* 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, 6627—Pampulha, 31270-901 Belo Horizonte MG Brasil. E-mail: mmtex{at}icb.ufmg.br
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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
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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.
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Reagents
Methylated bovine serum albumin (MBSA), concavalin A (Con A),
Dextran, Percoll, and Freund’s complete adjuvant (FCA) were purchased
from Sigma Chemical Co. (Poole, UK); Dulbeccos’s 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 (three–four 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 (0.1–10.0 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, 0.1–10
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 (0–4, 4–8, and 20–24 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 2–43 (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
(1–100 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
Hank’s buffered salt solution containing 0.25% BSA. Samples were spun
and cell numbers determined in a Neubauer haemocytometer after dilution
of samples in Turk’s 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.
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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.
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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).
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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)
.
 View larger version (38K): [in a new window] |
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.
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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 1–6 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 1–6 h and high levels of
IL-4 expression from 6–24 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).
 View larger version (37K): [in a new window] |
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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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.
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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)
.
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Figure 7. Effects of the pretreatment with anti-IL-4 or anti-IFN- antibodies
on the recruitment of 111In-eosinophils in Th1- and
Th2-predominant, DTH reactions. Control animals (open bars) and animals
that were immunized with (a) MBSA/FCA (Th1) or (b) S.
mansoni eggs(Th2) were pretreated with anti-IL-4 (closed
bars) or anti-IFN- (hatched bars) mAbs just prior to the challenge.
Skin sites 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. 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
antibody-treated 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 < 0.05, and
**P < 0.01 when compared with
control animals.
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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 seven–eight animals in each group.
*p < 0.05 when compared with sites treated
with control serum.
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 View larger version (18K): [in a new window] |
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.
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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 (20–30%), 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.
Received July 21, 2000; revised September 15, 2000; accepted September 20, 2000.
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