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Introduction
Asthma is an inflammatory disease in the respiratory
system, which is characterized by airway
inflammation hyperresponsiveness[1,2]. Clinical studies and a mouse model
of asthma showed leukocytes were involved in the
pathophysiological process of asthma and that eosinophils are
found to play a key role in the disease due to the toxic
granular proteins they secrete and the membrane products. In
response to the chemokines generated locally, eosinophils
from the microcirculation accumulate in the airway and
result in pulmonary inflammation[3,4].
Many small molecular proteins (8_10 kDa) have been
identified and characterized as chemokines by their actions on
distinct subtypes of leukocytes[5,6]. Chemokines regulate
immune cells by binding to 7-transmenbrane G
protein-coupled receptors, including C-C chemokine receptor (CCR)
1, CCR2, and CCR3. The distribution and expression of these
receptors vary in distinct cells.
CCR3 is a major chemokine receptor expressed on the
surface of eosinophils and is responsible for cell activation
and chemotaxis[7,8]. In addition to eosinophils, CCR3 also
exist in basophils, mast cell, and Th2
cell[9,10]. In in vitro and in vivo
studies, several C-C types of chemokines are shown
to be activated on eosinophils in allergic inflammation.
Chemokines that are selective for CCR3 include eotaxins,
eotaxin-2, and eotaxin-3, whereas RANTES, MCP-3, and
MCP-4 bind to other chemokine receptors other than
CCR3[11]. The chemokines that contribute to aberrant eosinophil
recruitment to the airways in asthma are not certain. It may be
that different chemokines operate in the process of asthma
by different immune mechanisms. It has been reported that
the binding of an oligodeoxynucleotide to the
complementary sequence of a messenger RNA, can prevent the
synthesis of the encoded protein[12]. So making the blockade of a
common receptor is more appealing. CCR3 have been
identified and characterized from other species, such as mice,
rats, guinea pigs, and non-human primates, and antibodies
of CCR3 are generated to achieve the aim at this attractive
biological target for therapeutic
intervention[13].
The treatment of asthma has been improved by the
implementation of management guidelines in recent years, with
further development in the study for the mechanism of
asthma. Many medicines, such as corticosteroids and
theophylline have been used to control asthma as
anti-inflammatory agents. However, the effects of those drugs are not
satisfied in the clinical practice. Therefore, a new agent for
asthma treatment is required[14,15]. For this reason, CCR3 has
been more important as a significant anti-inflammatory
pharmacological target, and CCR3 antagonists are currently
being developed for the treatment of asthma and other allergic
disorders. Administration of an anti-CCR3 monoclonal
antibody (mAb) was capable of inducing a targeted reduction of
eosinophils in peripheral blood. Inhibitors of CCR3 showed
these agents were effective in inhibiting eosinophil
recruitment in allergen models of
asthma[16,17]. We also reported that antimouse CCR3 mAb inhibited airway inflammation in
mice[18]. However, there is no report to evaluate the effects
of the antihuman CCR3 antibody on airway inflammation,
and mucus secretion in the mouse model of asthma.
Therefore, in the present study, we used the 30
amino acid synthetic peptide which is the NH2-terminal of CCR3 as
the immunizing antigen and generated murine mAb against
the human CCR3. Furthermore, the study was conducted on
mice sensitized and challenged with ovalbumin (OVA) to
determine the effects of this specific antibody. The
bronchoalveolar lavage fluid (BALF), histopathology, and
mucus secretion were examined. We found that the
antibody we generated was specific to CCR3. The allergic mice
treated with the antihuman CCR3 antibody exhibited a
significant reduction of pulmonary inflammation accompanied
by the alteration of cytokine in the BALF.
Materials and methods
Generation of mouse antihuman CCR3 mAb A peptide
was synthesized (Wolwo Biotech, Shanghai, China),
corresponding to the predicted NH2 terminus of the human CCR3
amino acid sequence (NH2-mttsldtvet fgttsyyddv gllcekadtr.
Accession No U49727 EMBL/GenBank). The similar
sequence could be found in Rhesus (NH2-mttsldtvet fgptsydddm gllcekadvg. Accession
No AF405535 EMBL/GenBank). The peptide conjugated to
keyhole limpet hemocyanin (KLH) was injected into BALB/c mice at 6 d
intervals. Mice sera were tested by ELISA and the spleen
cells of the mouse that produced the most potent serum titer
larger than 1:6250 were used for fusion with SP2/0 cells.
Positive hybridoma clones were selected by ELISA. Hybridoma
clones producing antihuman CCR3 mAb were injected into
mice for ascites production. The IgG fraction from the
ascites fluid was purified and dialyzed.
Western blot analysis of mouse antihuman CCR3 mAb
Daudi cell lysates (Wolwo Biotech, China) were prepared by
homogenization. Electrophoresis of secretions for CCR3 was
performed on 12% (w/v) agarose gels, including 0.1%
sodium dodecyl sulfate; 200 µg of the protein in each sample
was solubilized in electrophoresis sample buffer. For the
Western blot analysis, the membranes were blocked and the
antihuman CCR3 antibody we obtained was used as the
primary antibody at a dilution of 1:500. The membranes were
incubated with peroxidase-conjugated antimouse IgG
(Promega, Madison, WI, USA) at a dilution of 1:2000 with
dilution buffer for 1 h, and the signal was developed with
enhanced chemiluminescence system (ECL-Plus Amersham
Life Science, Heights, IL, USA).
Mice Eight to ten-week-old male BALB/c mice weighing
20_25 g were purchased from the Laboratory Animal Center
of the Medical School of Zhejiang University (Hangzhou,
China. Certificate No 220010129, conferred by the Zhejiang
Medical Laboratory Animal Administration Committee). The
mice were sensitized on d 0 and 14 by an intraperitoneal
injection of OVA mixed with aluminum. At d 24, 25, and 26,
the mice were challenged by aerosolized 1% OVA in saline
for 40 min. Thirty two mice were divided into 4 groups (8
mice in each group): the saline group, OVA group, anti-CCR3
group, and the IgG group. The antibody was administered
by ip injection before the OVA challenge at d 24, 25, and 26 at
the dose of 3 mg per kg, and the non-specific IgG (R&D,
Minneapolis MN, USA) was administered at the same time
point in the IgG group.
BALF of the lungs At d 28, BALF was performed using
phosphate-buffered solution (PBS) containing 2% Fetal
bovine serum (FBS). A small incision was made in the upper
trachea with a needle and a catheter was inserted into the
trachea through the incision. 0.5 mL 2% FBS/PBS was slowly
injected into the lungs of the mice and removed repeatedly 3
times; about 1.2 mL BALF was harvested. The total BALF
cellularity was determined with the use of a hemocytometer.
Cytospin slides were fixed and stained by Wright_Giemsa
staining. Differential cell counts by unbiased observers were
based on counts of 200_300 cells using standard
morphological criteria to classify individual leukocyte populations.
Pulmonary histology After BAL was performed, the
lungs were inflated with 1 mL of 10% neutral-buffered
formalin via a tracheotomy tube. After the instillation of the fixative,
the trachea was ligated and the lung was excised and fixed in
formalin for 24 h at 4 oC. These tissues were then embedded
in paraffin, cut in 4 µm frontal sections, mounted onto slides,
and stained with hematoxylin and eosin (HE). Periodic
acid-Schiff (PAS) was used to quantitate mucus. Image-Pro Plus
software (version 5.0.1, Media Cybernetics, Silver Spring,
MD, USA) was used to analyze histopathology. The goblet
cell hyperplasia ratio (HR) and the epithelial cell mucus
occupying ratio (MOR) were used to determine airway
mucus[19]: HR=( number of goblet cells/length of the airway
epithelial)×100%, MOR=( area of airway epithelium staining positive
for mucus/total area of airway epithelium)×100%.
Cytokine assay Interleukin-4 (IL-4) and
the IFN-γ levels in the BALF were determined using the mouse IL-4 and
IFN-γ ELISA kits (Jingmei BioTech, Shenzhen, China), as per the
manufacturer's instructions. The sensitivity for each
cytokine was 2.0 pg/mL for IL-4 and IFN-γ.
Statistical analysis Data are expressed as mean±SEM.
Statistical analysis was performed using one-way ANOVA.
Results
Generation and identification of the antihuman CCR3
mAb ELISA was employed to detect the affinity of the
generated antibody. Antibodies from the 3 different hybridoma
clones (PBND03, PBND04, and PBND05) showed high
affinity with the peptide. The affinities were
PBND03³1.28×109,
PBND04³2×107, and
PBND05³3.2×108, respectively. To
further identify the production of the antibody, the 3
antibodies were chosen as the primary antibodies for Western
blotting. The lysates of the Daudi cell expressing CCR3
were used to determine the specificity of the generated
antibody to CCR3. As shown in Figure 1, remarkable strips were
observed. The antibody from the PBND03 clone was
employed for further study in vivo because of its high affinity.
Antihuman CCR3 mAb decreases leukocytes in the
BALF of allergic mice To examine the effect of antihuman
CCR3 mAb on inflammatory cell accumulation responding
to the allergen challenge, the BALF was recovered. The
total cells of the BALF were counted and differentiated
using morphological criteria. The total number of leukocytes
and eosinophils in the OVA group was significantly greater
than that in the saline group
(P<0.01, n=4; Figure 2). A comparison of individual cell numbers per mL of BALF revealed
that these increases were mainly due to increases in
eosinophil populations. There were no differences in the total number
of leukocytes and eosinophils between the OVA group and
the IgG group. In comparison to the OVA group, the total
number of leukocytes and eosinophils in the antihuman CCR3
mAb groups decreased significantly. No significant
differences in neutrophils and lymphocytes were observed
between the groups.
Antihuman CCR3 mAb inhibits pulmonary
inflammation in allergic mice To determine if antihuman CCR3 mAb
inhibits the airway inflammatory infiltration, we observed
the pulmonary pathology stained with HE of the mice in all
groups. Compared with the mice treated with saline,
exposure to OVA resulted in the development of a characteristic
peribronchial and perivascular airway tissue infiltration. In
comparison to the mice in the OVA group, no obvious change
of pulmonary inflammation was observed in the mice
administered with non-specific IgG. However, the allergic mice
administered with antihuman CCR3 mAb exhibited
remarkably reduced inflammation in the lung tissues (Figure 3).
Antihuman CCR3 mAb reduces mucus overproduction
of allergic mice To evaluate the inhibitory effect of
antihuman CCR3 mAb on the hypersecretion of mucus in the
airways, PAS stain was performed. In allergic mice,
morphometric analyses of the PAS-stained lung sections revealed
an obvious increase in mucus cell hypertrophy and the
percentage of airway mucus, which is calculated by HR and
MOR, respectively. Antihuman CCR3 mAb significantly
reduced mucus secretion (Figure 4). Compared with the mice
in the OVA group, the mice treated with antihuman CCR3
mAb exhibited decreased HR and MOR. No reduction of
mucus hypersecretion induced by allergens was observed
in the mice treated with non-specific IgG.
Antihuman CCR3 mAb decreases the IL-4 level in the
BALF of allergic mice To determine the effect of antihuman
CCR3 mAb on the pulmonary immune response in allergic
mice, the concentration of IL-4 and IFN-γ in the BALF were
measured. The IL-4 level in the OVA group was significantly
greater than that in the saline group
(P<0.01, n=4). The mice treated with antihuman CCR3 mAb showed a significant
decrease of IL-4 compared to the allergic mice
(P<0.01). The IFN-γ level in the OVA group was less than that in the saline
group (P<0.05, n=4) and there were no differences between
the antihuman CCR3 mAb groups and OVA group at the
IFN-γ levels. Non-specific IgG had no effect on these
cytokine levels in the allergic mice.
Discussion
In the present study, we described the generation of a
murine monoclonal antibody against the human CCR3, which
was revealed as specific for CCR3 by Western blotting. This
antibody selected from the clones decreased the total
number of leukocytes and eosinophils in the BALF from allergic
mice. A pathology analysis found that the allergic
inflammatory infiltration and mucus secretion in the lung tissues were
reduced by the antibody. We also proved that the antibody
decreased pulmonary inflammation induced by the allergen
challenge, with the reduction of enhanced level of IL-4 in the
BALF. Collectively, all of the results demonstrated
the antihuman CCR3 mAb obtained in this study inhibited the
airway inflammation in the mouse asthma model, which
suggests that the development of potent antagonists for the
chemokine receptor is viable, such as the mAb against CCR3.
A synthesized peptide corresponding to the predicted
NH2 terminus of the human CCR3 amino acid sequence was
chosen as the immunizing antigen to generate the antibody.
To determine if the antibody generated is specific for human
CCR3, we performed a Western blot analysis using the
Daudi cell, which is a human B lymphoblast. CCR3 is one of
receptors expressed on the surface of Daudi
cells[20]. As shown in Figure 1, an obvious strip appeared in the lanes 1_3. To our
knowledge, this is the first report of an antihuman CCR3
monoclonal antibody being produced that employed the NH2
terminal peptide as the antigen. Depending on the binding
domain of the receptor, the monoclonal antibody against
chemokine receptors are usually divided into 2 groups: one
binds to the NH2 terminal region and the other recognizes
the second extracellular loop. The domain that our
generated antibody recognized was the NH2 terminal region, which
is in agreement with a reported monoclonal antimacaque
CCR3 antibody[13].
Although it is well known that eosinophil is critical in
airway damage and dysfunction in asthma, the role of the
cell is still questioned[21]. The results we obtained further
confirm the central role of eosinophils in the pathogenesis
of asthma. The infiltration of eosinophils was significantly
reduced by the administration of the antibody. The
reduction of eosinophils accounts for the total leukocyte decrease.
The antibody also remarkably decreased the
overproduction of mucus in the airways (another property of asthma) as
shown by histological analyses. The anti-inflammatory
activity of CCR3 blocking is supported by the existing
experimental studies. It was reported that the CCR3
antibody inhibited the eosinophil chemotaxis to various
chemokines[22]. Repeated antimouse CCR3 mAb treatment
can partially deplete eosinophils in Nippostrongylus
brasiliensis-infected lung tissue and
BALF[23]. A previous study also found that the anti-CCR3 antibody could inhibit
eosinophil accumulation in response to eotaxins
in vivo[24]. CCR3 is the predominant chemokine receptor on human
eosinophils by which many ligands bind to activate the cells.
For therapeutic purposes, blocking CCR3 will be more
efficient than blocking individual chemokines, such as eotaxins
or RANTES.
There is a lot of evidence that supports that
inflammatory cell infiltration and mucus hypersecretion are
associated with Th2 cytokines that regulate immune functions in
mice. IL-4 was shown to play a key role in mucus production
through the recruitment of Th2 cells to the lungs and the
induction of inflammation[25]. The blockage of the IL-4
receptor in mice sensitized and challenged with the antigen
markedly decreased airway goblet cells metaplasia. In the
absence of IL-4R alpha in recipient mice, there was no goblet
cell metaplasia or mucus hypersecretion in response to OVA,
even in the presence of Th2 cells and substantial
eosinophilic infiltration[23]. It was reported that the expression of
Th2 cytokines was unaffected as a consequence of the
CCR3-mediated depletion of eosinophils in
OVA-sensitized/challenged mice[26]. However, CCR3-mediated signaling can
rapidly mobilize IL-4 stored in human eosinophils for release by
vesicular transport to contribute to immune responses
[27]. An antibody to CCR3 abrogated the eotaxin-enhanced IL-4
release in vitro[28]. The cytokine analysis in our experiments
showed that the anti-CCR3 antibody significantly decreased
the elevated IL-4 level in the BALF of allergic mice with
reduced eosinophils. However, the antibody did not affect
the level of IFN-γ in the BALF, which identical to the
previous study[26]. Our results suggested that leukocytes,
including eosinophil, lymphocyte,, and other inflammatory
cells were inhibited by the CCR3 blocking pathway that
contributed to the reductive synthesis of cytokine in the lungs,
which leads to the decrease of airway inflammation and
mucus secretion.
In conclusion, our study demonstrates that we have
generated an anti-CCR3 antibody which is specific against
human CCR3. The antibody could significantly inhibit the
inflammation in the lungs. It is suggested that the blockade
of CCR3 is an appealing therapeutical target for asthma. The
present research may provide an experimental basis for the
further study of this agent.
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