© Birkhäuser Verlag, Basel, 2001
Inflamm. res. 50 (2001) 309– 316
1023-3830/01/060309-8 $ 1.50+0.20/0
Mechanisms of allergen- and LPS-induced bone marrow eosinophil
mobilization and eosinophil accumulation into the pleural cavity:
a role for CD11b/CD18 complex
A. P. Larangeira 1, A. R. Silva1, R. N. Gomes1, C. Penido1, M.G. M. O. Henriques 2,
H. C. Castro-Faria-Neto1 and P. T. Bozza1
1 Laboratório de Imunofarmacologia, Departamento de Fisiologia e Farmacodinâmica, IOC, Fundação Oswaldo Cruz, Av. Brasil 4365,
Manguinhos, Rio de Janeiro, RJ, CEP 21045-900, Brazil, Fax: 55 21 5909490, e-mail: pbozza@gene.dbbm.fiocruz.br
2
Laboratório de Farmacologia Aplicada, Instituto de Tecnologia Farmacêutica, FarManguinhos, Fundação Oswaldo Cruz, Rio de Janeiro, Brazil
Received 21 July 2000; returned for revision 13 September 2000; accepted by G. Letts 10 January 2001
Abstract. Objective: The mechanisms involved in bone mar-
row eosinophil emigration and recruitment to inflammatory
sites are not fully understood. The involvement of
CD11b/CD18 in marrow eosinophil release induced by
lipopolysaccharide (LPS) or allergen was investigated in
mice.
Methods: Eosinophil and neutrophil counts in the pleural
cavity, blood and bone marrow were performed at different
time intervals after the intrathoracic injection of LPS (250 ng/
cavity) or ovalbumin (OVA, 12 mg/cavity; into actively sen-
sitized mice) and compared to anti-CD11b/CD18 (5C6, 1 mg/
mouse) or anti-IL-5 (TRFK-5, 500 mg/kg) treated mice.
Results: LPS induced local eosinophil influx, that peaked
within 24 h and that was preceded by a decrease in marrow
eosinophils at 4 h. Antigenic challenge induced a decrease in
marrow eosinophils within 4 h, followed by a long lasting
pleural eosinophil accumulation and a persistent increase in
marrow eosinophil numbers. Pretreatment with anti-
CD11b/CD18 abolished LPS-induced neutrophil and eosino-
phil accumulation in the pleural cavity at 4 and 24 h, respec-
tively. This pretreatment failed to modify neutrophil emigra-
tion from bone marrow, but significantly inhibited marrow
eosinophil release at 4 h post-LPS or OVA challenge. Anti-
IL-5 pretreatment failed to inhibit LPS-induced pleural eo-
sinophil accumulation and mobilization from bone marrow,
but it abolished allergen-induced effects, indicating a role for
IL-5 in marrow eosinophil mobilization induced by antigen,
but not by LPS challenge.
Conclusions: Our results suggest that eosinophil migration
induced by antigen or LPS into the pleural cavity is preceded
by bone marrow eosinophil release through a mechanism that
depends on CD11b/CD18.
Key words: Eosinophils – Cell trafficking – Bone marrow –
Adhesion molecules
Correspondence to: P. T. Bozza
Inflammation Research
Introduction
Leukocyte migration from the circulation into surrounding
tissues is a critical stage of the inflammatory process, neces-
sary to host defense against pathogens. This process involves
sequential events that have been described as: leukocyte and
endothelial cell activation by chemoattractants, rolling of
leukocytes on vascular endothelium via selectin interaction
with carbohydrate ligands, firm adhesion mediated by inter-
action between integrins and their ligands and transendothe-
lial migration [1, 2].
The availability of cells in the circulation is also an
important regulatory factor for the process of leukocyte
accumulation into inflammatory sites. In this respect, the
bone marrow pool of leukocytes plays a crucial role by pro-
viding the circulation with a great number of newly gener-
ated cells. Indeed, approximately half of the nucleated cells
from human or rodent bone marrow consists of mature gra-
nulocytes, which are able to rapidly emigrate from the mar-
row upon stimulation [3–5]. Different factors have an in-
fluence in the egress of cells from bone marrow, such as: the
structural organization of the bone marrow, the action of leu-
kocyte-activating agents (as cytokines, chemokines and lipid
mediators) and adhesive interaction [6, 7]. It has been
demonstrated that granulocyte emigration is a highly selec-
tive process that can be obtained without the concomitant
release of other cell types, such as monocytes, erythrocytes
or platelets [8, 9]. Even among granulocytes, the selectivity
can be seen, since eosinophil marrow release can be elicited
by IL-5 or eotaxin without neutrophil egress [10]. Several
lines of evidence suggest a key role for adhesive interaction
in leukocyte egress from bone marrow. Adhesion molecules
may affect granulocyte release, either by anchoring granu-
locytes to the extracellular matrix (ECM) or to endothelial
cells, in order to allow cell adhesion and migration [11, 12].
Recently, a role for b2 integrins in eosinophil marrow release
has been suggested after the observation of increased expres-
sion of b2 integrins on IL-5-recruited marrow eosinophils
310
and after the observation of the inhibition of IL-5-induced
marrow release by anti-CD18 blocking antibody [13].
In the present study, we have investigated the role of the
b2 integrin, CD11b/CD18 (aMb2 , Mac-1, CR3), on marrow
eosinophil release in vivo triggered by the intrathoracic
administration of LPS or allergen. As a strategy, we blocked
the interaction between CD11b/CD18 and its ligand by the
use of a monoclonal antibody to CD11b/CD18. In order to
further delineate the involvement of CD11b/CD18 complex
in this model, the eosinophil alterations in the pleural cavity,
in blood and in bone marrow were analyzed and compared to
the alterations observed in the neutrophil counts in all three
compartments. We provided evidence for a critical role of b2
integrins in egress of marrow eosinophil, but not in marrow
neutrophil, induced either by LPS or antigen.
Materials and methods
Animals
Male Swiss mice (Oswaldo Cruz Foundation breeding unit) weighing
20 to 25 g were used. The animals were kept at a constant temperature
(25 °C) with free access to pelleted diet and water in a room with a
12-h light/dark cycle. Animals are maintained and treated according to
the animal care guidelines of the Council for International Organiza-
tions of Medical Sciences and the Brazilian College of Animal Exper-
imentation (COBEA).
Pleurisy
Pleurisy was induced in isofluorene (Forane™, Abbott, Brazil) anes-
thetized mice through the intrathoracic (i.t) injection of LPS
(250 ng/cavity) in a final volume of 0.1 ml. Each experiment included a
control group receiving the same volume of vehicle (sterile saline). Al-
lergic pleurisy and animal sensitization were performed as previously
described [14]. In brief, mice were subcutaneously immunized with a
mixture of ovalbumin (50 mg) and aluminium hydroxide (5 mg), in a
final volume of 200 ml. Allergic pleurisy was induced in ovalbumin-sen-
sitized mice by intrathoracic injection of 0.1 ml OVA (12 mg) 14 days
after sensitization. Sensitized animals that received saline were used as
negative control. The animals were killed in a CO2 chamber at different
timepoints after the challenge. The thoracic cavity was opened and rin-
sed with 1 ml of saline containing heparin (10 UI/ml). The pleural wash
was collected and its volume evaluated with a graduated syringe.
Blood and bone marrow analysis
Blood samples were obtained from the tail vein at different timepoints
after i.t. injection of LPS, allergen or saline. Bone marrow cells were
isolated from the right femur. The femoral head and condyles were
removed, and displaceable cells were recovered by flushing the marrow
cavity of the femur shaft with 3 ml of heparinized minimum essential
medium (MEM – pH 7.45).
Leukocyte analysis
Total leukocyte counts from blood, pleural wash and bone marrow were
performed in Neubauer chambers under optical microscope, after dilut-
ing the samples in Türk solution (2% acetic acid). Differential leukocyte
counts in bone marrow and pleural wash samples were performed on
cytospin smears stained by the May-Grünwald-Giemsa method. Differ-
ential counts in blood samples were performed on blood smears stained
as described for the cytospin preparation.
A. P. Larangeira et al. Inflamm. res.
Treatments and drugs
Lipopolysaccharide from E. coli (serotype 0127 :B8), ovalbumin (type
III) and minimum essential medium (MEM) were obtained from Sigma
(St Louis, MO). Anti-CD11b/CD18 mAb (5C6; Celltech LTD – Slough,
UK) or purified rat IgG control were intravenously administrated (i. v.)
10 – 1000 mg/animal 5 min before stimulus [15, 16]. The degree of 5C6
mAb binding to bone marrow cells was estimated by fluorescence
analysis in a FASCalibur flow cytometer (Becton Dickinson, San Jose,
CA). Animals were pretreated with 5C6 or IgG control as described
above and bone marrow analysis was assessed 4 h after LPS adminis-
tration. Samples of 106 cells recovered from one femur were labeled
with FITC conjugated mAb to rat IgG for 30 min at 4 °C after incuba-
tion with mouse serum to block non-specific binding. Cells were then
washed with PBS/0.1% azide and analysis performed using the Cell
Quest program. At least 104 bone marrow cells were acquired per sam-
ple. To determine the percentages of 5C6 binding, myelomonocytic
cells were gated and non specific labeling was discounted by the sub-
traction of values from rat IgG control injected animals. Neutralizing
anti-IL-5 mAb (500 mg/kg; TRFK5; Pharmigen, San Diego, CA) was
intraperitoneally administered 30 min before the i.t. injection of LPS or
allergen. Purified rat IgG was used as irrelevant antibody.
Statistical analysis
Data are reported as the mean SEM and were statistically analyzed by
means of analysis of variance, followed by Newman-Keuls-Student test,
with the level of significance set at p < 0.05.
Results
Kinetics of LPS-induced neutrophil and eosinophil
mobilization to the mice pleural cavity
The i.t. injection of LPS (250 ng/cavity) induced a marked
neutrophil influx into the mice pleural cavity within 4 to 24 h
(Fig. 1A). After 24 h, neutrophil numbers started to decline,
whereas the eosinophil counts reached maximum values
(Fig. 2A), returning to basal levels after 96 h (not shown).
Pleural neutrophil accumulation was accompanied by an
increase in neutrophil counts in the peripheral blood at 4
(Fig. 1B) and 12 h (not shown). No changes in blood eosino-
phil counts were observed at any of the timepoints analyzed
(Fig. 2B). At 4 h, the numbers of both neutrophils and eo-
sinophils present in the bone marrow were significantly
decreased if compared to those in the control group (Fig. 1C
and 2C, respectively), suggesting that these cells were being
released from bone marrow to blood before migrating into
the pleural cavity.
Kinetics of OVA-induced eosinophil mobilization to the
pleural cavity of actively sensitized mice
As shown in figure 3A, the i.t. injection of OVA (12 mg/cav-
ity) into actively sensitized animals induced a significant
and long-lasting pleural eosinophil accumulation, that could
still be observed 7 days after challenge. The sensitization
procedure and antigenic challenge induced marked alterati-
ons in the bone marrow eosinophil population (Fig. 3C). The
sensitization process induced a significant increase of mar-
row eosinophil counts, if compared to those in non-sensitized
Vol. 50, 2001 CD11b/CD18 in marrow eosinophil release
Fig. 1. Temporal analysis of neutrophil count alterations in the pleural
cavity (A), blood (B), and bone marrow (C) induced by i.t. injection of
LPS (250 ng/cavity). Results are expressed as the mean ± SEM from at
least eight animals. Statistically significant differences (p ≤ 0.05) be-
tween saline and LPS-stimulated groups are indicated by an asterisk.
animals (1.78 ± 0.19 eosinophils ¥ 106/ femur in non-sen-
sitized animals vs. 2.72 ± 0.25 eosinophils ¥ 106/femur in
sensitized animals, n = 8, p < 0.05). The antigenic challenge
in actively sensitized animals induced a biphasic change in
bone marrow eosinophil numbers. Initially, a significant
decrease in eosinophil marrow pool was observed at 4 and
12 h post-challenge (Fig. 3C), probably related to the mobi-
lization of marrow eosinophils to periphery (Fig. 3B). This
decrease in the marrow eosinophil content was followed by
an increase in the marrow eosinophil numbers within 24 h,
which was still observed 7 days after antigenic challenge
(Fig. 3C).
311
Fig. 2. Temporal analysis of eosinophil count alterations in the pleural
cavity (A), blood (B), and bone marrow (C) induced by i.t. injection of
LPS (250 ng/cavity). Results are expressed as the mean ± SEM from at
least eight animals. Statistically significant differences (p ≤ 0.05) be-
tween saline and LPS-stimulated groups are indicated by an asterisk.
Effect of anti-CD11b/CD18 mAb on LPS-induced neutro-
phil and eosinophil mobilization to the mice pleural cavity
To analyze the involvement of CD11b/CD18 on neutrophil
migration to the pleural cavity, mice were pretreated with an
i.v. injection (1 mg/animal) of a blocking monoclonal anti-
body (5C6) against this b2 integrin, 5 min before LPS ad-
ministration. The pretreatment with anti-CD11b/CD18 in-
hibited pleural neutrophil accumulation at 4 h (not shown)
and 24 h (Fig. 4A). Similarly, the pleural eosinophil ac-
cumulation induced by LPS at 24 h was abolished by anti-
CD11b/CD18 pretreatment (Fig. 4B).
312
Fig. 3. Temporal analysis of eosinophil count alterations in the pleural
cavity (A), blood (B), and bone marrow (C) induced by i.t. injection of
ovalbumin (12 mg/cavity) in actively sensitized mice. Results are expres-
sed as the mean ± SEM from at least eight animals. Statistically signi-
ficant differences (p ≤ 0.05) between sensitized mice stimulated with
saline and ovalbumin-stimulated groups are indicated by an asterisk.
Effect of anti-CD11b/CD18 mAb on LPS-induced marrow
eosinophil and neutrophil mobilization
The role of b2 integrin in marrow eosinophil and neutrophil
mobilization induced by LPS challenge was investigated.
Figure 5A shows the degree of anti-CD11b/CD18 mAb
(10–1000 mg per animal) binding to bone marrow cells as
assessed 4 h after a single injection. Saturation binding to
myelomonocytic cells was seen at doses of 100–1000 mg per
animal, with 61% and 77% binding, respectively.
A. P. Larangeira et al. Inflamm. res.
Fig. 4. Effect anti-CD11b/CD18 (1mg/mouse) treatment on neutrophil
(A) or eosinophil (B) count alterations in the pleural cavity induced
24 h after i.t injection of LPS (250 ng/cav). Results are expressed as the
mean ± SEM from at least eight animals. Statistically significant differ-
ences (p ≤ 0.05) between saline and LPS-stimulated groups are indicat-
ed by an asterisk, whereas (+) indicates differences between treated and
untreated animals stimulated with LPS.
Interestingly, the administration of anti-CD11b/CD18
dose-dependently inhibited the decrease of eosinophil num-
bers in the bone marrow, as observed 4 h after the LPS ad-
ministration (Fig. 5B). Nevertheless, anti-CD11b/CD18 fail-
ed to modify LPS-induced blood neutrophilia (not shown) or
bone marrow neutrophil mobilization (Fig. 5C). It should be
remarked that the i.v. administration of anti-CD11b/CD18 to
naive mice did not change basal levels of leukocytes in the
pleural cavity, blood or bone marrow (not shown). In addi-
tion, pretreatment with irrelevant mAb (purified rat IgG)
failed to modify LPS-induced changes in pleural, blood or
bone marrow granulocyte counts (not shown and Fig. 5).
Effect of anti-CD11b/CD18 mAb on allergen-induced
marrow eosinophil mobilization
Differently from the observed for LPS, the anti-
CD11b/CD18 pretreatment only slightly affected the pleural
eosinophil accumulation induced by OVA, suggesting that
other molecules are involved in OVA-induced eosinophil
migration to tissues (Fig. 6A). However, the administration
of anti-CD11b/CD18 significantly inhibited the decrease of
eosinophil numbers in the bone marrow, as observed 4 h after
OVA injection into actively sensitized mice (Fig. 6B).
Vol. 50, 2001 CD11b/CD18 in marrow eosinophil release
Fig. 5. Dose-dependent increase in anti-CD11b/CD18 (5C6 mAb,
10–1000 mg/mouse) binding to bone marrow cells (A). The degree of 5C6
mAb binding to bone marrow cells was estimated by fluorescence analy-
sis in a FASCalibur flow cytometer. Results were presented as percenta-
ges of anti-CD11b/CD18 binding to myelomonocytic cells and non spe-
cific labeling was discounted by the subtraction of values from rat IgG
control injected animals. Dose-dependent effect of anti-CD11b/CD18
(10–1000 mg/mouse) on eosinophil (B), but not neutrophil (C) bone mar-
row mobilization induced 4 h after i.t injection of LPS (250 ng/cav).
Results are expressed as the mean ± SEM from at least eight animals. Sta-
tistically significant differences (p ≤ 0.05) between saline and LPS-
stimulated groups are indicated by an asterisk, whereas (+) indicates
differences between treated and untreated animals stimulated with LPS.
313
Fig. 6. Effect of anti-CD11b/CD18 (1mg/mouse) treatment on eosino-
phil recruitment to pleural cavity (A) or mobilization from the bone
marrow (B) induced respectively at 24 or 4 h after i.t injection of oval-
bumin in actively sensitized mice. Results are expressed as the mean
± SEM from at least eight animals. Statistically significant differences
(p ≤ 0.05) between saline and stimulated mice are indicated by an aster-
isk, whereas (+) indicates differences between treated and untreated ani-
mals stimulated with ovalbumin.
Effect of anti-IL-5 mAb on LPS- and allergen-induced
marrow eosinophil mobilization
IL-5 has been identified as a key mediator in the develop-
ment of eosinophilic reactions. IL-5 does not only induce
eosinophil proliferation, differentiation and activation [17,
18], but also implies the emigration of eosinophils from bone
marrow [10, 19, 20]. We have previously demonstrated that
LPS-induced pleural eosinophil accumulation does not
depend on IL-5, since neutralizing antibodies to IL-5 were
shown to completely abrogate the mouse pleural eosinophilia
triggered by antigen, but not by LPS [14]. In order to further
study the mechanisms of marrow eosinophil release, we have
evaluated the effect of IL-5 neutralization on marrow eosino-
phil emigration induced either by LPS or antigenic chal-
lenge. As shown in Fig. 7, the administration of anti-IL-5
(TRFK-5, 500 mg/kg, i.p.) significantly inhibited the OVA-
induced marrow eosinophil release observed within 4 h,
whereas it failed to inhibit the marrow eosinophil mobiliza-
tion induced by LPS. The administration of purified rat IgG
314
Fig. 7. Effect of anti-IL-5 treatment (TRFK-5, 500 mg/kg) on eosino-
phil mobilization from bone marrow induced 4 h after i. t injection of
LPS (A) or ovalbumin in actively sensitized mice (B). Results are
expressed as the mean ± SEM from at least eight animals. Statistically
significant differences (p ≤ 0.05) between saline and stimulated mice
are indicated by an asterisk, whereas (+) indicates differences between
treated and untreated animals stimulated with LPS or ovalbumin.
had no effect on OVA- (1.45 ± 0.16 ¥ 106/femur in untreated
mice vs. 1.35 ± 0.24 ¥ 106/ femur in treated mice) or LPS-
induced marrow eosinophil mobilization (1.10 ± 0.11 ¥ 106/
femur in untreated mice vs. 1.48 ± 0.16 ¥ 106/femur in treat-
ed mice).
Discussion
Cell mobilization from the bone marrow is a paramount ear-
ly step in leukocyte trafficking during inflammation. In the
present study, we have investigated the role of the b2 integrin
CD11b/CD18 in bone marrow eosinophil emigration induc-
ed by two distinct and differentially regulated inflammatory
reactions triggered either by antigenic challenge or by LPS.
We have demonstrated that i.t. injection of LPS induces a
significant neutrophil infiltration followed by a late
mononuclear cell and eosinophil accumulation in the pleural
cavity of rats and mice [14, 21]. As previously observed in
rats, the LPS-induced pleural neutrophil influx occurs simul-
taneously to an increase in blood neutrophil counts and a
drop in the number of these cells in the bone marrow. Thus,
A. P. Larangeira et al. Inflamm. res.
suggesting that neutrophil accumulation is caused by mobi-
lization of neutrophils from bone marrow to blood, followed
by neutrophil transmigration into the pleural cavity after LPS
administration [22, 23]. Although we could not observe
blood eosinophil alterations after i.t. LPS administration, the
eosinophil accumulation also seems to depend on the bone
marrow pool of eosinophils. Indeed, the i.t. injection of LPS
induced an acute (4 h) release of eosinophils from the bone
marrow, followed by a pleural eosinophil accumulation with-
in 24–48 h.
Eosinophil tissue infiltration is a hallmark feature of the
allergic response. The i.t. injection of OVA into actively sen-
sitized mice caused a marked and long-lasting eosinophil
infiltration, that peaked at 48 h, with a selective circulating
eosinophilia. Both local and systemic eosinophilia persisted
until 120 h. Interestingly, OVA-challenge induced a biphasic
reaction of changes in the marrow eosinophil population. The
first phase was characterized by a drastic reduction in the
number of mature eosinophils in the bone marrow, as obser-
ved at 4 and 12 h post-challenge, indicating an acute marrow
eosinophil mobilization. Accordingly, the drop in the marrow
eosinophil population was followed by a significant and per-
sistent increase in marrow eosinophil numbers. In agree-
ment, several studies have indicated a strong association of
atopic disease with bone marrow response to allergen.
Indeed, myeloid progenitor cells are increased in response to
antigenic challenge in different experimental models [24].
Moreover, higher numbers of eosinophil-basophil colony-
forming units have been consistently detected in atopic and
asthmatic subjects and seem to play a role in the development
of allergic inflammation [25, 26]. Several pieces of evidence
suggest that recruitment of eosinophils during allergic reac-
tions requires a multi-mediated process of at least two com-
bined components [27, 28]. These include an IL-5-driven
systemic component, associated to in situ events working in
parallel, such as adhesion and locomotion, that are predomi-
nantly mediated by local generated factors including lipid
mediators, bradykinin and chemokines [19, 29–33]. Indeed,
we observed a significant inhibition of OVA-induced acute
marrow eosinophil release by the pretreatment with anti-IL-
5, in line with previous reports [20, 32].
In despite of the fact that in allergic models IL-5 has an
essential role in eosinophilic differentiation and mobilization
from bone marrow and in regulating eosinophil migration
into tissues [19, 32], we have demonstrated that LPS-induced
pleural eosinophil migration occurs through an IL-5 in-
dependent mechanism. Indeed, anti-IL-5 mAb (TRFK-4/
TRFK-5) inhibited pleural eosinophil accumulation induced
by antigen in actively sensitized mice but not by LPS [14].
Moreover, the i.t. administration of LPS significantly in-
duced eosinophil accumulation in IL-5 genetically deficient
animals (Pelled et al., unpublished observations). In agree-
ment, we have now demonstrated that anti-IL-5 treatment
failed to inhibit LPS-induced marrow eosinophil recruit-
ment, indicating that other factors beside IL-5 are capable of
controlling eosinophil egress from bone marrow.
Many reports have demonstrated the critical involvement
of b2 integrins, mainly CD11b/CD18, in neutrophil-endo-
thelial cell adhesion and recruitment into inflamed tissues in
different experimental models [15, 16, 34–37]. In order to
investigate the involvement of b2 integrin CD11b/CD18 on
Vol. 50, 2001 CD11b/CD18 in marrow eosinophil release
LPS-induced leukocyte accumulation in the mouse pleural
cavity, we used an anti-CD11b/CD18 mAb (5C6), which
blocked the a subunit (CD11b) of this complex. The pre-
treatment with anti-CD11b/CD18 drastically inhibited LPS-
induced neutrophil as well as eosinophil accumulation in the
mouse pleural cavity. In agreement, it has been demonstrated
that pulmonary neutrophil accumulation induced either by
aerolized or i.v. LPS is suppressed by mice pretreatment with
anti-CD11b/CD18, suggesting that b2 integrins, specifically
CD11b/CD18, play an important role in LPS-induced neu-
trophil accumulation [16]. Integrins are pleiotropic molec-
ules that might be involved in different stages of LPS-in-
duced cell activation and recruitment [38, 39]. b2 integrins
may act in cellular signaling and, like CD14, may be involved
in LPS recognition [40–42]. However, anti-CD11bCD18
treatment failed to block the release of LPS-induced eosino-
philotatic factors (data not shown), indicating that anti-
CD11b/CD18 blocked LPS-induced eosinophil mobilization
by directly inhibiting the eosinophil migration process but
not by preventing LPS recognition or signaling through b2
integrins.
Several pieces of evidence suggest an important role for
adhesion molecules in controlling leukocyte egress from
bone marrow. These molecules participate in cell migration
and/or cell anchoring to prevent their release into the blood
stream [7, 11, 43]. Interestingly, pretreatment with anti-
CD11b/CD18 significantly inhibited LPS-induced marrow
eosinophil mobilization, whereas it had no effect on neutro-
phil emigration. The lack of involvement of CD11b/CD18 on
marrow neutrophil release is in agreement with previous
reports that show that neutrophilia induced by several stim-
uli, including LPS, is not impaired by inactivation of
CD11b/CD18, as observed either in anti-CD11b/CD18 treat-
ed animals or in genetically deficient animals [15, 16, 44].
Although the mechanisms of LPS-induced neutrophil emi-
gration from bone marrow are not fully understood, PAF and
a1-adrenoceptors seem to be involved [22, 23]. To determine
whether b2 integrins are generally involved in acute bone
marrow eosinophil mobilization or whether it is a character-
istic of LPS-induced eosinophilia, we evaluated the effect of
anti-CD11b/CD18 in OVA-induced eosinophil marrow
egress and accumulation into the pleural cavity. The treat-
ment with anti-CD11b/CD18 also inhibited marrow eosino-
phil mobilization induced by OVA. However, differently
from what has been observed for LPS, OVA-induced pleural
eosinophil accumulation was partially but not significantly
inhibited by this treatment, suggesting that other adhesion
molecules are more important for tissue eosinophil migration
in the allergic response. Indeed, many reports have demon-
strated a critical role for the b1 integrin VLA-4 and its coun-
terligand VCAM-1 in supporting firm adhesion and trans-
migration of eosinophils during allergic reactions [ 45–48].
Our results indicating that b2 integrins are important
molecules in mediating eosinophil interactions in bone mar-
row are supported by recent findings by Palframan et al. [13],
demonstrating that b2 integrins are upregulated in marrow
eosinophils after IL-5 stimulation. Moreover, IL-5-induced
ex vivo marrow eosinophil release, using a marrow perfusing
system, also depended on CD11b/CD18 [13]. Although IL-
5-induced b2 integrin upregulation in marrow eosinophils
may play an important role in the recruitment of these cells
315
during allergic inflammation, other cytokine/mediators
might have overlapping functions, since IL-5 is not signifi-
cantly involved in LPS-induced eosinophil marrow release.
In conclusion, our results indicate that IL-5 is involved in
marrow eosinophil mobilization induced after antigen chal-
lenge, but not after LPS stimulation. Moreover, our results
established a role for b2 integrin CD11b/CD18 for the acute
mobilization of marrow eosinophils induced either by LPS or
allergen.
Acknowledgments. We thank Paul Hellewel for insightful discussion
and for kindly providing anti-CD11b/CD18 mAb, Dr. Marcelo Bozza
and Dr. João Viola for helpful contributive discussion of this work and
manuscript, Dr. Claudia Paiva and Ramza Cabral Harab for the valuable
assistance with the FACS analysis. The present work was supported by
Conselho de Desenvolvimento Científico e Tecnológico (CNPq, Bra-
zil), PAPES (Oswaldo Cruz Foundation, Brazil) and FAPERJ (Brazil).
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