Full text of DOI:10.1097/01.LAB.0000020417.13757.05

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LABORATORY INVESTIGATION
Vol. 82, No. 7, p. 929, 2002
Copyright © 2002 by The United States and Canadian Academy of Pathology, Inc.
Printed in U.S.A.
CCR3-Blocking Antibody Inhibits Allergen-Induced
Eosinophil Recruitment in Human Skin Xenografts
from Allergic Patients
Stéphanie Sénéchal, Olivier Fahy, Thibaut Gentina, Han Vorng, Monique Capron,
Andrew F. Walls, Alan R. McEuen, Mark G. Buckley, Qutayba Hamid,
Benoît Wallaert, André Bernard Tonnel, and Anne Tsicopoulos
INSERM U416 (SS, OF, TG, HV, MC, BW, ABT, AT), IFR 17, and INSERM U547 (MC), Institut Pasteur de Lille, and
Clinique des maladies respiratoires (TG, BW, ABT, AT), Hôpital Calmette, CHRU de Lille, France; Immunopharma-
cology Group (AFW, ARM, MGB), Southampton General Hospital, Southampton, United Kingdom; and Meakins
Christies Laboratories (QH), MacGill University, Montreal, Canada
SUMMARY: Eosinophil, basophil, and T helper 2 (TH2) cell recruitment into tissues is a characteristic feature of allergic diseases.
These cells have in common the expression of the chemokine receptor CCR3, which may represent a specific pathway for their
accumulation in vivo. Although animal models of allergic reactions are available, findings cannot always be extrapolated to man.
To overcome these limitations, we have developed a humanized mouse model of allergic cutaneous reaction using severe
combined immunodeficiency mice engrafted with skin and autologous peripheral blood mononuclear cells from allergic donors.
Intradermal injection of the relevant allergen into human skin xenografts from allergic individuals induced a significant recruitment
of human CD4ϩ T cells, basophils, and TH2-type cytokine mRNA-expressing cells, as well as murine eosinophils. Human skin
xenografts, atopic status, and autologous peripheral blood mononuclear cell reconstitution were all mandatory to induce the
allergic reaction. Next, we addressed the role of CCR3 in the endogenous mechanisms involved in the inflammatory cell
recruitment in this experimental model of allergic cutaneous reaction. In vivo administration of an anti-human CCR3-blocking
antibody selectively reduced accumulation of eosinophils but not that of CD4ϩ cells, basophils, or cells expressing mRNA for
TH2-type cytokines. These findings establish a new in vivo model of humanized allergic reaction and suggest that eosinophil
migration is mediated mainly through CCR3. Finally, these results suggest that this model might be useful to test human-specific
antiallergic modulators. (Lab Invest 2002, 82:929–939).
llergic diseases are characterized by a tissue infil-
tration of eosinophils, basophils and T helper 2
protein–coupled seven-transmembrane receptor that
mediates the action of several chemokines, including
eotaxin (CCL11), RANTES (CCL5), MCP-3 (CCL7), and
MCP-4 (CCL13) (Ponath et al, 1996; Stellato et al, 1997).
All these ligands have been shown to be overexpressed
in human allergen-induced cutaneous LPR (Ying et al,
1999), in atopic dermatitis (Yawalkar et al, 1999) and in
asthma (Lamkhioued et al, 1997; Ying et al, 1997),
reinforcing the potential role of CCR3 as a major path-
way of cell recruitment and therapeutic target in allergic
reactions (Bertrand and Ponath, 2000). Hitherto, only two
studies have evaluated the effect of CCR3 antagonism in
vivo, one on chemokine-induced eosinophil recruitment
(Sabroe et al, 1998) and the other in a parasitosis model
(Grimaldi et al, 1999). In both cases, an inhibition of
eosinophil recruitment was observed. However, no in
vivo study has evaluated the effect of CCR3 antagonism
on the other CCR3-bearing cells or in an allergy model.
Moreover, CCR3 seems to be differentially regulated in
the murine and in the human system (Lloyd et al, 2000).
In mice, anti-CD3 stimulation, which mimics antigen
effect, increases CCR3 expression on TH2 cells,
whereas in humans, it down-regulates it (Sallusto et al,
1999), questioning the relevance of murine studies to
human situations. For all these reasons, the develop-
A
(TH2) cells, which is thought to be related to the severity
and chronicity of the allergic reaction. This preferential
cell accumulation in tissue suggests that there are spe-
cific pathways used for their accumulation in vivo. Un-
derstanding these mechanisms could aid in developing
pharmacologic therapies that would block their recruit-
ment. This recruitment is orchestrated in part by chemo-
kines that act through specialized surface receptors. The
chemokine receptor CCR3 is of particular interest in the
context of allergic reactions, because it is expressed by
TH2 lymphocytes (Sallusto et al, 1997), eosinophils
(Ponath et al, 1996), basophils (Uguccioni et al, 1997),
and mast cells (Ochi et al, 1999; Romagnani et al, 1999),
all of which are found at sites of allergen-induced late-
phase response (LPR) (Frew and Kay, 1988; Gaga et al,
1991; Irani et al, 1998). The CCR3 receptor is a G-
DOI: 10.1097/01.LAB.0000020417.13757.05
Received March 19, 2002.
Address reprint requests to: Dr. Anne Tsicopoulos, U416, Institut Pasteur
de Lille, BP245, 59019 Lille cedex, France. E-mail: anne.tsicopoulos@
pasteur-lille.fr
Laboratory Investigation • July 2002 • Volume 82 • Number 7 929

Sénéchal et al
ment of a humanized model of allergic reaction was
needed. We therefore used the severe combined immu-
nodeficient (SCID) mouse and developed a model of
humanized cutaneous allergic reaction. These mice have
a defect in antigen receptors on the T and B lympho-
cytes and can be reconstituted with a functional human
immune system (Mosier et al, 1988). The model of SCID
mice grafted with skin alone (Christofidou-Solomidou et
al, 1996, 1997b; Juhasz et al, 1993; Nickoloff et al, 1995;
Yan et al, 1993) or with peripheral blood mononuclear
cells (PBMC) has been previously extensively used in the
field of skin diseases (Christofidou-Solomidou et al,
1997a; Gilhar et al, 1997) and to study different types of
T cell–mediated cutaneous inflammation (Petzelbauer et
al, 1996; Tsicopoulos et al, 1998) and graft rejection
(Murray et al, 1994; Sultan et al, 1997) but never as a
model of allergic reaction. In the present study, intrader-
mal injection of allergen in SCID mice xenografted with
human skin and autologous (PBMC) from the same
allergic donors reproduced the features of the human
allergen-induced LPR. In addition, we report in this
model that the blockade of human CCR3 with a mono-
clonal antibody selectively decreases eosinophil
infiltration.
these CD4 T cells, BB1ϩ basophils (Fig. 1E) and mainly
eosinophils were also recruited after allergen challenge.
These human skin-infiltrated eosinophils were of murine
origin (mMBPϩ) with no human EG2ϩ eosinophil infiltra-
tion (data not shown), which was not surprising, given
the reconstitution with PBMC only. However, when SCID
mice were reconstituted with both PBMC and eosino-
phils from allergic donors, no human eosinophil infiltra-
tion was observed in the allergen-challenged skin xeno-
grafts (not shown). When other potent eosinophil stimuli
were injected into the skin, such as IL-4 plus TNF-␣, or
eotaxin or RANTES, again no human eosinophil infiltra-
tion was obtained (not shown), although murine eosino-
phils were recruited. CD4 T cell and eosinophil recruit-
ment was not observed in SCID mice engrafted with
human skin and autologous PBMC from nonatopic do-
nors, suggesting that the cellular infiltration was strictly
related to the atopic status. Allergen challenge had no
effect on the number of CD8ϩ, HLA-DRϩ, CD68ϩ mono-
cytes/macrophages, or tryptaseϩ mast cells (not
shown).
Allergen Challenge into Human Skin Xenografts from
Allergic Donors Reproduces the Preferential TH2-Type
Cytokine Profile Observed in Human Allergic Reactions
Results
To determine the cytokine profile of the skin-infiltrated
cells, TH1-type (IL-2 and IFN-␥) and TH2-type (IL-4
and IL-5) cytokine mRNA expression were evaluated
by using in situ hybridization (Fig. 3). In the allergic
SCID mice group, significant increases in the number
of IL-4 (Fig. 1F) and IL-5 mRNAϩ cells were found in
the allergen-injected versus the diluent-injected sites.
The absence of IL-2 or IFN-␥ mRNAϩ cells indicated a
preferential recruitment of TH2-type cells. The ab-
sence of the latter cells in the nonatopic SCID mice
group was consistent with a specific immunoallergic
process involving human TH2 cells as observed in
humans.
Allergen Challenge into Human Skin Xenografts from
Allergic Donors Reproduces Both the Immediate and
Late-Phase Allergic Reactions
In a first series of experiments, skin prick tests and
intradermal injection of the house dust mite Dermato-
phagoides pteronyssinus (Dpt) into skin xenografts
obtained from allergic donors induced positive cuta-
neous reactions within 15 minutes (a photograph is
shown in Fig. 1A). No such reaction was observed in
human skin xenografts from nonatopic donors or in
mouse skin, which was consistent with an IgE-
dependent mechanism specific to human skin xeno-
grafts from allergic donors. No delayed reaction was
observed in allergic or nonatopic skin specimens. To
examine the allergen-induced late-phase reaction, au-
tologous PBMC were inoculated intraperitoneally in
SCID mice bearing two human skin grafts from allergic
donors and immediately followed by intradermal injec-
tion of the allergen. Twenty-four hours later, the chal-
lenged site was erythematous and swollen as com-
pared with the contralateral diluent-injected graft.
Here again, this delayed cutaneous reaction was only
observed in skin xenografts from allergic patients but
not in those from nonatopic donors.
In Vivo Inhibitory Effect of an Anti-Human CCR3-Blocking
Antibody and Functional Cross-Reactivity with
Murine Eosinophils
Before assessing the in vivo effect of CCR3-blocking
antibody in our model, we performed an ex vivo study
to check the efficiency of CCR3 blockade (Fig. 4).
Preliminary dose-response experiments showed that
the optimal in vivo inhibitory concentration of the
CCR3-blocking antibody was 25 ␮g/mouse. At this
dose, sera from anti-CCR3–injected mice were able to
inhibit human eotaxin-induced chemotaxis of human
eosinophils (anti-CCR3 sera: Ϫ85% at 24 hours, con-
trol IgG2a sera: Ϫ19%, versus the activity of the sera
before injection). Intraperitoneal coadministration of
anti-CCR3 antibody and PBMC led to a residual
inhibitory activity of 62% at 24 hours (versus 0% for
PBMC plus IgG2a), indicating the presence of a large
proportion of free anti-CCR3 antibody despite its
binding on the surface of PBMC (Fig. 4A). In addition,
anti-CCR3 antibody was also able to inhibit the che-
motaxis of murine eosinophils (anti-CCR3 Ϫ77% ver-
sus control IgG2a Ϫ21%). These results clearly indi-
Allergen Challenge into Human Skin Xenografts from
Allergic Donors Reproduces the Cellular Infiltration
Observed in Human Allergic Reactions
The human cellular infiltration in 24-hour skin xenografts
obtained from the allergic SCID mice group was exam-
ined using immunohistochemistry (Fig. 2). A significant
infiltration of human CD4ϩ cells was observed at the site
of allergen injection as compared with the diluent (Fig. 1,
C and D), including memory CD45ROϩ cells. Besides
930 Laboratory Investigation • July 2002 • Volume 82 • Number 7

CCR3 in Allergic Cutaneous Reaction
Figure 1.
A, Positive skin prick test to Dermatophagoides pteronyssinus (Dpt) in a severe combined immunodeficient (SCID) mouse grafted with human allergic skin. B,
May-Grünwald-Giemsa–stained skin graft section showing the junction between human (on the left) and murine skin (on the right of the arrow) (original magnification,
ϫ50). Cryostat sections of human allergic skin grafts 24 hours after allergen (C) or diluent (D) injections immunostained with anti-CD4 antibody (original
magnification, ϫ100). E, Anti-BB1 staining in an allergen-injected site (original magnification, ϫ250). F, Autoradiograph of cryostat sections of human allergic skin
graft from an allergen site hybridized with 35S-labeled antisense IL-4 (original magnification, ϫ250). Anti-mMBP staining of allergen-injected sites in allergic SCID
mice treated with IgG2a (G) or anti-CCR3 (H) antibody (original magnification, ϫ100). Immunostaining was performed by using the alkaline phosphatase anti-alkaline
phosphatase technique.
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Sénéchal et al
Figure 2.
Infiltration of CD4ϩ T cells, CD45ROϩ memory T cells, mMBPϩ eosinophils, and BB1ϩ basophils after intradermal allergen versus diluent injections in 52 skin grafts
obtained from allergic or nonatopic donors (each donor is represented by a different symbol), 24 hours after introduction of autologous human PBMC by ip injection.
Results are expressed as number of positive cells per field. Median values are represented by the solid bars. Within-group comparisons were performed using the
ranked Wilcoxon test. NS ϭ not significant.
cated a functional cross-reactivity between the anti-
Discussion
human CCR3 antibody and the murine CCR3 on
There is considerable evidence to support an impor-
eosinophils (Fig. 4B).
tant role for chemokines and their receptors in the
pathophysiology of allergic diseases such as asthma
Anti-CCR3 Antibody Treatment Inhibits In Vivo Eosinophil
Recruitment Induced by Allergen Challenge into Human
Skin Xenografts from Allergic Donors
and atopic dermatitis (Lukacs and Tekkanat, 2000;
Schroder and Mochizuki, 1999). A detailed under-
standing of the molecular mechanisms that govern the
inflammatory cell recruitment into tissues during in-
flammation is essential if chemokine-based therapies
are to be developed for treatment of allergic diseases.
Although animal models of allergic reactions are avail-
able, findings cannot always be extrapolated to man.
Therefore we took advantage of the SCID mouse
model to set up an in vivo model of humanized allergic
reaction, using SCID mice engrafted with skin and
PBMC from allergic donors.
Human cutaneous allergic reaction is usually di-
vided into two phases: a first immediate reaction
occurring within 15 minutes and a delayed or late-
phase reaction consisting of a leukocyte infiltration at
the site of allergen administration (Frew and Kay,
1988). In the humanized SCID mouse model of allergy,
we are able to reproduce both the immediate and late
cutaneous allergic reactions. The immediate allergic
reaction was only observed in skin xenografts from
allergic donors but not in skin from nonatopic donors
and not in mouse skin. No PBMC reconstitution was
needed, suggesting that the human skin xenografts
After intraperitoneal coadministration of anti-CCR3
antibody and PBMC and intradermal allergen chal-
lenge, skin biopsies performed 24 hours later revealed
a significant reduction of recruited murine eosinophils,
which was not observed with the use of the control
IgG2a antibody (Fig. 1, G and H, and Fig. 5). Surpris-
ingly, anti-CCR3 antibody treatment had no significant
effect on the numbers of recruited CD4 and BB1ϩ
cells, which were similar to the control IgG2a-treated
group. Also, there was no increase in numbers of
CD8ϩ, HLA-DRϩ, CD68ϩ, and tryptaseϩ cells after
allergen challenge, whatever the group (not shown).
Treatment with anti-CCR3 versus control IgG2a anti-
bodies did not affect the number of recruited IL-4 and
IL-5 mRNA-expressing cells. The same TH2-type cy-
tokine profile was observed in the IgG2a-treated
group and in the allergic group of the pilot study. There
was no expression of TH1-type cytokines in any
groups. These results strongly suggest that anti-CCR3
Ab affects specifically the recruitment of eosinophils in
the context of the cutaneous allergic challenge.
932 Laboratory Investigation • July 2002 • Volume 82 • Number 7

CCR3 in Allergic Cutaneous Reaction
Figure 3.
Cytokine mRNA expression profile after intradermal allergen versus diluent injections in 52 skin grafts obtained from allergic or nonatopic donors (each donor is
represented by a different symbol), 24 hours after introduction of autologous human PBMC by intraperitoneal injection. Analyses are as in Figure 2.
retained all the components required for the immedi-
ate allergic reaction. In our model, the late allergic
reaction occurred only with autologous PBMC recon-
stitution and allergenic challenge of skin xenografts.
Human skin and PBMC from allergic donors were both
necessary and sufficient for the development of aller-
gic LPR. No human cell migration was observed when
allergen was injected into mouse skin in the reconsti-
tuted SCID mouse model, and without PBMC, no LPR
occurred in allergen-injected human allergic skin of
grafted SCID mice. Taken together, the allergen chal-
lenge of skin xenografts from allergic donors in recon-
stituted SCID mice resulted in the development of an
immediate reaction and a delayed reaction exactly as
observed in skin from allergic patients.
In humans, allergen-induced cutaneous LPR is as-
sociated with an infiltration of CD4ϩ T cells, eosino-
phils, activated CD25ϩ cells (Barata et al, 1998; Tsi-
copoulos et al, 1994), and basophils (Irani et al, 1998;
Ying et al, 1999). A preferential TH2-type cytokine
mRNA expression with almost no TH1 expression is
found at the sites of such cutaneous allergic reactions
(Kay et al, 1991; Tsicopoulos et al, 1994). In the
humanized SCID mouse model of allergy, allergen-
injected skin xenografts from nonatopic individuals
did not exhibit cell infiltration, whereas those from
atopic subjects reproduced all the main findings in
human systems. This cellular infiltration was not a
result of MHC incompatibility, based first on the use of
autologous mononuclear cells and skin specimens
and second on the difference in cell recruitment ob-
served between the diluent- and allergen-injected
sites. The cellular infiltration after allergen challenge
was not observed in all mice. This was not related to
variability among donors, as can be observed from the
individual data presented for each donor. Rather,
these variations probably reflect the complexity of the
model, which requires a good chimerism between the
human and the murine systems to be functional. One
interesting difference is that the recruited eosinophils
were exclusively of murine origin. The accumulation of
murine eosinophils in the human skin suggests that
some human mediators may attract murine eosino-
phils. Such cross-reactivity has been demonstrated
for human IL-5 and eotaxin (Fattah et al, 1990; Teixeira
et al, 1997). There was no infiltration of human eosin-
ophils, in agreement with the mode of reconstitution of
SCID mice that involved only mononuclear cells. In a
separate set of experiments, we tried to attract human
eosinophils to the site of the cutaneous allergic reac-
tion by reconstituting SCID mice with both PBMC and
eosinophils from the same donors. No human eosin-
ophil infiltration was observed (not shown). The same
results were obtained when cytokines or chemokines
known to attract eosinophils were injected into the
skin (not shown). Taken together, these results sug-
gest either a lack of eosinophil survival mediators in
the vicinity of the peritoneal cavity or an absence of
cross-reactivity between human eosinophil adhesion
molecules and murine vessels counter receptors,
leading to an absence of migration to the skin.
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Sénéchal et al
Figure 4.
A, Kinetics of in vivo inhibitory effect of CCR3-blocking antibody on eosinophil chemotaxis. Human eosinophils were incubated with sera from SCID mice collected
before (0) and at 1, 6, and 24 hours after ip injection of 25 ␮g of anti-CCR3 or IgG2a antibodies with or without PBMC, and with human eotaxin as chemoattractant.
B, Functional chemotactic cross-reactivity between anti-human CCR3 and murine eosinophils. Eosinophils from recombinant human IL-5-SCID mice were incubated
with 20 ␮g/ml of anti-CCR3 or control IgG2a antibodies and chemoattracted using murine eotaxin. Results are expressed as mean Ϯ SEM number of recruited
eosinophils per field (n ϭ 3 for A and B).
One important chemoattractant receptor expressed
on infiltrating cells during allergic reactions is repre-
sented by CCR3. To investigate its role in allergic
reactions, we evaluated the effect of a CCR3-blocking
antibody in our humanized model of allergen-induced
LPR. A selective inhibition of mouse eosinophil recruit-
ment was observed after skin allergen challenge in the
anti-CCR3–treated group. This suggests that in re-
sponse to allergen, the human skin xenograft pro-
duced chemoattractant mediators, such as eotaxin, as
already shown in human allergen-induced cutaneous
LPR (Ying et al, 1999), able to trigger murine eosino-
phils through CCR3. The high level of CCR3 expres-
sion on both human and murine eosinophils (Ponath et
al, 1996) and their similar sensitivity to human eotaxin
are consistent with this hypothesis. Moreover, the high
amino-acid sequence homology (80%) between mu-
rine and human CCR3 explains the cross-reactive
inhibitory effect of the anti-human CCR3 antibody on
murine eosinophil chemotaxis. Thus, in this human-
ized SCID mouse model of allergy, we can conclude
that the recruitment of eosinophils into human skin
xenografts challenged with allergen is mainly medi-
ated by CCR3 and its ligands.
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CCR3 in Allergic Cutaneous Reaction
Figure 5.
Infiltration of CD4ϩ T cells, CD45ROϩ memory T cells, mMBPϩ eosinophils, BB1ϩ basophils and cytokine mRNAϩ cells after allergen versus diluent challenges in
28 skin grafts obtained from allergic donors (each donor is represented by a different symbol), 24 hours after ip introduction of IgG2a- or anti-CCR3-treated
autologous human PBMC. Results are expressed as number of positive cells per field. Median values are represented by the solid bars. Differences within the four
groups were first evaluated by using the Kruskal-Wallis test and were found statistically significant for CD4, BB1, mMBP, IL-4, and IL-5 mRNA but not for CD45RO,
IFN-␥, and IL-2 mRNA. When significant, comparisons within allergen and diluent values were performed by using the Wilcoxon test. Comparisons between IgG2a
and anti-CCR3-treated groups were performed by using the Mann-Whitney U test after subtraction of the diluent values from the allergen values. p values are indicated
and were considered significant when less than 0.05.
The role of CCR3 in eosinophil recruitment has been
studied in other animal models. In a guinea pig model
of eosinophil cutaneous recruitment, pretreatment of
labeled eosinophils in vitro with a blocking CCR3
antibody inhibited their accumulation in response to
eotaxin in vivo (Sabroe et al, 1998). In a model of
Nippostrongylus brasiliensis-infected mice, in vivo de-
pletion of CCR3-expressing cells led to a reduction in
lung eosinophilia (Grimaldi et al, 1999). In the present
study, we show for the first time that in a humanized
mouse model of cutaneous allergy, allergen-induced
recruitment of eosinophils is also dependent upon
CCR3. Hitherto, the effect of CCR3 antagonists has
only been evaluated on eosinophils, even though
CCR3 is also highly expressed on basophils (Uguc-
cioni et al, 1997). In the present study, there was no
inhibitory effect of anti-CCR3 treatment on basophil
migration, suggesting that these cells may be at-
tracted to the site of the reaction through additional
pathways. It is of note that in human allergen-induced
cutaneous LPR, it has been recently shown that the
expression of CCR3-binding chemokines correlated
with the presence of eosinophils but not basophils
(Ying et al, 1999). Basophils are known to express
CCR4 in particular (Power et al, 1995), which might be
another potential target involved in allergic inflamma-
tion (Lloyd et al, 2000).
TH2 cell recruitment in a murine model of allergic
airway disease. In this study, the involvement of the
eotaxin/CCR3 pathway was confined to early stages
of the TH2 response in vivo (Lloyd et al, 2000). It is of
note that CCR3 expression is differentially regulated in
the human and the mouse system. In mice, anti-CD3
stimulation increases CCR3 expression on TH2 cells
(Lloyd et al, 2000), whereas it down-regulates it in
humans (Sallusto et al, 1999). These species differ-
ences underline the interest of the human xenograft
model to assess effects of molecules devoted to
human usage. In our human skin xenograft model of
allergy, anti-CCR3 treatment had no effect on CD4
T-cell recruitment, which is in line with the very small
proportion of peripheral blood T cells expressing
CCR3 (Sallusto et al, 1997), but also had little or no
effect on TH2-type cytokine mRNA-expressing cells. It
is probable that, as for basophils, TH2-type mRNAϩ
cells may be recruited in vivo through additional
chemokine receptors, such as CCR4 and CCR8, ex-
pressed on the latter cells (Bonecchi et al, 1998). In
this context, it has been reported that in human
allergic cutaneous reactions, there is an overexpres-
sion of thymus activation regulated chemokine
(CCL17) (Kakinuma et al, 2001; Vestergaard et al,
2000) and macrophage-derived chemokine (CCL22)
(Galli et al, 2000), which bind CCR4 (Imai et al, 1997,
1998). The in vivo relevance of the CCR4 pathway on
TH2 cell recruitment in allergic inflammation has been
assessed indirectly using antibody against macro-
phage-derived chemokine in a murine model, where it
was mainly effective on the chronic recruitment of TH2
cells (Lloyd et al, 2000). The CCR8 receptor has also
been recently shown to be implicated in TH2 func-
CCR3 was the first chemokine receptor reported to
be preferentially expressed on human TH2 cells (Sal-
lusto et al, 1997), in particular cells producing IL-4
(Sallusto et al, 1998). No previous studies have directly
addressed the role of CCR3 in vivo on TH2 cell
recruitment. Indirect arguments have been provided
by assessing the effect of anti-eotaxin antibody on
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Sénéchal et al
tional responses in vivo. Interestingly, CCR8-deficient
mice display impaired TH2 cytokine production and
reduction in eosinophils in a model of ovalbumin-
induced allergic airway inflammation (Chensue et al,
2001). Taken together, these data suggest that che-
mokine receptors may play a redundant role in baso-
phil and TH2 cell recruitment in allergic reactions.
from human donors was obtained from plastic surgery
or from truncal operations where skin was discarded
and blood was collected on heparin 6 weeks later. The
protocol was approved by the Centre Hospitalier
Regional et Universitaire Ethical Committee (no. 96–
102). All donors signed an informed consent form.
Animals and Skin Grafting
In conclusion, the present study establishes an effi-
cient humanized model of allergic cutaneous reaction
by using human skin and PBMC xenografts in SCID
mice. In addition, the study demonstrates that CCR3
is a major mechanism of eosinophil recruitment in
allergic cutaneous LPR, although additional pathways
are likely to be involved in the recruitment of the
noneosinophilic cells. Finally, these data open new
insights in the evaluation of potential human-specific
antiallergic molecules.
Inbred mice with severe combined immunodeficiency
(CB-17 SCID mice) were maintained at the Institut Pas-
teur de Lille in sterilized isolators. Leaky mice (displaying
spontaneous IgG production) were discarded. The mice
were housed under pathogen-free conditions. Animals
were handled according to the ethical principles of
animal experimentation established by the European
Center of Tufts University. Skin grafting was performed
as previously described (Tsicopoulos et al, 1998; Yan et
al, 1993). Two full-thickness human skin grafts of the
same size were placed onto the wound beds on each
mouse. The transplants were held in place with 6-0 silk
suture material and covered with an adhesive wound
dressing and then with an adhesive bandage. The dress-
ing material and sutures were removed 10 days after
transplantation.
Materials and Methods
Reagents
Anti-CD4 monoclonal antibody was purchased from
Becton Dickinson (Pont de Claix, France). EG2 was
obtained from Pharmacia (St. Quentin en Yvelines,
France). Anti-CD8, anti-CD45RO (T-cell memory phe-
notype), anti-CD68 (macrophages), anti-HLA-DR, rab-
bit anti-mouse Ig, monoclonal alkaline phosphatase
anti-alkaline phosphatase (APAAP), and control IgG
antibodies were from Dako (Trappes, France). Anti-
tryptase antibody was from Chemicon (Temecula,
California). BB1, a human basophil-specific monoclo-
nal antibody, and rabbit antiserum specific for murine
eosinophil major basic protein (mMBP) were produced
as previously described (Filley et al, 1981; McEuen et
al, 1999). Biotin-labeled goat anti-rabbit antibody,
extravidin-alkaline phosphatase, and Fast Red TR/
naphthol ASMX tablets were from Sigma (St. Quentin
Fallavier, France). Dpt extract was obtained from Stal-
lergènes (Antony, France). The endotoxin concentra-
tion was less than 0.125 EU/ml for the dose of Dpt
used as assessed by the Limulus amebocyte lysate
test (BioWhittaker, Walkersville, Maryland). Anti-
human CCR3-blocking monoclonal antibody (7B11),
from Leukosite Inc., was obtained through the AIDS
Research and Reference Reagent Program, Division
of AIDS, National Institute of Allergy and Infectious
Diseases, National Institutes of Health (Heath et al,
1997; Ponath et al, 1996). A control monoclonal anti-
body of the same isotype (IgG2a) was purchased from
Diaclone (Besançon, France).
Study Design: Pilot, CCR3, and Eosinophil Studies
Six weeks after human skin transplantation, anti-asialo
GM1 (50 ␮l; Wako, Osaka, Japan) was injected ip to
neutralize murine natural killer activity. Twenty-four
hours later, SCID mice were reconstituted ip with 15 to
20 ϫ 106 PBMC (hu-PBMC-SCID mice), purified from
the donor’s blood using a Ficoll-Hypaque (Pharmacia,
Uppsala, Sweden) gradient after platelet depletion and
resuspended in PBS at a concentration of 40 ϫ 106
cells/ml. One IR unit of Dpt (index reactivity unit) in 20
␮l of endotoxin-free saline was then injected intrader-
mally immediately, with 5% Evans blue dye (Sigma) to
mark the site of injection. The contralateral graft of
each mouse was injected with endotoxin-free saline
diluent containing 5% Evans blue dye to serve as
internal control. In the CCR3 study, the same protocol
was followed, except that PBMC were incubated with
either the 7B11 anti-CCR3 antibody or with an irrele-
vant isotype-matched IgG2a antibody at the predeter-
mined optimal dose (see chemotaxis assays) of 25
␮g/mouse for 30 minutes before ip reconstitution.
In a different set of experiments, human eosinophils,
obtained from allergic donors and purified as described
below, were injected together with autologous PBMC at
a concentration of 106 cells. One graft was injected with
the diluent, and the second one with allergen, as de-
scribed above, or the cytokines IL-4 (2000 U) or IFN-␥
plus TNF-␣ (2000 U each), or chemokines (eotaxin or
RANTES at 4 ␮g). In all experiments, mice were killed 24
hours after the injections, and the skin grafts were cut
into two halves through the center of the injection site
marked by the Evans blue dye. One half was immedi-
ately embedded in OCT compound (Labonord, Ville-
neuve d’ascq, France), snap-frozen in isopentane pre-
cooled in liquid nitrogen, and stored at Ϫ80° C. Cryostat
Human Donors
The human donors were separated into two groups
according to their atopic status. Allergic donors (n ϭ 5)
had a history of perennial rhinitis or asthma and
exhibited positive skin prick tests to Dpt extracts and
positive radio allergo sorbent tests (RASTs). The total
IgE level was 632 Ϯ 284 KU/ml (mean Ϯ SEM). Nona-
topic donors (n ϭ 3) served as a control group, and
their mean total IgE level was 125 Ϯ 54 KU/ml. Skin
936 Laboratory Investigation • July 2002 • Volume 82 • Number 7

CCR3 in Allergic Cutaneous Reaction
sections (6 ␮m) were cut, air-dried, fixed in a mixture of
60% acetone and 40% methanol, dried, wrapped in foil,
and stored at Ϫ20° C until used for immunohistochem-
istry. The other half was fixed in 4% paraformaldehyde
(for in situ hybridization studies) and washed in 15%
phosphate-buffered saline/sucrose. Cryostat sections
(10 ␮m) were cut onto poly-L-lysine–coated RNase-free
slides and stored with silica gel at Ϫ80° C until use.
Mice with spontaneously activated grafts, as evi-
denced by leukocyte infiltration in the diluent-injected
site, were discarded. The pilot study included three
allergic donors and three nonatopic donors as a
control group, with a total of 13 mice in each group
representing 52 grafts. In the CCR3 study, two addi-
tional allergic donors were included, with a total of 14
mice representing 28 grafts.
Human and Murine Eosinophil Chemotaxis Assay
Highly purified human eosinophils (95.4 Ϯ 1.6%),
obtained from peripheral blood of hypereosinophilic
patients, were produced as previously described
(Fahy et al, 2000) using magnetic beads coated with
anti-CD16 (MACS; Miltenyi Biotech, Bergisch Glad-
bach, Germany) and resuspended in RPMI at a con-
centration of 106 cells/ml. Human eosinophils were
incubated with sera from SCID mice diluted 1:5 with
RPMI, which had been collected before and at 1, 6,
and 24 hours after ip injection of either anti-CCR3 or
IgG2a control antibody (10 or 25 ␮g/mouse) with or
without PBMC. The chemotaxis protocol was per-
formed with a 48-well microchemotaxis chamber
(Neuro Probe, Cabin John, Maryland) using 5-␮m pore
polycarbonate filters (Nucleopore Corporation, Pleas-
anton, California) during 1 hour at 37° C in 5% CO2.
Chemotaxis was induced using human eotaxin at
10Ϫ7 M (Tebu, Le Perray en Yvelines, France).
Immunohistochemistry
Murine eosinophils were obtained from recombinant
human IL-5-SCID mice as previously described (Fat-
tah et al, 1990). Briefly, SCID mice were treated using
25 ␮g of recombinant human IL-5 (R&D, Minneapolis,
Minnesota) delivered during 7 days through subcuta-
neous osmotic pumps (Charles River, St. Aubin,
France). After death, spleen eosinophils were resus-
pended at a concentration of 106 cells/ml and incu-
bated with medium, control IgG2a, or anti-CCR3 an-
Immunohistochemistry was performed as previously de-
scribed (Tsicopoulos et al, 1994) using a modified
APAAP method for anti-human monoclonal antibody
staining. Briefly, sections were incubated with the pri-
mary antibody for 30 minutes, washed in tris-buffered
saline, and successively incubated for 30 minutes with
rabbit anti-mouse and then APAAP diluted in 20%
normal human serum. For anti-mMBP staining, the sec-
ond reagent was a biotinylated goat anti-rabbit antibody,
and the third reagent was extravidin-alkaline phospha-
tase. The color was developed using Fast Red. Sections
were hematoxylin counterstained. Substitution of the
primary antibody with an irrelevant antibody of the same
species was used as negative control. No cross-
reactivity was observed with murine cells.
tibody at
a final concentration of 20 ␮g/ml.
Chemotaxis was induced using murine eotaxin at
10Ϫ7 M (Tebu).
Quantification and Statistical Studies
Slides were encoded and counted in a blinded fashion
at ϫ250 magnification using an eyepiece graticule.
The upper edge of the grid was placed at the epider-
mal junction. For each specimen, at least three sec-
tions were evaluated, from which three to nine fields
were counted for both immunohistochemistry and in
situ hybridization studies. The absolute number of
positive cells was counted per field (0.4 mm2). Statis-
tical analysis was performed using Statview 5 soft-
ware. Comparisons within allergen- and diluent-
injected sites were performed by using the Wilcoxon
matched-pairs signed rank test. Differences within
anti-CCR3- and IgG2a-treated groups were first eval-
uated for the four subgroups using the Kruskal-Wallis
test. When statistical significance was observed, dif-
ferences were subsequently analyzed by using the
Wilcoxon test for diluent/allergen pairs and the un-
paired Mann-Whitney U test for anti-CCR3- and
IgG2a-treated groups after subtraction of the diluent
value from the allergen value. Only p values less than
0.05 were considered significant.
In Situ Hybridization
In situ hybridization was performed as previously
described (Hamid et al, 1987; Tsicopoulos et al, 1998).
Briefly, sections were hybridized with 35S-labeled ri-
boprobes (2 ϫ 106 cpm per section) in hybridization
buffer containing 50 mM dithiothreitol (Promega, Mad-
ison, Wisconsin). Sections were hybridized overnight
at 45° C and washed in high-stringency conditions
(45° C, 0.1ϫ SSC). Human peripheral blood-derived
polarized TH1 or TH2 cells that expressed mRNA for
IL-2 and IFN-␥ or IL-4 and IL-5 were used as cytokine
expression positive controls. For negative controls,
skin sections were hybridized using sense probes for
the relevant cytokines. Additional sections were
treated with RNase-A before the prehybridization step
with antisense probes. Specific hybridization was rec-
ognized as clear dense deposits of silver grains in the
emulsion overlying cells in the tissue preparations.
Cells were identified as dense, discrete, well-
circumscribed areas of silver grains. When hybridized
cells were in close proximity, their numbers were
determined by visualizing the number of individual
nuclei, using dark field illumination.
Acknowledgements
The authors wish to thank Dr. P. Lassalle and Dr.
A. S. Charbonnier for critical review of the manuscript,
Mr. E. Fleurbaix and Mr. J. P. Decavel for breeding
Laboratory Investigation • July 2002 • Volume 82 • Number 7 937

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