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Introduction
Allergic asthma is a chronic disease characterized by airway hyperresponsiveness (AHR), airway inflammation, and
reversible airway obstruction. Since the mid-1980s, there has been a wealth of information documenting a potential
pathogenic and destructive role for eosinophils in
asthma[1]. A line of mice devoid of eosinophils was recently created to confirm
hypotheses that a causative link exists between eosinophils and asthma-related
pathogenesis[2]. Eotaxin is a potent
eosinophil chemoattractant causing selective infiltration of these cells into the lungs. It binds to CC-chemokine receptor-3 (CCR3)
that appears to be restricted to the eosinophil in
mice[3]. Therefore, the eotaxin/CCR3 pathway is a potentially important
target for asthma therapy.
We explored the effect of a CCR3 monoclonal antibody (CCR3 mAb) on airway eosinophilia and goblet cell hyperplasia
(GCH)/mucus overproduction, the 2 most important hallmarks in asthma. The MUC5AC gene expression is thought to be a
marker of GCH[4], and the epidermal growth factor receptor (EGFR) pathway is involved in mucin production in the
airways[5]. In this study, we also examined the effect of CCR3 mAb on MUC5AC and EGFR gene expression in the lungs of asthmatic
mice.
Materials and methods
Reagents Rat anti-mouse CCR3
mAb[6] was received as a gift from Dr James J LEE (Mayo Clinic, Phonex, Arizona, USA).
Nonspecific rat IgG (ns-IgG) was purchased from Chemicon Co (Temecula, CA, USA); chicken ovalbumin
(OVA, grade V) was from Sigma Chemicals (St Louis, MO,
USA);
inject alum was from Pierce Co (Rockford, IL, USA); IL-5 ELISA kit was from R&D Co (Minneapolis, MN, USA); Trizol for
extracting RNA was from Invitrogen Co (Carlsbad, CA, USA).
OVA sensitization and
challenge Male C57BL/6 mice, weighing 18_23 g, were purchased from the Shanghai Experimental
Animal Center of the Chinese Academy of Sciences (Shanghai, China). The mice were sensitized and challenged with chicken
OVA as previously described[7]. All the mice were sensitized by intraperitoneal injections (100 µL) of 80 µg OVA emulsified in
2 mg of Inject Alum on d 0 and d 14. The mice were exposed for 30 min to 1% OVA aerosol generated by an ultrasonic nebulizer
on d 24_26. Blank control mice were sensitized and challenged with normal saline. In the preliminary experiment, we found
that 6 µg/kg-mice of CCR3 mAb intraperitoneal administration resulted in over 90% reduction of eosinophils in the peripheral
blood and bronchial alveolar lavage fluid (BALF). In order to strengthen the inhibition effect of CCR3 mAb on eosinophils,
the mice were injected intraperitoneally with 150 µg of CCR3 mAb, then 50 µg of CCR3 mAb was mixed with the OVA solution
and administered with each of the 3 OVA challenges in our formal experiment. Negative control mice were administered the
same amounts of rat ns-IgG by both routes.
Cell count in the BALF Mice were sacrificed for sample collection on d 28. The tracheas were cannulated, the right lungs
were ligated with a suture thread, and the left lobes of lungs were lavaged 3 times with 0.3 mL of ice-cold phosphate buffered
saline (PBS). BALF 10 µL was used for the total cell counts and the residual BALF was centrifuged. The cell pellet was used
to prepare cell slides by cytospin and stained with methylene blue/eosin. Four hundred cells were counted for estimating the
differential counts of various cell types afterwards. The supernatant was preserved at -70 ºC for cytokine detection. The right
lungs without lavage were stored at -70 ºC for total RNA isolation.
Lung histology The left lobes of the lungs were fixed in 10% buffered formalin. After embedded in paraffin, the tissues
were cut into 4 µm thick sections. The lung sections were stained with hematoxylin/eosin (HE) and Alcian blue/periodic
acid-Schiff (AB/PAS), respectively. Infiltrating lung eosinophils in parasagittal sections with HE staining were counted under
microscopy. The goblet cell percentage (GCP) and airway mucus index (AMI) were measured as follows: GCP=the amount of
goblet cells/total number of airway epithelial cells; AMI=the area of airway epithelium with AB/PAS staining/total area of the
conducting airway epithelium. The analysis of the mucus content of the airway epithelium of mice from different groups was
preformed using the imaging program of Leica Co (Wetzlar, Hessen, Germany).
Cytokine assay Interleukin (IL)-5 levels in the BALF
were measured by ELISA method according to the manufac-turer's
protocol for users. The limit of IL-5 assay was 10 pg/mL.
Semi-quantitative RT-PCR The total RNA was extracted from about 0.2 mg of the lung tissues with Trizol reagents. For
cDNA synthesis, 20 µL RT mixture containing total RNA 2 µg, dNTP 1 mmol/L,
Olig(dt)18 prime 0.2 µg, RNasin 20 U, M-MLV
reverse transcriptase 200 U was incubated at 42 ºC for 60 min, then the reverse transcriptase was inactivated by heating the
reaction mixture at 72 ºC for 15 min. The expression of MUC5AC and EGFR mRNA in the lungs was measured by RT-PCR.
Oligonucleotide primers specific for mouse MUC5AC, EGFR and glyceraldehyde phosphate dehydrogenase (GAPDH, an
internal control) were synthesized according to published sequences: MUC5AC: sense: 5' CAG CCG AGA GGA GGG TTT
GAT CT 3', anti-sense: 5' AGT CTC TCT CCG CTC CTC TCA AT
3'[4]; EGFR: sense: 5' GTG TGA AGA AGT GCC CCC GAA AC
3', anti-sense: 5' AAC GAC CGC CAA AGA AAA CTG ACC
3'[8]; GAPDH: sense: 5' CTG GTG CTG AGT ATG TCG TG 3',
anti-sense: 5' CAG TCT TCT GAG TGG CAG TG
3'[4], with the product sizes 389 bp, 452 bp
and 296 bp, respectively. PCR was performed as follows:
1 µL of cDNA mixture was subjected to amplification in 50µL of final volume with
MgCl2 1.5 mmol/L, dNTPs 0.2 mmol/L, 20 pmol of each primer, and 2 U of
Taq DNA polymerase in the reaction buffer on a PE2400 cycler
(Perkin-Elmer, Foster City, CA, USA). The condition of PCR reactions was used below: 94 ºC for 5 min; then 94 ºC for 1 min; 56 ºC for
MUC5AC, 58 ºC for EGFR and 60 ºC for GAPDH for 1min; 72 ºC for 45 s for 30
cycles (GAPDH) or 35 cycles (MUC5AC and EGFR) and 72
ºC for 10 min to end the reaction. PCR products were electrophoresed by 1.5% agarose gel containing ethidium
bromide. The density of each band was measured by Gel-pro analyzer 4.0 (Exon-Intron, Inc, Loganville, PA, USA). The
results were normalized as ratios compared with GAPDH.
Statistical analysis All data were presented as mean± SEM. One-way ANOVA was used for statistical analysis of the
differences between the groups. A value of
P<0.05 was considered statistically significant.
Results
Effect of CCR3 mAb on airway eosinophilic
inflammation Numerous eosinophils appeared around the bronchioles and
blood vessels in asthmatic mice whereas smooth airway walls and regular epithelium without inflammatory cell infiltration
were seen in the saline control group. CCR3 mAb treatment obviously attenuated eosinophil infiltration, however, no
significant difference was observed between the ns-IgG-treated mice and OVA-challenged mice (Figure 1).
Eosinophils significantly increased and became the predominant inflammatory cells in the BALF and lung tissues of the OVA-challenged
mice. CCR3 mAb treatment significantly reduced the number of total cells and eosinophils in the BALF and eosinophils
number in the lung parenchyma (Table 1), but the administration of CCR3 mAb had no obvious effect on other cell types
including lymphocytes, neutrophils and macrophages (data not shown).
Effect of CCR3 mAb on GCH and mucus
overproduction The pathological results from the lung tissues showed that CCR3
mAb treatment significantly alleviated epithelial hypertrophy and mucus production in the airways of OVA-challenged mice
compared with the ns-IgG treatment mice (Figure 2). However, GCP and AMI of the airway epithelium of CCR3 mAb-treated
mice were still higher than those of the saline-challenged control mice (Table 2).
Effect of CCR3 mAb on IL-5 levels in the
BALF The increased IL-5 levels were detected in the BALF of
OVA-challenged mice vs the saline-treated mice. However, the
IL-5 concentration in the BALF in CCR3 mAb and the ns-IgG-treated mice
exhibited no obvious changes compared with the OVA-treated animals (Table 3).
MUC5AC and EGFR mRNA expression in the lungs of
mice The upregulated MUC5AC and EGFR mRNA expression was
found in the OVA-challenged mice. CCR3 mAb treatment decreased the expression of MUC5AC and EGFR mRNA. No
obvious difference of MUC5AC and EGFR mRNA expression was observed in the ns-IgG-treated mice compared with the
OVA-challenged mice (Figures 3, 4).
Discussion
Although the key role of eosinophil in allergic pulmonary diseases is challenged by the clinical trials of the anti-IL-5
monoclonal antibody[9], more and more data from both asthma patients and mouse models have demonstrated that airway
eosinophilia infiltration is one of the most important characteristics of asthma. Human eosinophils mediate
pathogenic effects from several independent mechanisms that allow the eosinophil to modulate the intensity of pulmonary inflammation,
as well as elicit cell death and loss of structural integrity of the lung, leading to pulmonary
dysfunction[10]. Recently, a line of mice devoid of eosinophils was created to confirm hypotheses that a causative link exists between eosinophils and
asthma-related pathogenesis such as airway epithelial pathology, mucus hypersecretion and AHR in Lee Laboratory (Mayo Clinic,
Phonex, Arizona, USA)[2,7]. Therefore, blocking the onset and progression of asthma by specifically depleting eosinophils is
effective in asthma therapy.
The chemokine receptor CCR3 seems to play a major role in allergic diseases which is supported by the detection of this
receptor on human eosinophils, Th2 cells, mast cells and
basophils[11_13]. However, Grimaldi
et al revealed that mouse CCR3 was expressed on eosinophils, but not on stem cells, dendritic cells or cells from the thymus, lymph node, or spleen of normal
mice by flow cytometry. Unlike human Th2 cells, mouse Th2 cells did not express detectable levels of CCR3 nor did they give
a measurable response to eotaxin[3]. The reactivity of CCR3 mAb in our experiment was proven to be restricted to
eosinophils[3, 6].
Our study showed that dual
administration (intraperi-toneal injection and aerosol inhalation) of CCR3 mAb can
effectively ablate eosinophils in the BALF and significantly reduce lung eosinophilia relative to OVA-challenged mice. In addition,
CCR3 mAb was found to have no observable effect on the number of other cell types (lymphocytes, macrophages and
neutrophils) in the BALF and lung tissues, indicating the specificity of mouse CCR3 mAb treatment for only eosinophils.
Using the CCR3-deficient mouse model, Humbles
et al found that CCR3 depletion significantly abrogated eosinophil
recruitment to the lung after allergen challenge, with the majority of the eosinophils being arrested in the subendothelial
space[14]. Similarly, a CCR3 monoclonal antibody and small molecule CCR3 antagonists were demonstrated to be capable of reducing
pulmonary eosinophilic inflammation in mouse allergenic models of airway
inflammation[15]. Taken together, CCR3
antagonism has the potential to influence eosinophil infiltration into the lungs of asthmatics and is therefore a promising therapeutic
target.
Shi and colleagues pointed out that eosinophils were able to express major histocompatibility complex II complex and
costimulatory molecules and function as antigen-presenting cells
(APC)[16]. Eosinophils have the potential to activate lung
Th2 cells to release disease-modulating
cytokines[17], but the investigation from van Rijt
et al did not support any role for airway eosinophils as APC to naive T cells, despite their migration to the draining lymph nodes at times of allergen
exposure[18]. In our study, Th2 cytokine (IL-5)
levels in the BALF from CCR3 mAb-treated animals
were unaffected compared with the OVA-treated mice. Justice
et al also proved that the anti-CCR3-mediated loss of eosinophils from the lung did not have a
demonstrable effect on the activities of T and B
lymphocytes[6]. So we think that eosinophils function as APC in the
modulation of T cell activities which may be relatively limited and can be compensated by other APC (dendritic cells,
macrophages and B lymphocytes) in the absence of eosinophils because dendritic cells are clearly superior in activating T
cells in the draining lymph nodes of the
lung[18].
Globlet cell metaplasia (GCM), GCH and mucus hypersecretion are prominent features of allergic
asthma[19]. Numerous inflammatory mediators produced in asthmatic airways, together with neural mechanisms, can theoretically increase mucin
secretion, induce plasma exudation, upregulate MUC gene expression (MUC5AC gene expression is thought to be a marker
of GCH[5]), increase mucin synthesis and cause
GCH[20,21]. Most of the above pathways operate through the
EGFR and its tyrosine kinase intracellular signaling
cascade[22]. EGFR and MUC5AC expression at both gene and protein levels is upregulated
in goblet cells in bronchial biopsies from asthmatic patients compared with non-asthmatic
persons[23]. Burgel et al demonstrated that activated human eosinophils induced mucin synthesis in human airway epithelial cells via EGFR
activation[24]. We analyzed MUC5AC and EGFR gene expression in the lungs of asthmatic mice. Over-expression of MUC5AC and EGFR
mRNA in the lungs was found in the OVA-treated mice compared with the saline-treated animals, and CCR3 mAb treatment
significantly reduced MUC5AC and EGFR mRNA levels in the lungs of OVA-challenged mice by the ablation of eosinophils
relative to ns-IgG-treated mice. Therefore, we deduce that CCR3 mAb may downregulate MUC5AC gene expression, at least
partly, through the EGFR pathway.
Our results show that CCR3 mAb-mediated ablation of eosinophils in OVA-treated mice can significantly reduce
GCM/GCH and mucus overproduction compared with ns-IgG-treated mice, indicating that the products of eosinophils are capable
of stimulating an increase in goblet-cell number and mucus secretion. However, the GCP and AMI of CCR3 mAb-treated mice
were still higher than those of the normal saline-treated mice, suggesting the existence of both eosinophil-independent and
eosinophil-dependent pathways leading to allergen-mediated goblet cell metaplasia/mucus
production[6].
In summary, CCR3 mAb can effectively ablate lung eosinophilia and significantly inhibit mucus overproduction in a
mouse model of asthma. Interfering with the CCR3 receptor may represent a potential new therapy for the treatment of
asthma.
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