The Journal of Experimental Medicine
© 2008 Walsh et al.
The Rockefeller University Press $30.00
J. Exp. Med. Vol. 205 No. 6 1285-1292 www.jem.org/cgi/doi/10.1084/jem.20071836
BRIEF DEFINITIVE REPORT
Eosinophilia of the lung and airways has been
observed in concurrence with other symptoms
in models of allergic asthma, as well as in hu-
mans, and has been regarded as a cardinal fea-
ture of asthmatic responses ( 1, 2 ). However,
the importance of eosinophils to the generation
of allergic asthma has remained ambiguous de-
spite the quantity of research that has been per-
formed on the subject. The cytokine IL-5 has
been shown to be important for eosinophil
development ( 1, 3 ), and levels are elevated dur-
ing asthmatic responses; however, studies in
IL-5 – defi cient mice, which have reduced num-
bers of eosinophils, have yielded inconsistent re-
sults. On a C57BL/6 background lacking IL-5,
airway hyperresponsiveness (AHR) is abolished,
whereas, on the BALB/c background, AHR is
either aff ected or not; this depends on the model
used, perhaps because of residual numbers of
eosinophils that may remain in the lungs ( 4 – 8 ).
These studies suggest that in addition to IL-5,
other factors may be required for regulation
of eosinophils, and that perhaps eotaxins, in-
cluding eotaxin-1, a chemokine that attracts
eosinophils to sites of infl ammation, may need
to be blocked in combination with IL-5 to
counteract the function of eosinophils in the
lungs ( 3, 9 ).
T cells, particularly IL-4 – , IL-5 – , and IL-
13 – producing Th2 cells, have been shown to
be important in allergic asthma, as introducing
antigen-specifi c Th2 cells followed by antigen
challenge is suffi cient to cause AHR ( 10, 11 ).
The independent administration of Th2 cyto-
kines IL-4, -5, or -13 can also induce AHR
( 11 – 14 ). Evidence from mouse models suggests
that IL-13 is necessary for mucous hypersecre-
tion and AHR, and has also been shown to aid
in eosinophil induction by eotaxin-1 – and IL-
5 – dependent mechanisms ( 14 – 16 ). The rela-
tionship among these three factors is still under
investigation, but studies in double-transgenic
The online version of this article contains supplemental material.
Strain-specifi c requirement for eosinophils
in the recruitment of T cells to the lung
during the development of allergic asthma
Elizabeth Rose Walsh , 1,2 Nisebita Sahu , 1,3 Jennifer Kearley , 4
Ebony Benjamin , 4 Boo Hyon Kang , 5 Alison Humbles , 4 and Avery August 1
1 Center for Molecular Immunology and Infectious Disease and Department of Veterinary and Biomedical Sciences,
2 Pathobiology Graduate Program, 3 Department of Biochemistry and Molecular Biology, Pennsylvania State University,
University Park, PA 16802
4 Respiratory, Infl ammation and Autoimmunity, Medimmune, Inc., Gaithersburg, MD 20878
5 Non-clinical Pathology Research Center, Medvill Co., Seoul, South Korea
Eosinophils have been implicated as playing a major role in allergic airway responses.
However, the importance of these cells to the development of this disease has remained
ambiguous despite many studies, partly because of lack of appropriate model systems. In
this study, using transgenic murine models, we more clearly delineate a role for eosinophils
in asthma. We report that, in contrast to results obtained on a BALB/c background, eosino-
phil-defi cient C57BL/6 ? dblGATA mice (eosinophil-null mice via the ? DblGATA1 mutation)
have reduced airway hyperresponsiveness, and cytokine production of interleukin (IL)-4, -5,
and -13 in ovalbumin-induced allergic airway infl ammation. This was caused by reduced
T cell recruitment into the lung, as these mouse lungs had reduced expression of CCL7/MCP-3,
CC11/eotaxin-1, and CCL24/eotaxin-2. Transferring eosinophils into these eosinophil-
defi cient mice and, more importantly, delivery of CCL11/eotaxin-1 into the lung during the
development of this disease rescued lung T cell infi ltration and airway infl ammation when
delivered together with allergen. These studies indicate that on the C57BL/6 background,
eosinophils are integral to the development of airway allergic responses by modulating
chemokine and/or cytokine production in the lung, leading to T cell recruitment.
T CELL MIGRATION TO LUNG IN ASTHMA IS RELIANT ON EOSINOPHILS | Walsh et al.
ways of lungs from the C57BL/6 ? dblGATA mice ( Fig. 1 B ,
Fig. 2 B , and Fig. S2 B). These data indicate that on the
C57BL/6, but not BALB/c, background, eosinophils are criti-
cal for the development of allergic airway infl ammation, sug-
gesting that diff erent mouse strains have diff ering requirements
for development of AHR.
Reduced CD4 + T cell recruitment and Th2 cytokines
in the lungs of C57BL/6 mice lacking eosinophils
after airway challenge
CD4 + T cells are recruited to the lung during chronic asth-
matic responses, producing Th2 cytokines such as IL-4, -5,
and -13 that perpetuate and exacerbate the pathology of this
disease ( 11, 13, 14, 21 ). To determine if the underlying cause
of the observed reduced AHR and pathology in the lungs of
C57BL/6 ? dblGATA mice was reduced recruitment of in-
fl ammatory cells to the lungs, we analyzed bronchoalveolar
lavage fl uid (BALF) from these mice for the presence of
CD4 + T cells. We found that C57BL/6 ? dblGATA mice
have signifi cantly reduced numbers of CD4 + T cells in the
BALF ( Fig. 3 A ). This correlated with reduced expression of
mice lacking IL-5 and eotaxin-1 have shown a defect in T cell
IL-13 production ( 17 ).
Most recently, two research groups published confl icting
data on the importance of eosinophils to the development
of this disease. Using a transgenic cell ablation approach on a
C57BL/6 background, Lee et al. found that eosinophils are
integral to the development of airway infl ammation and AHR
( 18 ). In contrast, Humbles et al. used the eosinophil-null mice
via the ? DblGATA1 mutation ( ? dblGATA) mouse, which
lacks eosinophils ( 4 ), on a BALB/c background and deter-
mined that the absence of eosinophils did not protect mice
from AHR development in an acute model of allergic in-
fl ammation, but are required for extensive airway remodeling
( 19 ). It is possible that diff erent backgrounds have dissimilar
responses to those observed for other genes, such as IL-4 and -5,
and the development of allergic asthma ( 20 ). In this study, we
have performed a more detailed analysis of the ? dblGATA
mice on C57BL/6 and BALB/c backgrounds. Our results
show that the hallmarks of allergic asthma, including T cell
infi ltration of the lungs, Th2 cytokine production, and chemo-
kine production, are reduced in C57BL/6 ? dblGATA mice.
Also unique to our study, we reconstituted ? dblGATA mice
with eosinophils to determine whether the characteristics ob-
served were, indeed, caused by these mice lacking eosinophils.
We determined that eosinophils are required for T cell infi l-
tration as well as cytokine production in the lungs during al-
lergic airway responses in C57BL/6 mice. Finally, we show
that intranasal (i.n.) delivery of CCL11/eotaxin-1 rescued T
cell recruitment and the development of AHR in C57BL/6
? dblGATA mice.
RESULTS AND DISCUSSION
Eosinophils are required for the development of AHR
and lung infl ammation in C57BL/6, but not BALB/c,
We used OVA in a standard sensitization protocol in WT and
? dblGATA mutant mice to induce allergic airway infl amma-
tion. 24 h after the last i.n. challenge, mice were subjected to
mechanical ventilation for analysis of AHR. We found that
WT mice on both C57BL/6 and BALB/c backgrounds, as well
as BALB/c ? dblGATA mice, showed an increase in AHR by
mechanical ventilation, whereas C57BL/6 ? dblGATA mice
did not show a signifi cant increase in this parameter by me-
chanical ventilation or whole body plethysmography ( Fig. 1 A ,
Fig. 2 A , and Fig. S1, available at http://www.jem.org/cgi/
content/full/jem.20071836/DC1). Analysis of lung sections
from these mice stained with hematoxylin and eosin (HE)
showed that both WT mice and BALB/c ? dblGATA mice
exposed to OVA i.n. had elevated infl ammation ( Fig. 1 B and
Fig. 2 B ). In contrast, lungs from the C57BL/6 ? dblGATA
mice had signifi cantly reduced infl ammation ( Fig. 1 B and
Fig. S2 A). To determine if infl ammation was accompanied
by goblet cell mucous production in the airways, we analyzed
similar sections stained with PAS. Again, as expected, there was
mucous in the airways of WT and BALB/c ? dblGATA lungs;
however, there was little if any mucous detected in the air-
Figure 1. Eosinophils are required for the development of AHR,
lung infl ammation, and mucous production in response to allergic
asthma induction in C57BL/6 ? dblGATA mice. (A) C57BL/6 WT and
? dblGATA mice were immunized and challenged i.n. with OVA. Some
? dblGATA mice were given 1.5 × 10 6 eosinophils or neutrophils, followed
by analysis of AHR by mechanical ventilation. *, P < 0.05 for WT versus
? dblGATA alone ( n = 4 mice/group, repeated 3 times). (B) Fixed and sec-
tioned mouse lungs from the above groups were analyzed by HE or PAS
stain. Bars: (WT) 50 μ m; (all other panels) 20 μ m.
JEM VOL. 205, June 9, 2008
BRIEF DEFINITIVE REPORT
ated similar levels of total IgE, as well as OVA-specifi c IgE,
to WT mice (Fig. S3, B and C). Thus, T cell populations from
these mice are capable of mounting an immune response,
but are not able to migrate into the lungs to respond to
Reduced expression of CCL7/MCP-3, CCL11/eotaxin-1,
and CCL24/eotaxin-2 in lungs of C57BL/6 ? dblGATA mice
after airway challenge
One reason that T cells may not be able to migrate to
the lungs during OVA challenge is reduced expression of
chemokines critical for their migration into tissues ( 11 ).
In particular, CCL7/MCP-3 and CCL11/eotaxin-1 have
been shown to be important for recruitment of T cells into
the lung during the development of allergic asthma ( 24 –
26 ). Eosinophils can induce proliferation and cytokine
secretion from T cells ( 11, 17, 22, 23 ), as well as secrete
T cell growth and chemotactic factors themselves, such as
CCL11/eotaxin-1 and CCL24/eotaxin-2 ( 4 ). Analysis of
lung RNA shows that C57BL/6 ? dblGATA mice had a
greatly reduced expression of these three chemokines ( Fig.
4 A ), which suggests that T cells are not recruited into
the lungs of C57BL/6 ? dblGATA mice because of a defi -
ciency in chemokines able to aid migration of these cells to
RNA for cytokines IL-4, IL-13, and IFN- ? in the lungs of
C57BL/6 ? dblGATA mice ( Fig. 3 B ). Protein levels of Th2
cytokines IL-4, -5, and -13 were also reduced in the BALF
of C57BL/6 ? dblGATA mice ( Fig. 3 C ). Thus, C57BL/6
? dblGATA mice are defective in recruitment of CD4 + T
cells and the production of Th2 cytokines required for the
induction of the disease.
Normal systemic immune responses in C57BL/6 ? dblGATA
mice upon OVA immunization and challenge
Reduction in Th2 cytokines in the lung could be the result
of ineffi cient T cell activation or diff erentiation to Th2 cells.
Because eosinophils have been suggested to serve as antigen-
presenting cells under certain conditions ( 11, 17, 22, 23 ), we
determined if splenic T cells from C57BL/6 ? dblGATA
mice were able to respond to OVA restimulation. We found
that splenic T cells from both C57BL/6 WT and ? dblGATA
mice were able to respond to OVA stimulation similarly
(Fig. S3 A, available at http://www.jem.org/cgi/content/full/
jem.20071836/DC1). We also found that C57BL/6 ? dblGATA
mice were able to generate a Th2 response by class switch-
ing responding antibodies to the IgE isotype, as they gener-
Figure 2. Eosinophils are not required for the development of
AHR, lung infl ammation, and mucous production during allergic
asthma induction in BALB/c ? dblGATA mice . (A) BALB/c WT and
? dblGATA mice were treated as in Fig. 1 A , followed by analysis of AHR by
mechanical ventilation ( n = 7 – 8 mice/group for OVA-challenged mice;
n = 4 Sham). *, P < 0.02 for WT and ? dblGATA ± Eos versus PBS groups.
(B) Mouse lungs from WT, ? dblGATA, or ? dblGATA + eosinophils treated
as in Fig. 1 A , and analyzed by HE or PAS stain. Bars, 50 μ m.
Figure 3. Eosinophils are required for the recruitment of CD4 + T
cells to the lung in C57BL/6 ? dblGATA in response to allergic air-
way infl ammation. (A) Lungs from immunized and OVA-challenged
C57BL/6 WT and ? dblGATA mice ( n = 3 mice/group, repeated 3 times)
were analyzed for CD4 + T cells. (B) Lungs from WT and ? dblGATA mice
treated as in A were analyzed for mRNA for the indicated cytokines ( n = 8
mice/group). (C) BALF from WT and ? dblGATA mice treated as in A was
analyzed for the indicated cytokines by ELISA ( n = 3/group, repeated 3
times). *, P < 0.05 WT versus ? dblGATA. Error bars are ± the SEM.
T CELL MIGRATION TO LUNG IN ASTHMA IS RELIANT ON EOSINOPHILS | Walsh et al.
required to rescue these responses. There were small numbers
of T and B cells, as well as neutrophils, in our purifi ed eo-
sinophil population (unpublished data). We addressed this by
transferring 1.5 × 10 6 neutrophils into C57BL/6 ? dblGATA
mice and challenging with OVA, which did not result in
increases in AHR ( Fig. 1 A ), demonstrating that increased
numbers of infl ammatory cells are not suffi cient to rescue
allergic airway responses. In addition, we transferred IL-5
transgenic T cells into C57BL/6 ? dblGATA mice. This did
not result in increased airway infl ammation or mucous pro-
duction, indicating that rescue of lung airway infl ammation
is not caused by contaminating populations of T cells (Fig. S4,
A and B, available at http://www.jem.org/cgi/content/full/
Transfer of eosinophils, but not neutrophils or IL-5 trans-
genic T cells, followed by OVA challenge was also able to
rescue the recruitment of T cells into the BALF and lungs of
C57BL/6 ? dblGATA mice ( Fig. 4, B and C ). In contrast,
Rescue of T cell recruitment and lung infl ammation
by transfer of eosinophils to C57BL/6 ? dblGATA mice
To determine whether eosinophils are, indeed, required
for recruitment of T cells and generation of allergic asthma
symptoms on the C57BL/6 background, we performed i.v.
transfers of 1.5 × 10 6 eosinophils, which were purifi ed from
the peritoneum of IL-5 transgenic mice, into OVA-immu-
nized ? dblGATA mice, followed by i.n. challenges with
OVA. These mice were then analyzed for the development
of AHR 24 h after the last challenge. We found that transfer
of eosinophils into the C57BL/6 ? dblGATA mice 6 h be-
fore the fi rst i.n. challenge was able to rescue the develop-
ment of AHR ( Fig. 1 A ), as well as lung infl ammation and
mucous production ( Fig. 1 B ). Transfer of eosinophils into
C57BL/6 ? dblGATA mice followed by challenge with
PBS did not lead to AHR, the development of lung infl am-
mation, or mucous production (unpublished data), indicating
that eosinophil transfer in tandem with OVA challenge was
Figure 4. Eosinophils are required for expression of CCL7, CCL11, and CCL24, and CD4 + T cell recruitment in the lungs during allergic air-
way infl ammation in C57BL/6 ? dblGATA mice. (A) WT or ? dblGATA mice were immunized. Some ? dblGATA mice were given eosinophils, and then
challenged with OVA or PBS; lungs were analyzed for mRNA for CCL7, CCL11, or CCL24 ( n = 4 – 5 mice/group, repeated 2 times). *, P < 0.05 WT versus
? dblGATA + OVA. (B) Lungs from WT or ? dblGATA mice treated as in A were analyzed for CD4 + T cells. *, P < 0.05 WT versus ? dblGATA + OVA ( n = 3
mice/group, repeated 3X). (C) BALF from mice treated similarly as in A ( n = 3 mice/group, repeated 3 times). (D) Lung cytokine mRNA analysis from mice
treated similarly to those in A. *, P < 0.05 WT versus ? dblGATA + OVA ( n = 4 – 5 mice/group repeated 3X). (E) BALF from mice treated as in A analyzed for
the indicated cytokines by ELISA. *, P < 0.05 WT versus ? dblGATA + OVA ( n = 4, repeated 3 times). Error bars are ± the SEM.
JEM VOL. 205, June 9, 2008
BRIEF DEFINITIVE REPORT
treatment also rescued T cell migration into the lungs, which
did not occur in mice given i.n. CCL11/eotaxin-1 in combi-
nation with CCL-11/eotaxin-1 blocking antibody ( Fig. 5 B ).
As expected, OVA plus i.n. CCL11/eotaxin-1 did not recruit
eosinophils in the C57BL/6 ? dblGATA mice because these
mice lack these cells (Fig. S5 C). The chemokine receptor
CCR3 most likely mediates this migration because this is the
only receptor that has been reported to interact with these che-
mokines. Our data suggest that, in the absence of eosinophils,
exposing mice to an allergic airway challenge results in the lack
of production of appropriate chemokines; particularly eotax-
ins, which allow recruitment of T cells into the lung and con-
tribute to the pathology of the disease.
In this investigation, we have provided evidence that eo-
sinophils are required for the development of allergic airway
responses and recruitment of T cells into the lungs after allergen
challenge in C57BL/6 ? dblGATA mice. These results add
weight to other studies that have shown abrogation of the
symptoms of allergic asthma in the absence or suppression
of eosinophil function ( 4, 8 ). Lee et al. have provided evidence
C57BL/6 ? dblGATA mice that received eosinophils but were
challenged with PBS had signifi cantly fewer CD4 + T cells
than those challenged with OVA ( Fig. 4, B and C ). Transfer
of eosinophils into C57BL/6 ? dblGATA mice followed by
OVA challenge resulted in the appearance of eosinophils in
the lungs of C57BL/6 ? dblGATA mice (unpublished data),
and also rescued the expression of RNA for cytokines IL-4,
-5, and -13. These mice actually displayed higher levels of
IL-13 than WT mice challenged with OVA ( Fig. 4 D ).
Analysis of cytokine protein levels in the BALF of C57BL/6
? dblGATA mice showed that when these mice received eosin-
ophils and were challenged with OVA, they produced levels
of IL-4, -5, and -13 similar to those of WT mice ( Fig. 4 E ).
C57BL/6 ? dblGATA mice transferred with eosinophils and
challenged with PBS had low levels of these cytokines, com-
parable to C57BL/6 ? dblGATA mice challenged with OVA
( Fig. 4 E ). Eosinophil transfer-mediated rescue of CD4 + T
cell recruitment into the lung was also accompanied by rescue
of CCL7/MCP-3, CCL11/eotaxin-1, and CCL24/eotaxin 2
expression ( Fig. 4 A ). WT C57BL/6 lung CCL17 levels were
signifi cantly higher than ? dblGATA, but expression of this
chemokine was not rescued by eosinophil transfer into these
mice, indicating that eosinophils may not directly regulate
CCL17 (Fig. S5 A, available at http://www.jem.org/cgi/
content/full/jem.20071836/DC1), and CCL22 levels were
equivalent in all groups (Fig. S5 A). Indeed, CD4 + T cells that
were recruited to the lungs of WT mice expressed as CCR3
(Fig. S5 B). These results suggest that eosinophils may modu-
late the expression of CCL11/24-eotaxin1/2, which are needed
for recruitment of T cells into the lung during allergic airway
infl ammation. Our data imply that eosinophils may be required
for low levels of secretion of CCL11/24-eotaxin1/2-Th2
cytokines in the lung, which could induce T cell migration and
secretion of eff ector cytokines by these cells that can further
amplify the recruitment of eosinophils and T cells into the lung
in a feed-forward mechanism.
i.n. delivery of CCL11/eotaxin-1 rescues CD4 + T cell
recruitment to the lung and the development of AHR
in C57BL/6 ? dblGATA mice
Our aforementioned experiments revealed signifi cantly re-
duced expression of the chemokine CCL11/eotaxin-1 in the
lungs of OVA-challenged C57BL/6 ? dblGATA mice. The
reduction of this chemokine suggests a possible mechanism for
the lower responses in these mice. To test if CCL11/eotaxin-1
is able to rescue T cell recruitment and the development of
AHR, we delivered this chemokine to the lungs of C57BL/6
? dblGATA mice previously immunized with OVA over the
4 d of i.n. challenge, along with OVA. We found that CCL11/
eotaxin-1 delivered with OVA was suffi cient to induce AHR
in C57BL/6 ? dblGATA mice ( Fig. 5 A ). CCL11/eotaxin-1
delivery with CCL11/eotaxin-1 blocking antibody did not
induce AHR in C57BL/6 ? dblGATA mice challenged with
OVA, indicating that the rescue was specifi c to this chemokine
(Fig. S5 D). Analysis of the lungs of the C57BL/6 ? dblGATA
mice given CCL11/eotaxin-1 and OVA i.n. showed that this
Figure 5. CCL11/eotaxin-1 can rescue AHR and T cell recruitment
to the lung in C57BL/6 ? dblGATA mice. (A) Immunized ? dblGATA
mice were given CCL11/eotaxin-1 during challenge with OVA. Alterna-
tively, immunized WT C57BL/6 or ? dblGATA mice were just given CCL11/
eotaxin. This was followed by AHR analysis by mechanical ventilation
( n = 4, repeated 2 times). *, P < 0.05 for ? dblGATA + Eot/OVA versus WT
or ? dblGATA + Eot alone. (B) Lungs from immunized and i.n. OVA-chal-
lenged WT and ? dblGATA mice or ? dblGATA mice delivered eotaxin-1
with OVA, were analyzed for CD4 + T cells. Some mice challenged with
eotaxin/OVA also received anti-CCL11 blocking antibody i.n. ( n = 4 mice/
group, repeated 2 times). *, P < 0.05 WT versus ? dblGATA + OVA. Error
bars are ± the SEM.
T CELL MIGRATION TO LUNG IN ASTHMA IS RELIANT ON EOSINOPHILS | Walsh et al.
in T cells during allergic airway responses ( 17 ). This latter study
did not investigate whether CD4 + T cells were actually re-
cruited to the lungs in the eotaxin-1/IL-5 double knockout
mice, and only determined that the levels of Th2-type cyto-
kines in the lung were reduced. Together with these data,
our fi ndings indicate that instead of IL-13 production by T
cells causing up-regulation of eotaxin that selectively recruits
eosinophils to the lungs, as has been previously suggested
( 21 ), eosinophils are required to provide a stimulus, perhaps
CCL11/24-eotaxin-1/2, for T cell migration and secretion
of cytokines in the lungs.
While this work was under review, Jacobsen et al. reported
similar fi ndings in C57BL/6 PHIL mice, suggesting that T cell
recruitment to the lung during generation of allergic airway
responses is via an eosinophil-dependent mechanism ( 29 ).
However, there were some diff erences between that study and
our fi ndings. Jacobsen et al. report that eosino phil transfer into
primed and OVA i.n. challenged PHIL mice is not able to res-
cue recruitment of T cells to the lungs or Th2 cytokine pro-
duction. This work is in direct contrast to data presented here
in that eosinophil transfer to sensitized and i.n. OVA-chal-
lenged ? dblGATA mice promotes T cell recruitment, as well
as subsequent Th2 cytokine production in the lung. Jacobsen
et al. attribute this defect in the PHIL mice to an inability to
generate Th2 cells in the absence of eosinophils; however, they
do not address whether other Th2 responses, such as induction
of B cells to class switch for IgE production, are preserved as
they are in the ? dblGATA mice, nor do they attempt to trans-
fer eosinophils during the initial OVA sensitization process to
determine whether this could aff ect the generation of Th2 cells.
This potential inability for PHIL mice to eff ectively generate
a Th2 T cell response is an interesting fi nding that warrants
further study of T cell responses in these mice to determine
whether this observation is, indeed, eosinophil dependent or
whether it indicates an underlying defect in antigen-specifi c
T cell responses. Lastly, the mechanism by which T cells are
recruited to the lung appears to diff er in these two models of
allergic airway responses. Although we fi nd that eosinophils do
not contribute to lung RNA levels of MDC or TARC in
C57BL/6 ? dblGATA mice, Jacobsen et al. show that PHIL
mice have a defect in the production of these chemokines
in the lung that can be rescued with transfer of in vitro – diff er-
entiated OT-II T cells and eosinophils to these mice ( 29 ).
Conversely, Jacobsen et al. ( 29 ) found similar BAL levels of
eotaxin-1 and 2 in PHIL and WT mice, whereas our data sug-
gest that there is reduced production of these chemokines in
the lungs of ? dblGATA mice. In addition, we show that ad-
ministration of eotaxin-1 with OVA to the lungs of ? dblGATA
mice rescues AHR and T cell recruitment to the lung. The
reason for the discrepancy between the two mechanisms is not
clear, but may perhaps be caused by the diff ering nature of the
defects in these two mouse models. Regardless, data from both
groups support the central hypothesis that eosinophils are re-
quired for the recruitment of T cells to the lung, and thus are
not only terminal eff ector cells but also important modulators
of allergic asthma.
in a transgenic eosinophil ablation approach in C57BL/6 mice
that eosinophils are required to develop AHR and airway
infl ammation, as well as mucus secretion ( 18 ). This is in
contrast to data provided by Humbles et al. in ? dblGATA
mutant BALB/c mice, suggesting that eosinophils are re-
quired only for airway remodeling ( 19 ). Our data in the
? dblGATA mutant mice on the BALB/c and C57BL/6 back-
grounds now indicate that the discrepancies observed be-
tween these two groups were most likely caused by strain
diff erences in C57BL/6 and BALB/c mice. Indeed, our studies
fi nd that systemic Th2 responses are intact in the ? dblGATA
mutant mice on the C57BL/6 background, similar to that
observed by Humbles et al. ( 19 ). The main diff erence we
observe is the requirement for eosinophils in the acute model
of allergic airway infl ammation, and the recruitment of
T cells into the lung. Indeed, we observed a reduction in re-
cruitment of the number of T cells into the lungs of both
C57BL/6 and BALB/c ? dblGATA mice ( Fig. 4 C and Fig.
S6 B, available at http://www.jem.org/cgi/content/full/jem
.20071836/DC1). However, although recruitment of T cells
into the BALB/c ? dblGATA mouse lungs was reduced
compared with WT, the number of T cells in these lungs
was signifi cantly greater than controls, and the percentage
of CD4 + T cells was similar to WT (Fig. S6, A and B). Data
from Voehringer et al. support this, showing in the Th2
Nippostrongylus model that T cell recruitment to BALB/c
? dblGATA lungs is intact ( 27 ). Also, reduced T cell numbers
did not prevent BALB/c ? dblGATA from producing the
Th2 cytokines IL-4 and -13 (Fig. S6, C and D). Although
this is in agreement with Voehringer et al., Fulkerson et al.
reported a reduction in IL-4 and -13 production in the lung
in an Aspergillus model of asthma ( 28 ). Whether these diff er-
ences are caused by the BALB/c strain having lower chemo-
kine/cytokine requirements for recruitment of infl ammatory
cells in the airways and subsequent production of Th2 cyto-
kines remains to be seen.
In contrast to the aforementioned research groups, our
data also address the infl uence that eosinophils have over
CD4 + T cells in allergic asthma, suggesting that eosinophils
are not just terminal eff ector cells, but are actively involved in
the adaptive immune response by assisting in the recruitment
of T cells to the lungs; this supports data that propose eosino-
phils can modulate the function of T cells in the allergic lung.
Eosinophils resident in the lung during allergic responses are
able to present antigen and traffi c to local lymph nodes, where
they colocalize with T cells; they can also induce prolifera-
tion and cytokine secretion from T cells ( 4, 11, 22, 23 ). In
allergic mice defi cient in eotaxin-1 and IL-5, there is reduced
T cell production of cytokine IL-13, although these cells
have normal cytokine production in general ( 17 ). Accompa-
nying this, transfer of T cells defective in IL-13 production
into eotaxin-1/IL-5 double knockout mice does not induce
AHR, whereas transferring in vitro – diff erentiated, IL-13 –
producing T cells can overcome defects in eotaxin-1/IL-5
double knockout mice and induce asthma, suggesting that
eosinophils may be linked to induction of IL-13 production
JEM VOL. 205, June 9, 2008
BRIEF DEFINITIVE REPORT
have no defect in in vitro splenocyte proliferation or IgE production in vivo.
Fig. S4 shows that transfer of IL-5 transgenic T cells to ? dblGATA mice
does not induce lung airway infl ammation and T cell recruitment to the lung.
Fig. S5 shows that levels of chemokine CCL17/CCL22 message in the lungs
of ? dblGATA and WT mice are not statistically diff erent, that the CD4 +
T cells recruited into the lungs express CCR3, and that ? dblGATA mice
administered anti-CCL11/-CCL11/-OVA have reduced AHR compared
with ? dblGATA that receive CCL11/OVA alone. Fig. S6 shows that CD4 +
T cell recruitment to the lung and Th2 cytokine production are intact in
BALB/c WT and ? dblGATA mice. The online version of this article is avail-
able at http://www.jem.org/cgi/content/full/jem.20071836/DC1.
We thank Drs. Jamie and Nancy Lee for the kind gift of IL-5 transgenic mice. We
also thank Dr. Wayne Mitzner (Johns Hopkins University) for help in analysis of AHR
by mechanical ventilation. We thank members of the August laboratory, the Center
for Molecular Immunology and Infectious Disease in the Department of Veterinary
and Biomedical Sciences at Penn State for comments, and Elaine Kunze, Susan
Magargee, and Nicole Bem in the Center for Quantitative Cell Analysis at Penn State
University for fl ow cytometry analysis.
This work was supported by grants from the National Institutes of Health
(AI051626, AI065566, and AI073955) and the American Heart Association to
A. August. E.R. Walsh is the recipient of a Penn State College of Agricultural
Sciences Graduate Fellowship, a NASA Space Grant Fellowship, and the American
Academy of Asthma, Allergy, and Immunology ST*AR Award.
The authors have no confl icting fi nancial interests.
Submitted: 27 August 2007
Accepted: 24 April 2008
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MATERIALS AND METHODS
Mice. WT C57BL/6, BALB/c, ? dblGATA on both backgrounds ( 19 ), and
IL-5 transgenic mice on both backgrounds (a gift from J. Lee and N. Lee,
The Mayo Clinic, Scottsdale, Arizona) ( 30 ) were used for these experiments.
All experiments were approved by the Offi ce of Research Protection ’ s Insti-
tutional Animal Care and Use Committee at Pennsylvania State University.
OVA-induced allergic asthma model. Groups of mice (WT or ? dblGATA)
were immunized i.p. on day 0 and 5 with 50 μ g/ml OVA (Sigma-Aldrich)
complexed with aluminum hydroxide (10 μ g OVA/1 mg alum; Thermo
Fisher Scientifi c). Mice were exposed daily i.n., with 30 μ l OVA (2 mg/ml)
on day 12 – 15, and killed 24 h later for analysis. In experiments where
? dblGATA mice received eosinophil transfers, ? dblGATA mice received
1.5 × 10 6 eosinophils i.v. on day 12, and were then challenged with OVA 6 h
later. In some experiments, ? dblGATA or WT mice received 0.75 μ g
CCL11/eotaxin-1 combined with the normal dose of 30 μ l (2 mg/ml) OVA
on days 12 – 15. WT and ? dblGATA mice primed with OVA/alum and i.n.
challenged with PBS were used as controls in these experiments.
Determination of AHR and analysis of airway infl ammation. AHR
was determined using a custom-made mechanical ventilator ( 31 ) or a Flexi-
vent mechanical ventilator (SciReq). Mice were anesthetized, a cannula was
placed in the trachea, and mice were ventilated at 120 breaths/min, V T = 0.2
ml, fl ow rate 1.5 ml/s at 2 – 3 cm H 2 O PEEP. Airway pressure in response to
methacholine was determined using a diff erential pressure transducer. Fixed
and sectioned lungs were stained with HE or PAS to detect mucous (per-
formed by the Animal Diagnostic Laboratories, Penn State University).
Adoptive transfer of eosinophils. Peritoneal eosinophils were obtained
from IL-5 transgenic mice by peritoneal lavage with RPMI media, sorted by
MACS negative bead selection, and washed 2 times in 1XPBS, and then 1.5 ×
10 6 cells were resuspended in 100 μ l 1XPBS and injected i.v. into ? dblGATA
mice. Typical purity was 85 – 90% as determined by CCR3 antibody (R & D
Systems) positive fl ow cytometric analysis.
Adoptive transfer of neutrophils. Peripheral blood neutrophils were pu-
rifi ed by Histopaque gradient centrifugation (1.119, 1.083, and 1.077). Cells
from the 1.119/1.083 interface were harvested and washed three times. On
the fi rst day of i.n. challenge, 1.5 × 10 6 cells were transferred i.v. into im-
munized ? dblGATA mice as outlined in the previous section.
Determination of T cell recruitment into the lungs and BAL. BAL
was collected from lungs of mice in PBS. In other mice, whole lungs were
dissociated using collagenase (Roche), and isolated cells from BAL or lungs
were either analyzed on an Advia Blood Analyzer or stained with monoclo-
nal antibodies to identify CD4 + T cells (eBioscience), and then analyzed by
fl ow cytometry.
Quantitative RT-PCR analysis of gene expression. RNA was isolated
from lung tissue, and total RNA (1 μ g) was reverse transcribed to cDNA.
PCR was performed in triplicate with commercially available primers and
probes as per manufacturer protocol (Applied Biosystems).
Analysis of cytokine levels. BAL or supernatants from T cell cultures
were analyzed for levels of IL-4, -5, and -13 by a Luminex multiplex bead
system kit (Lincoplex) on a Bioplex system (Bio-Rad Laboratories).
Data analysis . Statistical evaluation was conducted for all repetitions of
each experiment using Student ’ s t test with a probability value P ≤ 0.05 con-
sidered statistically signifi cant.
Online supplemental material. Fig. S1 demonstrates that ? dblGATA mice
have reduced lung function as assessed by plethysmograph. Fig. S2 shows the
quantitative analysis of the reduced lung pathology observed in ? dblGATA
mice compared with WT mice. Fig. S3 demonstrates that ? dblGATA mice
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