinophils remained high at 6 hours post-exposure
but declined considerably by 24 hours, although re-
maining significantly elevated compared to nonimmu-
nized nonexposed animals (p Ͻ 0.001 for all three time
points, Fig. 2). There was no evidence of any inflam-
matory response in the lungs of these mice.
In contrast, sensitized animals that received a single
uncontrolled exposure to aerosolized ovalbumin had
few eosinophils in the tracheal epithelium at 3 hours.
The numbers of these cells then increased rapidly,
reaching levels similar to those in chronically exposed
animals at 6 hours post-exposure, but subsequently
declined (Fig. 2). At 24 hours post-exposure, the lungs
of these mice exhibited a florid perivascular and
peribronchiolar inflammatory response, with accumu-
lation of numerous eosinophils (not shown), although
intraepithelial eosinophils were conspicuously absent.
In nonsensitized mice, virtually no recruitment of eo-
sinophils was observed following a single high expo-
sure to aerosolized antigen (Fig. 2).
(
Gonzalo et al, 1996, 1998; Mould et al, 1997). These
studies also provide support for the regulation of
eotaxin expression by so-called Th2 cytokines, nota-
bly interleukin-13 (IL-13) (Li et al, 1999; Teran et al,
1999; Zhu et al, 1999). However, it may be inappro-
priate to extrapolate to human asthma from studies
using these experimental models, because the
chronic inflammation and airway wall remodeling that
characterize human asthma are poorly replicated by
most rodent models of allergic bronchopulmonary
inflammation (Griffiths-Johnson et al, 1997; Nakamura
et al, 1999). Moreover, in most models, the inflamma-
tory response is characterized by a high density of
lymphocytes and eosinophils surrounding the airways
and extending to the blood vessels, which is unlike
chronic asthma (Cohn, 2001).
We have described an improved model of chronic
asthmatic inflammation of the airways in mice (Te-
melkovski et al, 1998). This involves systemic sensiti-
zation of high immunoglobulin E (IgE)-responder
BALB/c mice to ovalbumin and inhalational challenge
with low mass concentrations of aerosolized antigen
for 30 minutes/day, 3 days/week for 6 weeks. This
approach consistently elicits lesions typical of chronic
human asthma, including chronic inflammation in the
lamina propria with accumulation of lymphocytes,
plasma cells, and macrophages, as well as epithelial
hypertrophy, mucous cell hyperplasia/metaplasia, and
subepithelial fibrosis. In contrast to previously re-
ported mouse models of asthma, there are fewer total
inflammatory cells and no evidence of pulmonary
parenchymal inflammation, but numerous intraepithe-
lial eosinophils are seen in the upper airways. In
addition, the mice exhibit marked AHR to an inhaled
cholinergic agonist. Thus, this model replicates most
of the features that are typical of the chronic human
disease.
Expression of Eotaxin in Chronic and Acute
Exposure Models
In the chronic exposure model, immunohistochemical
staining for eotaxin revealed not only moderate-to-
strong expression in the airway epithelium but also
intense immunoreactivity in the cytoplasm of inflam-
matory cells in the lamina propria (Fig. 1B). These
latter cells had the morphological appearance of
plasma cells or plasmacytoid lymphocytes. No stain-
ing was observed when the primary antibody was
omitted. Furthermore, both the epithelial and the in-
flammatory cell reactivity could be completely inhib-
ited by preincubation of the antibody with the corre-
sponding blocking peptide (Fig. 1C). In the acute
exposure model, immunoreactivity was only observed
in the epithelium.
Semiquantitative grading of immunoreactivity in the
tracheal epithelium of chronically exposed, sensitized
acutely exposed, and nonsensitized acutely exposed
mice revealed striking differences in the intensity and
time course of expression. In chronically exposed
mice, high levels of expression were obvious at 3
In this study, we examined the relationship between
eosinophil recruitment and expression of eotaxin in
this long-term, low-exposure model of chronic exper-
imental asthma and compared the findings with those
obtained in a short-term, high-exposure model of
allergic bronchopulmonary inflammation. We also in-
vestigated eotaxin expression in fatal human asthma.
In addition, we examined the regulation of eotaxin
expression by IL-13 in gene-targeted mice deficient in
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Eotaxin Expression in Chronic Asthma
Figure 1.
A, Eosinophils within the tracheal epithelium of a sensitized chronically exposed BALB/c mouse at 3 hours following exposure to ovalbumin. B, Immunoperoxidase
staining for eotaxin in the airway wall of a sensitized chronically exposed BALB/c mouse at 3 hours following exposure to ovalbumin. Using a concentration of
antieotaxin four-fold lower than that required for optimal staining, there is still moderate immunoreactivity of airway epithelial cells and strong staining of the
cytoplasm of plasmacytoid inflammatory cells in the lamina propria. C, Same region from a consecutive section, demonstrating virtually complete inhibition of staining
by preincubation of the antibody with the corresponding blocking peptide. D, Absence of immunoreactivity for eotaxin in the airway wall of a sensitized chronically
exposed BALB/c mouse at 24 hours following exposure to ovalbumin. E, Immunoperoxidase staining for eotaxin in the trachea of a sensitized chronically exposed
IL-13 Ϫ/Ϫ mouse at 3 hours following exposure to ovalbumin, demonstrating strong immunoreactivity of both airway epithelial cells and plasma cells in the lamina
propria.
hours post-exposure (p Ͻ 0.05 compared with normal
animals), but these declined sharply at 6 hours, and
virtually no staining was detectable after 24 hours (Fig.
1D). In contrast, the levels of expression in the epithe-
lium of sensitized, acutely exposed mice were com-
parably high at 3 hours, but hardly declined thereafter.
Unexpectedly, we found that eotaxin expression pro-
gressively increased in the epithelium of nonsensitized
acutely exposed mice, so that at 24 hours post-
exposure it was similar to that observed in sensitized
mice at 3 hours (p Ͻ 0.05 compared with normal
animals) (Table 1).
To confirm the identity of the eotaxin-expressing
cells in the lamina propria of the airways in chronically
exposed mice at 3 hours post-exposure, we per-
formed double immunofluorescence staining for
Figure 2.
Numerical density of intraepithelial eosinophils (mean Ϯ SEM) in the tracheas
of nonsensitized acutely exposed, sensitized acutely exposed, and sensitized
chronically exposed mice. Significant differences compared to normal nonex-
posed control animals are shown as * ϭ PϽ0.05, ** ϭ PϽ0.01, *** ϭ
PϽ0.001.
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Kumar et al
Table 1. Eotaxin Expression in the Airway Epithelium
Time (h)
Nonsensitized Acute
Sensitized Acute
Sensitized Chronic
IL-13 Ϫ/Ϫ Chronic
3
6
1.17 Ϯ 0.17
1.50 Ϯ 0.42
2.17 Ϯ 0.48*
2.00 Ϯ 0.52*
2.00 Ϯ 0.45*
1.67 Ϯ 0.42
2.17 Ϯ 0.31*
0.83 Ϯ 0.31
0.33 Ϯ 0.21
2.17 Ϯ 0.17**
2.50 Ϯ 0.34**
1.50 Ϯ 0.22
2
4
Data are grades of immunoreactivity for eotaxin, expressed as mean Ϯ SEM. The mean grade in normal nonexposed control animals was 0.33 Ϯ 0.21. Significant
differences compared to these control animals are shown as * ϭ p Ͻ 0.05, ** ϭ p Ͻ 0.01.
eotaxin and mouse immunoglobulins. As shown in
Figure 3, A to C, there was intense expression of
eotaxin by inflammatory cells in the lamina propria,
which colocalized with expression of cytoplasmic
immunoglobulins, indicating that these were
immunoglobulin-producing, B-lymphocyte–derived
plasma cells. In addition, staining for eotaxin was also
observed in the tracheal epithelium, whereas staining
for immunoglobulins was also observed in blood
vessels.
ular, whether plasma cells in the airway wall exhibited
immunoreactivity for eotaxin. We found that immuno-
staining of the epithelium was prominent, together
with staining of a variety of inflammatory cells in the
lamina propria (Fig. 4A). There was minimal nonspe-
cific staining when the primary antibody was omitted
(Fig. 4B). Notable among the immunoreactive cells in
the airway wall were cells with abundant cytoplasm
and eccentric nuclei, which had the characteristic
“clock-face” appearance of plasma cells (Fig. 4C).
The intensity of immunoreactivity of plasma cells for
eotaxin was semiquantitatively assessed using a sim-
ilar approach to that employed for the epithelium. The
intense staining observed at 3 hours post-exposure
declined with time, but the decline was less marked
than that observed for the epithelium (Table 2), al-
though some animals exhibited no staining at 24 hours
Discussion
In this study, we investigated eosinophil recruitment
and the potential role of eotaxin, using a long-term
inhalational exposure model of asthma in mice, which
replicates the chronic inflammation and airway wall
remodeling that characterize the human disease. We
wish to emphasize that, because these animals do not
develop a significant alveolitis, relatively few inflam-
matory cells are present in bronchoalveolar lavage
fluid and lavage is thus an inappropriate method of
quantifying inflammation in this model. We therefore
employed morphometric quantification of eosinophils
within the airway epithelium and of chronic inflamma-
tory cells within the lamina propria. Although the
absolute numbers of eosinophils recruited into the
airways are smaller than in short-term, high-level ex-
posure models, the increase is significant at p Ͻ 0.001
compared with controls. Our results demonstrate that
there are significant differences in the pattern of
recruitment of eosinophils between this model and the
short-term, high-level exposure models of allergic
bronchopulmonary inflammation that have been ex-
tensively used to examine pathogenetic mechanisms
of asthma. Furthermore, our data raise important
questions about the biology of eosinophil recruitment
and the role of eotaxin as a chemoattractant for these
cells.
With respect to tissue accumulation of eosinophils,
in the chronic exposure model the number of intraepi-
thelial eosinophils in the airways was strikingly in-
creased at 3 hours post-exposure. Eosinophil num-
bers remained high at 6 hours but declined to lower
levels by 24 hours. These observations were in con-
trast to our findings in an acute exposure model, in
which intraepithelial eosinophils are usually said to be
absent (Korsgren et al, 1997). We found that, although
there was no accumulation of eosinophils in the epi-
thelium of the intrapulmonary bronchioles, large num-
bers of eosinophils were identified in the tracheal
epithelium of these animals. However, the timing of
(
Fig. 1D).
Eosinophil Recruitment and Eotaxin Expression in IL-13
/؊ Mice
؊
In IL-13 Ϫ/Ϫ mice that had been sensitized and
chronically exposed to low levels of aerosolized anti-
gen for 6 weeks, there was little evidence of recruit-
ment of eosinophils into the tracheal epithelium at any
of the time points studied (Fig. 2), which was consis-
tent with our previous observations in these mice at 18
hours post-exposure (Kumar et al, 2002). Surprisingly,
however, there was strong expression of eotaxin both
by tracheal epithelial cells and by plasma cells in the
lamina propria in these animals (Fig. 1E). Quantifica-
tion revealed significantly greater expression of
eotaxin in tracheal epithelium by IL-13 Ϫ/Ϫ mice
compared with wild-type mice at 6 hours post-
exposure, with persisting elevated expression at 24
hours (Table 1). Expression of eotaxin by plasma cells
in the airway wall of IL-13 Ϫ/Ϫ mice was at least
comparable to that in wild-type mice (Table 2), with a
tendency for increased expression at 6 hours, al-
though the observed difference was not statistically
significant.
Eotaxin Expression in Fatal Human Asthma
Because the antibody to mouse eotaxin that we em-
ployed in these studies cross-reacts with human
eotaxin, we were also able to examine expression of
eotaxin in human asthmatic airways. As a pilot inves-
tigation, we immunostained sections from a single
patient with fatal asthma, to assess whether the pat-
tern of eotaxin expression was similar to that observed
in the murine chronic exposure model and, in partic-
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Eotaxin Expression in Chronic Asthma
accumulation of these cells was quite different from
that observed in the chronic exposure model, in that
there was little evidence of eosinophil recruitment at 3
hours, although this reached comparable levels by 6
hours post-exposure. Recruitment of eosinophils was
clearly dependent upon prior sensitization, because
no cellular accumulation was observed in nonsensi-
tized control animals. Our findings suggest that differ-
ent mechanisms for eosinophil recruitment are oper-
ating in the chronic, as opposed to the acute,
exposure models, possibly related to the much higher
dose of antigen deposited in the airways of the latter.
The data also imply that chronic exposure to allergen
somehow primes the airways for much more rapid
recruitment of eosinophils following challenge.
Table 2. Eotaxin Expression by Plasma Cells
Time (h)
Sensitized Chronic
IL-13 Ϫ/Ϫ Chronic
3
6
2.83 Ϯ 0.17
1.83 Ϯ 0.48
1.83 Ϯ 0.60
3.00 Ϯ 0.0
2.83 Ϯ 0.17
1.67 Ϯ 0.21
2
4
Data are grades of immunoreactivity for eotaxin, expressed as mean Ϯ SEM.
exposure model might be that there is up-regulation of
pathways for the generation of chemoattractants. A
likely candidate in this regard is eotaxin, which is a
relatively specific chemoattractant for eosinophils. We
therefore examined expression of eotaxin in the airway
epithelium in these two models by immunohistochem-
ical staining with a polyclonal goat primary antibody to
eotaxin, which was generated by immunization with a
A possible mechanism for the rapid recruitment of
eosinophils in the chronic, compared with the acute,
19-amino acid peptide from mouse eotaxin that ex-
Figure 3.
A, Double label immunofluorescence staining demonstrating eotaxin-
immunoreactive epithelium and inflammatory cells in the lamina propria of the
trachea at 3 hours following exposure to ovalbumin, using an Alexa 568-
labeled reporter antibody which fluoresces red. B, Same field demonstrating
immunoglobulin-containing plasma cells, using an fluorescein isothiocyanate
Figure 4.
A, Immunoperoxidase staining for eotaxin in a patient who died of acute severe
asthma. In this peripheral airway, epithelial cells are strongly immunoreactive,
as are numerous inflammatory cells in the lamina propria. B, Same region from
a consecutive section, demonstrating virtually complete absence of staining
when the primary antibody was omitted. C, High magnification of a region of
the lamina propria from the section shown in A, demonstrating cells with the
characteristic “clock-face” nuclear morphology of plasma cells that were
strongly immunoreactive for eotaxin.
(
FITC)-labeled antibody which fluoresces green. C, Digitally merged image of
fields in A and B, demonstrating colocalization of eotaxin in inflammatory cells
in the lamina propria with cytoplasmic immunoglobulins, seen as yellow. Note
non-colocalizing staining for eotaxin in the tracheal epithelium and for
immunoglobulins in blood vessels.
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Kumar et al
hibits no significant homology to other members of the
eotaxin family or to other chemokines. At 3 hours
following antigen challenge, expression of eotaxin was
strikingly up-regulated in airway epithelial cells and
plasmacytoid B-lymphocytes in the lamina propria in
sensitized, chronically exposed mice. The identity of
the latter cells was established by double immunoflu-
orescent labeling. Semiquantitative assessment of im-
munoreactivity for eotaxin revealed that expression of
the protein declined rapidly in the tracheal epithelium
of chronically exposed mice. This finding was in con-
trast to observations in the acute exposure model of
allergic bronchopulmonary inflammation, in which
sensitized animals exhibited rapid induction of eotaxin
expression in the epithelium, but a much slower de-
cline. The duration of expression of eotaxin may be
related to antigen dose, and the results suggest that
different regulatory pathways for induction of eotaxin
expression are operative in the low- and high-level
exposure models. This possibility is supported by the
unexpected finding that expression was induced at a
lower level in nonsensitized acutely exposed mice and
rose noticeably after 24 hours, despite the lack of
eosinophil recruitment into the airway epithelium in
these animals. Together, these data imply that expres-
sion of eotaxin may be a nonspecific response follow-
ing high-level exposure, but that the time course of
induction might be modified by sensitization.
is required for recruitment of eosinophils into the
airways. Furthermore, recurrent exposure appears to
generate or up-regulate the additional signal or sig-
nals. Although the findings do not exclude an impor-
tant role for eotaxin, high eotaxin levels alone are
clearly not sufficient to cause eosinophil chemotaxis.
This inference is supported by the observation that
there was virtually complete absence of eosinophil
recruitment in the nonsensitized acutely exposed
mice, even though these animals did exhibit modest
expression of eotaxin. Even more persuasive is the
finding that in the IL-13 Ϫ/Ϫ mice, which also had
almost no intraepithelial eosinophils, expression of
eotaxin was comparable with wild-type mice and was
sustained for a longer period. The additional signal or
signals might include rapidly generated mediators,
such as eosinophil-selective arachidonic acid metab-
olites (Guilbert et al, 1999), which may be particularly
important in the early recruitment of eosinophils; a
variety of other eosinophil chemoattractant cytokines,
eg, macrophage inflammatory protein-1␣ (Campbell et
al, 1998; Gonzalo et al, 1998); as well as molecules
involved in the adhesion of eosinophils to vascular
endothelium (Burke-Gaffney and Hellewell, 1998; Na-
kajima et al, 1994). In this context, IL-13 is able to
up-regulate the expression of eosinophil-selective ad-
hesion molecules such as VCAM-1 and P-selectin on
endothelial cells (Bochner et al, 1995; Woltmann et al,
2000), and it is possible that absence of such up-
regulation accounts for the lack of eosinophil recruit-
ment in IL-13 Ϫ/Ϫ mice. It may also be noteworthy
that proinflammatory cytokines such as tumor necro-
sis factor-␣ can contribute to the up-regulation of
expression of both eosinophil-selective chemokines
(Teran et al, 1999) and relevant adhesion molecules
(Yamamoto et al, 1998).
In complete contrast to what might have been
predicted on the basis of published reports (Li et al,
1999; Matsukura et al, 2001; Zhu et al, 1999), we
found that in sensitized chronically exposed IL-13
Ϫ/Ϫ mice, eotaxin expression was undiminished, both
in epithelial cells and plasmacytoid cells. Indeed the
intensity of expression in the epithelium at both 6 and
24 hours was higher than in wild-type mice, despite
the virtual absence of intraepithelial eosinophils in the
knockout mice.
As has long been recognized, plasma cells consti-
tute a significant subpopulation in the airway wall in
human asthmatics (Laitinen et al, 1993). Our novel
demonstration of eotaxin expression by plasma cells
in the chronic exposure model of asthma appears to
be relevant to the human disease, because similar
expression of eotaxin was observed in a case of fatal
human asthma. However, because we do not have
access to a bank of tissue from human asthmatics, our
findings should be regarded as preliminary, and fur-
ther investigation of this response would be appropri-
ate. We also note that among the inflammatory cells in
the airway wall are macrophages, lymphocytes, and
eosinophils, all of which can produce eotaxin. A pres-
ently unresolved issue is the relative importance of
expression of eotaxin by different populations of in-
flammatory cells and by airway epithelial cells in
asthma. Another interesting question is whether
eotaxin might be expressed by plasma cells at other
sites, eg, the gut.
In the chronic exposure model of asthma, a sub-
stantial proportion of inflammatory cells in the lamina
propria is comprised of antigen-specific immunoglob-
ulin G–producing B-lymphocytes with the morphology
of plasma cells (Kumar et al, 2000a). In sensitized
chronically exposed wild-type mice, expression of
eotaxin by plasmacytoid cells in the lamina propria of
the trachea declined more slowly than in the epithe-
lium, but the induction of immunoreactivity following
antigen challenge and the decline at 24 hours post-
exposure both indicate that this response was related
to inhalational exposure to ovalbumin. Although there
was no statistically significant difference compared to
wild-type mice, the intensity of expression of eotaxin
in plasma cells was again higher at 6 hours post-
exposure in the IL-13 Ϫ/Ϫ mice. These observations
could imply that in the IL-13 Ϫ/Ϫ animals, a negative
feedback mechanism operative in wild-type mice is no
longer functional.
The results of this study highlight some important
differences between the chronic and acute exposure
models of asthmatic inflammation in mice. The time
course of eosinophil recruitment, as well as both the
time course and the intensity of expression of eotaxin
in the epithelium, are notably divergent. Moreover, the
The different time course of eosinophil accumula-
tion in the chronic, compared with the acute, exposure
models, together with the data on eotaxin expression
in the airway epithelium in the two models and in IL-13
Ϫ/Ϫ mice, strongly suggest that more than one signal
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Eotaxin Expression in Chronic Asthma
demonstration that plasma cells express eotaxin fol-
lowing antigen challenge is not possible in acute
exposure models because accumulation of plasma
cells does not occur in such models. In the context of
the potential importance of recruitment of eosinophils
into the epithelium of the airways in asthma (Henricks
et al, 1997), it is clearly desirable that appropriate
experimental models are employed to achieve a better
understanding of this process.
For the acute exposure model, sensitization was
performed as above. Both sensitized and nonsensi-
tized animals received a single exposure to a high
mass concentration of aerosolized ovalbumin for 30
minutes. No regulation of nebulizer airflow was em-
ployed for this exposure, and particle concentration
was estimated to be Ն100 mg/m under these condi-
tions. Groups of 6 animals were killed 3, 6, and 24
hours after exposure.
3
Materials and Methods
Collection of Tissues
Animals
Animals were killed by exsanguination following an
overdose of pentobarbital, and the lungs were per-
Specific pathogen-free female BALB/c mice were ob-
tained from the Biological Resources Centre, Univer-
sity of New South Wales, Sydney, Australia. Mice on a
BALB/c background, genetically deficient in their abil-
ity to produce IL-13 (IL-13 Ϫ/Ϫ mice) (McKenzie et al,
fused with 0.9% saline under a pressure of 40 cm H O
2
for 60 seconds to remove blood from the pulmonary
capillary bed. The trachea was then cannulated with a
blunted #19 needle, and the lungs were inflated with
10% buffered formalin. After fixation overnight, the
1998), were obtained from the Animal Services Divi-
longitudinally oriented trachea and a horizontal slice
from the mid-zone of the single-lobed left lung were
embedded in paraffin. Sections were cut at 5 ␮m and
stained with hematoxylin and eosin for assessment of
eosinophil recruitment, or immunostained for assess-
ment of eotaxin expression. Sections of human air-
ways from a patient who died as a result of acute
severe asthma were obtained from archival paraffin-
embedded blocks.
sion, Australian National University, Canberra, Austra-
lia. Animals were aged approximately 8 weeks at the
commencement of experimental studies. They were
maintained in a laminar flow holding unit (Gelman
Sciences, Sydney, Australia) and housed in auto-
claved cages on autoclaved bedding in an air-
conditioned room on a 12-hour light/dark cycle. Irra-
diated food and acidified water were provided ad
libitum throughout. All experimental procedures com-
plied with the requirements of the Animal Care and
Ethics Committee of the University of New South
Wales (ref. no. 98/29).
Immunohistochemistry
Expression of eotaxin was demonstrated using a goat
polyclonal antibody to a 19-amino acid peptide corre-
sponding to an epitope at the carboxy terminus of
mouse eotaxin (sc-6182; Santa Cruz Biotechnology,
Santa Cruz, California). The amino acid sequence of
this peptide exhibits no significant homology to other
members of the eotaxin family or to other chemokines.
The antibody recognizes mouse, rat, and human
eotaxin, but does not cross-react with other chemo-
kines. Immunostaining was performed on paraffin sec-
tions with antigen retrieval by boiling in 0.01 M citrate
buffer (pH 6.0) for 10 minutes in a microwave. Non-
specific binding was blocked by incubation for 15
minutes with 50% normal rabbit serum diluted in
phosphate-buffered saline (PBS). For routine immuno-
staining, this antibody was applied to sections at a
concentration of 2.5 ␮g/ml in PBS containing 0.075%
Brij-35 detergent for 60 minutes at room temperature.
To demonstrate the specificity of labeling with anti-
eotaxin, a four-fold lower concentration of antibody
was used. This was preincubated for 10 minutes with
either 175 ␮g/ml of the corresponding blocking pep-
tide (sc-6182P, Santa Cruz) or with 1% bovine serum
albumin, and the preincubated samples were applied
to consecutive sections. Slides were washed four
times with PBS after this and each subsequent
incubation.
Sensitization and Inhalational Exposure
For the chronic exposure model, these were per-
formed essentially as previously described (Temelk-
ovski et al, 1998). In brief, mice were sensitized by an
intraperitoneal injection of 50 ␮g of alum-precipitated
chicken egg ovalbumin (Grade V, Ն98% pure, Sigma,
St. Louis, Missouri; unless otherwise specified, all
chemicals were obtained from this source) 21 days
and 7 days before inhalational exposure. Animals were
exposed to aerosolized ovalbumin for 30 minutes/day
on 3 days/week for 6 weeks in a whole-body inhalation
exposure system (Unifab Corporation, Kalamazoo,
Michigan). During the exposures, the animals were
held in wire flow-through cage racks, and filtered air
3
was drawn through the 0.5 m inhalation chamber at a
flow rate of 250 l/minutes. A solution of 2.5% ovalbu-
min in normal saline was aerosolized by delivery of
compressed air to a sidestream jet nebulizer (Trimed,
Sydney, Australia) and injected into the airstream
entering the chamber. The concentration and size
distribution of particles within the breathing zone of
the mice were continuously and cumulatively moni-
tored using a light scatter device (PCAM-TX; PPM,
Knoxville, Tennessee). Particle concentration was
3
maintained in the range 10 to 20 mg/m by controlling
For immunoperoxidase staining, bound antibody
was detected by sequential 30 minutes incubations
with a 1:200 dilution of rabbit anti-goat immunoglobu-
lins (Dako, Sydney, Australia) in PBS-Brij containing
the airflow into the nebulizer. Experimental groups of 6
animals were killed 3, 6, and 24 hours after the last
exposure.
Laboratory Investigation • April 2002 • Volume 82 • Number 4 501

Kumar et al
1
% normal mouse serum, followed by a 1:100 dilution
California) was used for all data analysis and prepara-
tion of graphs.
of goat peroxidase-antiperoxidase complex (Dako).
Diaminobenzidine was used as the substrate, and
sections were lightly counterstained with hematoxylin
for 30 seconds.
Acknowledgement
We thank Angelina Enno and Gavin McKenzie for
invaluable assistance in the preparation and staining
of tissue sections.
For double-label immunofluorescence staining, par-
affin sections were stained essentially as previously
described (Kumar et al, 2000b). Slides were dewaxed,
blocked, and incubated with goat anti-mouse eotaxin
as above. Bound antibody was detected by incubating
for 45 minutes with 10 ␮g/ml of rabbit anti-goat
immunoglobulins conjugated to Alexa 568 (Molecular
Probes, Eugene, Oregon), which has excitation/emis-
sion maxima of 573/596 nm, similar to lissamine
rhodamine. Slides were then incubated for 45 minutes
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