(Journal of Leukocyte Biology. 2001;69:353-360.)
© 2001
by Society for Leukocyte Biology
Eosinophil recruitment into sites of delayed-type hypersensitivity reactions in mice
Mauro M. Teixeira*,
André Talvani*,
Wagner L. Tafuri
,
Nicholas W. Lukacs
and
Paul G. Hellewell
* Immunopharmacology Laboratory, Department of Biochemistry and Immunology, and
Department of Pathology, Instituto de Ciências Biológicas, Universidade Federal de Minas Gerais, Belo Horizonte, Brazil;
Department of Pathology, University of Michigan, Ann Arbor, Michigan; and
Cardiovascular Research Group, University of Sheffield, United Kingdom
Correspondence: Mauro Martins Teixeira, M.D., Ph.D., Immunopharmacology, Departamento de Bioquímica e Imunologia, Instituto de Ciencias Biologicas, Universidade Federal de Minas Gerais, Av. Antonio Carlos, 6627Pampulha, 31270-901 Belo Horizonte MG Brasil. E-mail: mmtex{at}icb.ufmg.br
 |
ABSTRACT
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The selective accumulation of eosinophils in tissue is a characteristic
feature of allergic diseases where there is a predominance of
lymphocytes expressing a Th2 phenotype. In an attempt to define factors
determining specific eosinophil accumulation in vivo, we
have used a radiolabeled technique to assess the occurrence and the
mechanisms underlying 111In-eosinophil recruitment into
Th1- and Th2-predominant, delayed-type hypersensitivity (DTH)
reactions. Eosinophils were purified from the blood of IL-5 transgenic
mice, labeled with 111In and injected into nontransgenic
CBA/Ca mice. Th1- and Th2-predominant, DTH reactions were induced in
mice by immunization with methylated bovine serum albumin (MBSA) in
Freunds complete adjuvant or with Schistosoma mansoni
eggs, respectively. In these animals, 111In-eosinophils
were recruited in skin sites in an antigen-, time-, and
concentration-dependent manner. Depletion of CD4+
lymphocytes abrogated 111In-eosinophil recruitment in both
reactions. Pretreatment of animals with anti-IFN-
mAb abrogated
111In-eosinophil recruitment in MBSA-immunized and
-challenged animals, whereas anti-IL-4 inhibited
111In-eosinophil recruitment in both models. Local
pretreatment with an anti-eotaxin polyclonal antibody inhibited the
MBSA and SEA reactions by 51% and 39%, respectively. These results
demonstrate that, although eosinophilia is not a feature of
Th1-predominant, DTH reactions, these reactions produce the necessary
chemoattractants and express the necessary cell adhesion molecules for
eosinophil migration. The control of the circulating levels of
eosinophils appears to be a most important strategy in determining
tissue eosinophilia.
Key Words: chemokines bone marrow interleukin-4 lymphocytes
 |
INTRODUCTION
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Tissue eosinophilia is a marked characteristic of allergic
diseases and is often associated with organ dysfunction. For example,
in asthma, the presence of activated eosinophils in the respiratory
mucosa is associated with significant lung dysfunction, and effective
anti-inflammatory therapies are usually accompanied by resolution of
pulmonary eosinophilia [1
, 2
]. Similarly,
in atopic dermatitis, deposits of eosinophil-derived granules have been
demonstrated extensively and associated with diseased skin, although
eosinophils are not seen in histological sections routinely
[3
]. These studies support the idea that eosinophils
play a central role in the pathophysiology of allergic diseases and
suggest that the development of strategies aimed at inhibiting
eosinophil recruitment to sites of inflammation in vivo may
be a relevant therapeutic approach in the treatment of allergic
diseases [4
5
6
].
In response to an appropriate inflammatory stimulus, circulating blood
eosinophils interact with endothelial cells initially and then enter
the tissue. The process of eosinophil migration is regulated tightly by
the expression of cell adhesion molecules (CAMs) on endothelial cells
and on leukocytes. In addition, eosinophils must be activated by
chemoattractant molecules [e.g., chemokines, leukotriene
B4 (LTB4)] acting on seven-transmembrane
receptors on the leukocyte surface prior to their entry into the tissue
[4
, 7
]. However, there are other factors
that control the accumulation of eosinophils in vivo, and
these include their release from the bone marrow and circulation in
blood, and survival in the tissue [4
]. For example, the
pretreatment of guinea pigs with interleukin (IL)-5 increases blood
levels of primed eosinophils and facilitates the recruitment of these
cells in the skin [8
]. Similarly, the recruitment of
eosinophils in the lungs of IL-5 transgenic mice after challenge with
the chemokine eotaxin is increased markedly compared with nontransgenic
animals [9
, 10
]. Finally, the importance of
survival for the accumulation of leukocytes in tissues is illustrated
by studies demonstrating the efficacy of apoptosis-inducing strategies
at reducing eosinophilic inflammation in sensitized and challenged
animals [11
, 12
].
A marked characteristic of allergic diseases, such as asthma and atopic
dermatitis, is the predominance of cytokines with a Th2 phenotype
secreted mainly by activated T lymphocytes [13
]. It is
thought that the production of these Th2 cytokines, and especially IL-4
and IL-5, drives the local expression of cell adhesion molecules and
chemokines necessary for eosinophil accumulation usually seen in these
diseases [4
, 14
]. In contrast, tissue
eosinophilia is not usually observed in diseases or models associated
with a predominant Th1 cytokine response, although Th1 cytokines may be
expressed in allergic tissues [15
, 16
]. The
explanations for the lack of tissue eosinophilia in inflammatory
responses associated with a Th1 response are not entirely known and
include the lack of the local expression of the necessary
chemoattractant molecules [17
] and CAMs
[18
, 19
]. However, the possibility that the
IL-5-driven blood eosinophilia plays a more central role in determining
tissue eosinophilia has not been examined in detail. The present study
was undertaken to examine the ability of eosinophils to migrate into
sites of Th1- or Th2-predominant, delayed-type hypersensitivity (DTH)
reactions. To evaluate the central role of primed blood eosinophils for
eosinophil recruitment in vivo, radiolabeled eosinophils
purified from the blood of IL-5-transgenic mice [20
,
21
] were injected into nontransgenic, immunized mice, and
their recruitment was assessed into discrete sites of cutaneous
inflammation following challenge with antigen.
 |
MATERIALS AND METHODS
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Animals
Female CBA/Ca mice (1820 g) were obtained from Harlan
(Bicester, UK) and René Rachou Reseach InstituteFiocruz (Belo
Horizonte, Brazil). CBA/Ca mice overexpresssing the murine IL-5 gene
(Tg1 mice) were a gift of Glaxo Wellcome (Stevenage, UK) and were bred
in the Bioscience Unit of the National Heart and Lung Institute,
Imperial College of Science Technology and Medicine (London, UK).
Reagents
Methylated bovine serum albumin (MBSA), concavalin A (Con A),
Dextran, Percoll, and Freunds complete adjuvant (FCA) were purchased
from Sigma Chemical Co. (Poole, UK); Dulbeccoss phosphate-buffered
saline (PBS; calcium- and magnesium-free, pH 7.4) was from Life
Technologies (Paisley, UK); LTB4 was from Cascade (Reading,
UK); and Schistosoma mansoni eggs and antigen were kindly
provided by Dr. Corrêa-Oliveira (René Rachou Research
Institute).
Purification and radiolabeling of murine eosinophils
Eosinophils (>97% purity and viability) were purified from the
blood of IL-5 transgenic mice, labeled with 111In, and
injected into nontransgenic CBA/CA mice as previously described
[21
, 22
]. Briefly, blood was obtained by
cardiac puncture (threefour donor mice/experiment), and red cells
were sedimented using Dextran (T500, one part blood to four parts
Dextran 1.25%). The leukocyte-rich supernatant was removed,
centrifuged (300 g, 7 min), and layered onto a
discontinuous, four-layer, Percoll gradient (densities: 1.070, 1.075,
1.080, and 1.085 g/ml). The gradients were centrifuged at 1500
g for 25 min at 20°C, and eosinophils and lymphocytes were
collected from the 1.080/1.085 interface. Lymphocytes were removed by
using negative immunoselection with rat anti-mouse CD2 and B220 mAbs
(Pharmingen, San Diego, CA). Purified eosinophils were then labeled
with 111In chelated to
2-mercaptopyridine-N-oxine.
DTH reactions
A Th1-predominant, DTH reaction was prepared by immunizing
animals with MBSA in FCA, as previously described [22
,
23
]. Briefly, mice received two intradermal (i.d.)
injections in the abdominal skin of 50 µl MBSA (5 mg/ml) that had
been emulsified in FCA. For the elicitation of the reaction, animals
were injected i.d. with MBSA (0.110.0 µg/site, dissolved in PBS) 6
or 7 days after immunization. A Th2-predominant was elicited by
immunizing mice with 1000 eggs of S. mansoni
[intraperitoneally (i.p.) in 100 µl PBS], as previously described
[24
]. After immunization (6 or 7 days), animals were
challenged i.d. with Schistosoma egg antigen (SEA, 0.110
µg/site, dissolved in PBS).
Measurement of 111In-eosinophil recruitment in murine
skin
The purified, 111In-labeled eosinophils were
injected intravenously (i.v.; 1x106 cells/animal) into
recipient mice that had been anesthetized previously with a mixture of
Hypnorm/Hypnovel/distilled water (1/1/4). After 10 min to allow the
circulation of the radiolabeled cells, i.d. injections of MBSA, SEA, or
LTB4 were given in 50 µl vol into the shaved dorsal skin
of the immunized mice (up to four injections/animal).
111In-labeled eosinophil accumulation was assessed at
various 4-h periods (04, 48, and 2024 h) following the i.d.
injections. A blood sample was then obtained via cardiac puncture, the
animals were sacrificed with an overdose of sodium pentobarbitone, the
dorsal skin was removed, cleaned free of excess blood, and the sites
were punched out. The samples were counted in an automatic 5-head
gamma-counter (Canberra Packard Ltd., Pangbourne, Berks, UK).
Eosinophil numbers in the skin sites were expressed as the number
111In-eosinophils per skin site.
Pretreatment with antibodies
For the neutralization of IL-4, animals were treated with the
anti-IL-4 monoclonal antibody (mAb) 11B11 (2 mg/mouse) i.p. 1 h
prior to challenge with antigen. For the neutralization of
interferon-
(IFN-
), animals were pretreated i.p. with the
anti-IFN-
mAb R4-6A2 (1 mg/mouse) 1 h prior to challenge.
Control animals received a similar dose of rat immunoglobulin G (IgG).
For the depletion of CD4+ lymphocytes, animals were
pretreated with the mAb GK1-5 (1 mg/mouse, i.p.) 2 h prior to
challenge. CD8+ lymphocyte depletion was carried out by
pretreating animals with mAb 243 (2 mg/mouse, i.p.) 2 h prior to
challenge. The depletion of CD4 or CD8 was confirmed by performing
fluorescein-activated cell sorter (FACS) analysis (B&D FACScan) of the
lymphocyte population in blood and spleen using anti-CD4 or anti-CD8
fluorescein isothiocyanate (FITC)-labeled antibodies (Sigma). To
evaluate the possible role of eotaxin as a mediator of eosinophil
recruitment, skin sites were injected with an anti-eotaxin polyclonal
antibody (20% dilution in saline) 15 min prior to the i.v. injection
of 111In-eosinophils. Control sites in the same animal
received an i.d. injection of pre-immune rabbit serum (20% dilution in
saline). This dose of anti-eotaxin polyclonal antibody has been shown
previously to abrogate the migration of eosinophils induced by 150
pmoles of eotaxin [21
].
Activation of spleen lymphocytes and detection of cytokines
Spleens were collected aseptically from naïve animals
and animals immunized with FCA/MBSA or Schistosoma eggs and
teased into single-cell suspension. A pool of spleens from three
animals in each group was used for each experiment. Red blood cells
were then removed by spinning the cell suspension (15 min, 800
g) over a Ficoll-Paque gradient (Pharmacia, Upsala, Sweden;
1.077). Cells obtained at the top of the gradient were collected,
washed thrice, and resuspended in a final solution of 3 x
106 cells/ml in RPMI containing 10% fetal bovine serum
(FBS). Cell suspension (500 µl) was added to each well of 24-well
plates and incubated with buffer, SEA (1, 5, and 25 µg/ml), MBSA
(1100 µg/ml), or Con A (5 µg/ml). Samples were collected and
stored 48 or 72 h later at -70°C until the measurement of
cytokines. IL-4, IL-5, IL-10 (72 h cultures), and IFN-
(48 h
cultures) were measured using sandwich enzyme-linked immunosorbent
assay (ELISA) with antibody pairs purchased from Pharmingen and
according to the protocol of the supplier.
Reverse transcriptase-polymerase chain reaction (RT-PCR) assay for
measuring in vivo expression of cytokine mRNA
To investigate the expression of cytokine/chemokine mRNA,
immunized or naive mice were challenged with specific antigen, and skin
sites were removed 1, 3, 6, and 24 h later. Total RNA was
extracted from each sample by the acid guanidinium,
thiocyanate-phenol-chloroform extraction method. Total RNA (1 µg) was
reverse-transcribed by the addition of 2.5 U RNAsin (Promega Corp.,
Madison, WI), 2.5 mM deoxynucleotides (dNTPs) (Boehringer Mannheim
Biochemicals, Mannheim, Germany), 0.1 M dithiothreitol (Gibco BRL Life
Technologies, Inc., Grand Island, NY), IX Moloney murine leukemia virus
RNAase H- RT buffer (Life Technologies), 25 ng Random
hexamer oligonucleotides (Boehringer Mannheim), and 200 U Moloney
murine leukemia virus RNAse H- RT (Life Technologies) in
20 µl total vol. The reaction proceeded for 1 h at 37°C and
was terminated by boiling for 5 min after the addition of 175 µl
H2O. The cDNA obtained (5 µL) was used for amplification
in a 30 µl PCR reaction containing 2.5 mM dNTPs (Pharmacia), a 0.2 mM
concentration of the 3' and 5' external primers, 1.5 mM
MgCl2, IX GeneAmp PCR buffer, and 5 U Taq DNA polymerase
(Promega). PCR conditions were executed as follows: denaturation
(95°C for 3 min and 94°C for 1 min), annealing (52°C for 1 min),
and extension (72°C for 2 min). The number of PCR cycles is given
after each sequence below, and this was followed by a final extension
of 7 min at 72°C. PCR products and molecular weight markers were run
on 6% polyacrylamide gel and stained with silver nitrate. Computer
images of gels were obtained for semiquantitation using a densitometer.
The primers (sense and anti-sense) were selected from the published
cDNA sequences [25
] and commercially synthesized
(MWG Oligo Synthesis, London, UK). Eotaxin,
CAC-GAA-GCT-TTA-GGT-AAG-CAG-TAA-CTT-CCA-TCT-GTC-TC//GCG-GCT-AGC-TGA-CTA-AAT-CAA-GCA-GTT-CTT-AGG-CTC-TG
(35 cycles); IL-4,
CTC-AGT-ACT-ACG-AGT-AAT-CCA//GAA-TGT-ACC-AGG-AGC-CAT-ATC (35 cycles);
IFN-
, AAC-GCT-ACA-CAC-TGC-ATC-TTG-G//GAC-TTC-AAA-GAC-TCT-GAG-G (30
cycles); and hipoxanthine phosphorybosyl transferase,
GTT-GGA-TAC-AGG-CCA-GAC-TTT-GTT-G//GAT-TCA-ACT-TGC-GCT-CAT-CTT-AGG-C
(30 cycles).
Quantitation of bone marrow eosinophils
The number of eosinophils in the bone marrow of mice was
quantified from bone marrow cell suspension of femurs as previously
described [26
, 27
]. Left femurs from
immunized or naïve mice were isolated and flushed with 5 ml
Hanks buffered salt solution containing 0.25% BSA. Samples were spun
and cell numbers determined in a Neubauer haemocytometer after dilution
of samples in Turks stain. The percentage of mature eosinophils was
determined using standard morphologic criteria in cytospin preparations
of bone marrow samples.
Statistical analysis
Experiments were analyzed by using two-way analysis of variance
(ANOVA) on normally distributed data. P values were assigned
using Newman-Keuls procedure and values of P < 0.05
were considered statistically significant. Percentage inhibition was
calculated subtracting background values. Results are presented as the
mean ± SE.
 |
RESULTS
|
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Recruitment of 111In-labeled eosinophils in sites of
DTH reactions
The i.d. administration of SEA in animals immunized with S.
mansoni eggs induced a time- and dose-dependent recruitment of
111In-labeled eosinophils (Figs. 1
and
2). There was no significant 111In-eosinophil
recruitment from 04 or 48 h, but a large number of cells were
recruited at the late time point of 2024 h (Fig. 1)
. In a similar
manner, the i.d. administration of MBSA in animals immunized with MBSA
in FCA (MBSA/FCA) resulted in a time- and dose-dependent recruitment of
111In-labeled eosinophils (Figs. 1
and 2)
. In this
reaction, 111In-eosinophil recruitment was already
noticeable from 48 h, but a significantly greater number of cells
were recruited in the 2024 h measurement period (Fig. 1)
. The
specificity of both reactions was assessed by the i.d. injection of the
relevant antigen in naive, Schistosoma egg- or
MBSA/FCA-immunized animals. As clearly demonstrated in Figure 2
, the
sensitization procedure imparted a great degree of specificity to the
reactions. A large number of 111In-eosinophils migrated to
sites injected with antigen in the relevant immunized animal but not in
naive animals or those immunized with the other irrelevant antigen
(Fig. 2)
. For comparison, the i.d. administration of LTB4,
an effective eosinophil chemoattractant [7
,
21
], induced a similar recruitment of
111In-eosinophils in naive and immunized animals, although
the response was substantially smaller (Fig. 2)
.

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Figure 1. Kinetics of the recruitment of 111In-eosinophils in Th1-
and Th2-predominant, DTH reactions. Animals that had been immunized
with MBSA/FCA (Th1) or S. mansoni eggs (Th2) received i.d.
injections of antigen (MBSA, 10 µg/site; SEA, 1 µg/site) at 20 h, 4 h, and immediately before the i.v. administration of
111In-eosinophils. LTB4 (150 pmol/site) was
given immediately before the 111In-eosinophils. The number
of 111In-eosinophils that migrated to skin sites was
assessed 4 h after their i.v. administration. Results are the
mean ± SE of six animals in each group.
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Figure 2. Dose- and antigen-dependent recruitment of
111In-eosinophils in Th1- and Th2-predominant, DTH
reactions. Animals that were not immunized (open bars) or that had been
immunized with MBSA/FCA (Th1, hatched bars) or S. mansoni
eggs (Th2, closed bars) received i.d. injections of antigen (MBSA, 1 or
10 µg/site; SEA, 1 or 10 µg/site) 20 h before the i.v.
administration of 111In-eosinophils. The effects of the
i.d. injection of PBS and LTB4 (150 pmol/site) immediately
before the i.v. administration of 111In-eosinophils are
shown for comparison. The number of 111In-eosinophils that
migrated to skin sites was assessed 4 h after their i.v.
administration. Results are the mean ± SE of six
animals in each group.
|
|
Histological studies of skin sites in naïve mice
The administration of SEA to S. mansoni, egg-immunized
animals induced a marked inflammatory infiltrate at 24 h in the
injected skin sites. The majority of the migrating cells was
mononuclear, but eosinophils comprised about 2030% of the
inflammatory infiltrate (Fig. 3C
and D). The administration of MBSA to animals
immunized with MBSA/FCA also induced a marked mononuclear cell
infiltrate in the injected skin sites (Fig. 3A
and 3B)
. Nevertheless,
and in contrast to the ability of 111In-labeled eosinophils
to migrate to sites of MBSA challenge, only occasional endogenous
eosinophils were noticed (Fig. 3B)
.

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Figure 3. Histological sections of skin sites of Th1- and Th2-predominant, DTH
reactions. Animals that had been immunized with MBSA/FCA (A and B) or
S. mansoni eggs (C and D) received i.d. injections of
antigen (MBSA, 10 µg/site; SEA, 1 µg/site, respectively) 24 h
prior to sacrifice. Note the marked inflammatory infiltrate seen in
both reactions at 24 h (A and C; original size, x100). In greater
magnification (original size, x400), note the large number of
infiltrating eosinophils in Th2 predominant, DTH reactions (D) but not
in Th1-predominant, DTH reactions (B).
|
|
Cytokine profile of spleen cells and challenged tissue of immunized
animals
Spleen cells were obtained from naive, S. mansoni, egg-
and MBSA/FCA-immunized animals and stimulated with Con A, MBSA, or SEA.
Con A-stimulated spleen cells from naive animals secreted a significant
amount of IFN-
and IL-10 but not IL-5 or IL-4 (Fig. 4
). Neither SEA nor MBSA induced significant cytokine secretion from
splenocytes of naive animals (Fig. 4)
. Upon stimulation with specific
antigen (SEA) or Con A, splenocytes from S. mansoni,
egg-immunized mice secreted significant amounts of IFN-
, IL-10, and
IL-5 (Fig. 4)
. Splenocytes from MBSA/FCA-immunized animals produced a
large amount of IFN-
spontaneously, but this was enhanced further in
the presence of Con A or specific antigen (MBSA; Fig. 4
). In splenocyte
cultures, little IL-4, IL-5, or IL-10 was produced in the presence of
MBSA (Fig. 4)
.

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Figure 4. Profile of the production of (a) IFN- , (b) IL-4, (c) IL-5, and (d)
IL-10 by splenocytes obtained from naïve animals or animals
immunized with MBSA/FCA or S. mansoni eggs. Splenocytes from
naïve or immunized animals were stimulated with vehicle (open
bars), Con A (5 µg/ml, closed bars), MBSA (10 µg/ml, hatched bars),
or SEA (5 µg/ml, stippled bars). Supernatants were collected for
cytokine measurements by ELISA 48 h (IL-4, IL-5, and IL-10) or
72 h (IFN- ) later. Results are representative of two similar
experiments and are the mean of triplicates from pooled cells of three
mice in each group.
|
|
The kinetics of the expression of mRNA for the Th2 cytokine IL-4 and
the Th1 cytokine IFN-
in skin sites of MBSA/FCA-immunized and
S. mansoni, egg-immunized and -challenged animals are shown
in Figure 5
. In MBSA/FCA-immunized animals, the challenge with specific
antigen induced a significant expression of IFN-
early (from 16 h)
in the course of the reaction and low-level expression of IL-4 at 3 and
24 h (Fig. 5a)
. The challenge of egg-immunized animals with SEA
induced low-level expression of IFN-
from 16 h and high levels of
IL-4 expression from 624 h after challenge (Fig. 5b)
. There was no
detectable expression of IL-4 or IFN-
in immunized animals
challenged with PBS, and injection of antigen in naive animals had
little effect on cytokine mRNA expression (unpublished results).

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Figure 5. Expression of mRNA for eotaxin, IL-4, and IFN- in (a) Th1- and (b)
Th2-predominant, DTH reactions. Mice were immunized with MBSA/FCA (Th1)
or S. mansoni eggs (Th2) and challenged i.d. with the
relevant antigen (MBSA, 10 µg/site; SEA, 1 µg/site, respectively).
Before (time 0) and at various times after challenge (1, 3, 6, and
24 h), skin sites from three animals were removed, pooled,
mRNA-extracted, and reverse-transcribed using specific primers for
HPRT, eotaxin, IFN- , and IL-4. The data are representative of two
similar experiments.
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|
Effects of the depletion of CD4+ and CD8+ cells
Pretreatment of animals with the anti-CD4 mAb GK1.3 resulted in
virtually complete depletion of circulating and spleen CD4+ cells in
S. mansoni, egg- and MBSA/FCA-immunized animals, as assessed
by flow cytometry (unpublished results). As seen in Figure 6
, anti-CD4 treatment abrogated the recruitment of
111In-eosinophils in SEA- and MBSA-induced DTH reactions.
In contrast, pretreatment with the anti-CD8 mAb R4-6A2, which depleted
CD8+ cells (unpublished results), had no significant effect on the
numbers of infiltrating 111In-eosinophils in both reactions
(Fig. 6)
. For comparison, neither antibody treatment had any
significant effect on LTB4-induced,
111In-eosinophil recruitment (Fig. 6c)
.

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Figure 6. Effects of the depletion of
CD4+ or CD8+ cells on the recruitment of
111In-eosinophils in Th1- and Th2-predominant, DTH
reactions. Control animals (open bars) and animals that had been
immunized with (a) MBSA/FCA (Th1) or (b) S. mansoni eggs
(Th2) were depleted of CD4+ (closed bars) or
CD8+ (hatched bars) cells. Skin sites were then challenged
i.d. with antigen (MBSA, 10 µg/site; SEA, 1 µg/site, respectively)
20 h before the i.v. administration of
111In-eosinophils. The effects of the i.d. injection of PBS
and (c) LTB4 (150 pmol/site) immediately before the i.v.
administration of 111In-eosinophils into control and
CD4+ or CD8+ cell-depleted animals are shown
for comparison. The number of 111In-eosinophils that
migrated to skin sites was assessed 4 h after their i.v.
administration. Results are the mean ± SE of six
animals in each group. *p < 0.01 when
compared with control animals.
|
|
Effects of the neutralization of IL-4 and IFN-
The next series of experiments assessed whether pretreatment with
blocking antibodies against IL-4 and IFN-
would affect the
recruitment of 111In-eosinophils in sites of late-onset
allergic reactions. The antibodies were given after the immunization
procedure and 1 h prior to challenge. As demonstrated in
Figure 7a
, pretreatment with anti-IL-4, but not anti-IFN-
, partially
inhibited 111In-eosinophil recruitment into sites of DTH
reactions induced by SEA in S. mansoni, egg-immunized
animals. Surprisingly, anti-IL-4 and anti-IFN-
abrogated the
recruitment of 111In-eosinophils in sites of DTH reactions
induced by MBSA in MBSA/FCA-immunized animals (Fig. 7b)
. Neither
antibody treatment affected the recruitment of
111In-eosinophils induced by the i.d. injection of
LTB4 (Fig. 7c)
.
Effects of the neutralization of eotaxin
We have shown previously that the chemokine eotaxin induced
significant 111In-eosinophil recruitment in murine skin and
was released endogenously in sites of allergic inflammation
[21
]. When the expression of eotaxin was assessed by
RT-PCR, significant message was detected early (from 16 h) in the
course of both reactions (Fig. 5)
. Moreover and in agreement with the
significant expression of eotaxin, an anti-eotaxin polyclonal antibody
used at a dilution (20% in PBS) previously shown to abrogate the
effects of eotaxin [21
], inhibited
111In-eosinophil recruitment in MBSA/FCA and egg/SEA DTH
reactions (Fig. 8
).

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Figure 8. Effects of the local pretreatment with anti-eotaxin antibody on the
recruitment of 111In-eosinophils in Th1- and
Th2-predominant, DTH reactions. Animals that had been immunized with
MBSA/FCA (Th1) or S. mansoni eggs (Th2) were challenged i.d.
with antigen (MBSA, 10 µg/site; SEA, 1 µg/site, respectively)
20 h before the i.v. administration of
111In-eosinophils. Control, pre-immune serum (20% dilution
in saline, open bars) or anti-eotaxin polyclonal antibody (20%
dilution in saline, closed bars) was injected i.d. just prior to the
i.v. administration of 111In-eosinophils. The number of
111In-eosinophils that migrated to skin sites was assessed
4 h after their i.v. administration. Results are the mean ±
SE of seveneight animals in each group.
*p < 0.05 when compared with sites treated
with control serum.
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Bone marrow eosinophils in S. mansoni, egg- and
MBSA/FCA-immunized animals
The results above demonstrate that endogenous eosinophils fail to
migrate into sites of Th1 predominant, MBSA/FCA reactions, whereas
these cells are found in abundance in S. mansoni,
egg-immunized and SEA-challenged animals (Fig. 3)
. In contrast,
exogenously added 111In-eosinophils appear to migrate to
both reactions upon challenge with specific antigen. To gain further
insights into the mechanisms explaining the recruitment of endogenous
eosinophils, we evaluated the total number of leukocytes and mature
eosinophils present in the bone marrow of MBSA/FCA and S.
mansoni, egg-immunized mice. As seen in Figure 9
, immunization with S. mansoni eggs induced a
significant increase in the total number of mature eosinophils in the
bone marrow of mice, whereas MBSA/FCA immunization enhanced the total
number of cells but failed to affect eosinophil numbers. The increase
in total leukocytes in the bone marrow of MBSA/FCA mice was explained
to some extent by an increase in the number of mature neutrophils
(unpublished results).

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Figure 9. Number of total cells and mature eosinophils in bone marrow obtained
from naïve animals or animals immunized with MBSA/FCA or
S. mansoni eggs. Left femurs were obtained from
naïve mice or mice that had been immunized with MBSA/FCA or
S. mansoni eggs six days before the experiment and prior to
challenge. Bone marrows were flushed with RPMI as described in
Materials and Methods, and the number of (a) total leukocytes and (b)
mature eosinophils was evaluated. Results are the mean ±
SE of five animals in each group.
*p < 0.05 when compared with control.
|
|
 |
DISCUSSION
|
|---|
The recruitment of activated eosinophils into skin sites appears
to play a major role in the pathophysiology of allergic disorders
[1
2
3
]. The recruitment of eosinophils to sites of
inflammation in vivo is largely dependent on the local
expression of chemoattractant molecules (e.g., chemokines and
LTB4) and CAMs [4
]. Nevertheless, there is
some evidence to support a role for IL-5-driven blood eosinophilia in
determining eosinophil recruitment in sites of inflammation
[8
9
10
, 28
, 29
]. In the
present study, we have investigated whether the IL-5-driven blood
eosinophilia plays a determinant role for the recruitment of
eosinophils in vivo. To evaluate this central role of primed
blood eosinophils for eosinophil recruitment in vivo,
radiolabeled eosinophils purified from the blood of IL-5 transgenic
mice were injected into nontransgenic, immunized mice, and their
recruitment was assessed into discrete sites of cutaneous inflammation
following challenge with antigen.
Two immunization procedures were used in the experiments described
herein. A set of animals was immunized with S. mansoni egg
and challenged with SEA, and another set of animals was immunized with
MBSA in CFA and challenged with MBSA [22
23
24
]. In the
former reaction, a significant proportion of endogenous migrating cells
were eosinophils (2030%), and splenocytes cultured in the presence
of specific antigen produced significant amounts of IL-4, IL-5, IL-10,
and IFN-
, characterizing a Th0/2 phenotype. It is unclear whether
the production of IFN-
, in addition to Th2-type cytokines, by
SEA-activated lymphocytes is important for the eosinophil migration
observed or just represents the great ability of parasite antigens to
stimulate the immune system. However, these results are in marked
agreement with inflammatory reactions induced in other tissues by a
similar immunization and challenge procedure [24
,
30
]. In the MBSA-induced, DTH reaction, a marked
inflammatory infiltrate characterized by the infiltration of
mononuclear cells was observed, and very rare eosinophils or
neutrophils were seen throughout the lesions at 24 h. Splenocytes
from these animals produced a significant amount of IFN-
spontaneously, and this was increased in response to specific antigen.
In contrast to the findings for endogenous eosinophils described above,
111In-eosinophils migrated into sites of MBSA/FCA,
delayed-onset, hypersensitivity reactions in an antigen- and
CD4-dependent manner. Similarly, in animals immunized with S.
mansoni eggs and challenged with SEA,
111In-eosinophils were also recruited in an antigen- and
CD4-dependent manner. Depletion of CD8+ cells was not
accompanied by any significant change of 111In-eosinophil
migration. These data are in good agreement with other studies
demonstrating a fundamental role for CD4+ T cells in the
control of eosinophil migration in sites of late-onset, allergic
inflammation [18
, 31
, 32
] and
demonstrate that, when present in sufficient amounts in blood,
eosinophils derived from IL-5-transgenic animals migrate to sites of
both Th1 and Th2-predominant reactions.
The challenge of S. mansoni, egg-immunized animals with SEA
induced the local expression of IL-4 mRNA. In agreement with this
finding, pretreatment of these animals with anti-IL-4 mAb was
accompanied by a significant inhibition of 111In-eosinophil
recruitment after challenge. In contrast, little IFN-
was expressed
after i.d. challenge with SEA, and treatment with anti-IFN-
mAb had
little effect on 111In-eosinophil migration. A central role
for IL-4 in mediating eosinophil influx into the lung or skin in
Th2-predominant, allergic inflammation has also been described
previously [33
, 34
]. In MBSA/FCA-immunized
animals, there was a significant expression of IFN-
early in the
course of the reaction, and treatment with anti-IFN-
mAb abrogated
111In-eosinophil recruitment into skin sites of challenged
animals. Moreover, pretreatment with anti-IL-4 also abrogated
111In-eosinophil recruitment in MBSA/FCA animals challenged
with specific antigen. This was an unexpected finding, inasmuch as
splenocytes from these animals produced little IL-4 upon stimulation
with specific antigen, and previous studies have demonstrated the
dependence of this Th1-predominant reaction on IL-12 production
[23
]. Nevertheless, it is worth noting that, albeit in
low levels, IL-4 mRNA was detected following challenge of immunized
animals with MBSA. Furthermore, a role for IL-4 in mediating eosinophil
influx in sites of contact hypersensitivity reactions has been
described previously [35
]. Overall, these studies argue
for an essential role for IL-4 in mediating eosinophil recruitment
following antigen challenge in cutaneous hypersensitivity reactions.
Moreover, they imply that mechanisms, in addition to the local
expression of IL-4, drive the specific recruitment of endogenous
eosinophils observed in both hypersensitivity reactions. The means by
which IL-4 drives eosinophil recruitment in our model has not been
investigated in detail. Nevertheless, our previous studies suggest that
the modulation by IL-4 of the expression of very late antigen (VLA)-4
ligands, such as vascular cell adhesion molecule 1 (VCAM-1), may
underlie the ability of this cytokine to regulate eosinophil
recruitment in mouse skin [22
]. Alternatively, IL-4 may
be driving the local production of eosinophil chemoattractants,
specially eotaxin [17
, 36
,
37
], by skin cells (e.g., fibroblasts), which may then
drive local eosinophil recruitment.
We and others have demonstrated previously the important role of the
chemokine eotaxin for the recruitment of eosinophils into sites of
allergic inflammation in vivo [21
,
38
39
40
]. Here, we show that eotaxin mRNA is expressed in
late-onset hypersensitivity reactions and, more importantly, that an
anti-eotaxin polyclonal antibody partially inhibits eosinophil
recruitment when administered just prior to antigen challenge. These
experiments are in agreement with previous studies demonstrating the
expression of eotaxin by Th1 and Th2 lymphocytes [41
].
Overall, these results argue that the local expression of the chemokine
eotaxin cannot explain the differential recruitment of endogenous
eosinophils observed in MBSA/FCA- and S. mansoni-immunized
and -challenged mice.
Next, we investigated whether the availability of mature
eosinophils in bone marrow and blood could provide an explanation for
the specificity of the migration of endogenous eosinophils to sites of
Th2-, but not Th1-predominant, DTH reactions. Our results clearly
show that, in mice immunized with S. mansoni eggs, there is
an increase in the number of eosinophils in the bone marrow. In
contrast, in MBSA/FCA-immunized mice, neutrophil, but not eosinophil,
numbers increased. These results demonstrate a correlation between
availability of eosinophils in bone marrow and the ability of
endogenous eosinophils to migrate into sites of DTH reactions. When
exogenous, IL-5-derived eosinophils are added, these cells are readily
available for migration in vivo. In support of this
hypotheses, Nagai et al. [42
] have shown
previously that the overproduction of IL-5 enhanced eosinophil
migration in a model of dinitrofluorobenzene-induced contact
hypersensitivity. Together, these results corroborate the idea that the
bone marrow may play an active role in the recruitment of eosinophils
in vivo [43
].
In conclusion, we demonstrate the ability of exogenously added,
IL-5-primed eosinophils to migrate into sites of late onset Th1- or
Th2-predominant reactions in vivo. Exogenous eosinophil
migration was CD4+ T cell- and IL-4-dependent and partially
blocked by an anti-eotaxin antibody. Nevertheless, endogenous
eosinophils only migrated when found in increased numbers in the bone
marrow [present data; 42], suggesting an important role for the bone
marrow in controlling eosinophil migration in vivo.
Strategies aimed at modulating eosinophil production and/or release
from the bone marrow may be of benefit in the therapy of
eosinophil-associated diseases, such as asthma and atopic dermatitis.
 |
ACKNOWLEDGEMENTS
|
|---|
We are grateful for the National Asthma Campaign, Novartis
(Switzerland), Fapemig, and CNPq for financial support. The helpful
comments of Dr. Sefik Alkan (Novartis) are greatly acknowledged.
Received July 21, 2000;
revised September 15, 2000;
accepted September 20, 2000.
 |
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