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*
Department of Allergy and Clinical Immunology, National Heart and Lung Institute, Imperial College School of Medicine, London, United Kingdom; and
Antenne d’Immunopathologie Respiratoire, Service de Pneumologie, Institut Paris Sud sur les Cytokines, Hôpital Antoine Béclère, Clamart, France
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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RI+ cells
(5, 6, 7, 8). The only difference seemed to be a strong
macrophage signal, i.e., there were increased numbers of
CD68+ cells and cells mRNA+
for GM-CSF receptor 
-chain in intrinsic asthma compared with
extrinsic asthma (9). In the present study we have extended these observations by measuring the expression of eotaxin, eotaxin-2, MCP-4, and CCR3 in bronchial biopsies from intrinsic and extrinsic asthma. Unlike RANTES, MCP-3, and MCP-4, which ligate several C-C chemokine receptors, eotaxin and eotaxin-2 bind exclusively to CCR3. The two C-C chemokines are also selective in their biological activity on eosinophils and basophils, although CCR3 may also be transiently expressed on Th2 cell lines. For these reasons we have tested the hypothesis that the range of C-C chemokines, including eotaxin and eotaxin-2, as well as the eotaxin receptor, CCR3, is similar in both atopic and nonatopic asthma, indicating a common mechanism of local eosinophilia in both subgroups.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The study was approved by the ethics committees of the Royal
Brompton Hospital (London, U.K.) and the Hochgebirgsklinik (Davos
Wolfgang, Switzerland). Each participant gave informed consent before
entering the study. Bronchial biopsy specimens were obtained from 10
atopic asthmatics (AA), 10 nonatopic asthmatics (NAA), 10 nonasthmatic
atopic controls (AC), and 10 nonatopic nonasthmatic controls (NC) as
previously characterized (5). The patients studied were
recruited from the Royal Brompton Hospital (London, U.K.) and the
Hochgebirgsklinik. Asthmatic subjects had a clear history of asthma,
requiring intermittent inhaled ß2-agonist
therapy, and documented reversible airway obstruction (20% improvement
in forced expiratory volume is 1 s (FEV1) either
spontaneously or after administration of inhaled
ß2-agonist), and a histamine
PC20 provocation test result 
6 mg/ml in the
previous 2 wk. Atopy was defined as a positive skin prick test (wheal
at 15 min >3 mm in diameter in the presence of positive histamine and
negative diluent controls) to more than one of the extracts of the
common local aeroallergens (i.e., mixed grass, tree pollen, mixed
moulds, cat and dog dander, and house dust mite). By use of the CAP
system (Pharmacia Diagnostics, Uppsala, Sweden) all atopic patients
demonstrated a positive radioallergosorbent (RAST) test (>0.70 IU/ml)
to one or more common allergens. Nonatopic subjects were defined by
negative skin test to a wide range of local common aeroallergens in the
presence of a positive histamine-positive control. The nonatopic
subjects also exhibited a serum IgE concentration within the normal
range and negative RAST test to 25 common aeroallergens. For all
subjects, total serum IgE and RAST measurements were performed in the
same laboratory. All subjects were nonsmokers and had not taken oral (2
mo) or inhaled (2 wk) glucocorticoids before the study. Exclusion
criteria included age <18 years or >65 years,
FEV1 <60% of the predicted value on the study
day, evidence of acute or chronic infection, pregnancy, or any chronic
medical illness other than asthma.
Fiberoptic bronchoscopy
The bronchoscopy procedure was performed using an Olympus model IT30 bronchoscope (Olympus, Tokyo, Japan) at the same time of day (9 a.m.) in all the subjects. Bronchoscopy was performed in all subjects following premedication with 2.5 mg of salbutamol by nebulizer, 0.6 mg of atropine, and 5–10 mg of midazolam administered i.v. Biopsies were taken from the right middle and lower bronchi using Olympus alligator forceps (model FB15C). All asthmatics were given an additional 2.5 mg of nebulized salbutamol immediately after the procedure. Bronchoscopy was well tolerated, and the safety of our protocol was prospectively monitored in all subjects. Bronchial biopsies were fixed immediately in freshly prepared 4% paraformaldehyde (BDH, Poole, U.K.) in PBS (pH 7.4) for 2 h, washed twice (1 h) with 15% sucrose (Sigma, Poole, U.K.) in PBS, embedded in OCT (optimum cutter temperature) compound (Bayer, Basingstoke, U.K.), snap-frozen, and stored at -80°C to be used later.
In situ hybridization (ISH)
Sections (6 µm, freshly cut from frozen bronchial biopsies)
were prepared onto slides coated with 0.15 poly-L-lysine
(BDH) and air-dried for 2 h at room temperature. All reagents used
for ISH were obtained from Sigma, unless otherwise indicated. Briefly,
308 bp of human eotaxin (10) (3' untranslated region,
893-1201 bp, a gift from Dr. J Rottman, LeukoSite, Cambridge, MA) and
1056 bp of human CCR3 (11) (encoding region, a gift from
Dr. B. L. Daugherty, Merck Research Laboratories, Rahway, NJ) cDNA
fragments were inserted into PCR Bluescript and pSP72 vectors,
respectively (12). A total of 188 bp of human eotaxin-2
(13) (encoding region 104–286, a gift from Drs. M.
Uguccioni and M. Baggiolini, Theodor Kocher Institute, Bern,
Switzerland) was inserted into pT/T3-18 vector (Life Technologies,
Basel, Switzerland). A total of 303 bp of PCR product encoding human
MCP-4 (encoding region 26–329) (14) was inserted into
pT7T3 18 vector (Ambion, Austin, TX). A total of 916 bp of human RANTES
(encoding region 21–937, a gift from Dr. P. Nelson, Department of
Pediatrics, Stanford University, Stanford, CA) was inserted into pGEM-3
(7). The Bluescript vector containing 600 bp of human
MCP-3 cDNA was provided by Drs. J. Van Damme and G. Opdenakker (Rega
Institute for Medical Research, University of Leuven, Leuven, Belgium)
(7, 15). Riboprobes were prepared from cDNA for these
chemokines and CCR3 as previously described (6, 7, 12)
Briefly, riboprobes (antisense or sense) were synthesized in the
presence of ATP, GTP, CTP, and [35S]UTP and
appropriate RNA polymerases (T7, SP6, or T3), respectively.
Permeabilization, prehybridization, and hybridization protocols were described previously (6, 7, 12). Incubation in N-ethylmaleimide, iodoa-cetamide, and triethanolamine reduced nonspecific binding of the [35S]UTP-labeled probes. Furthermore, the experiments were performed under very high stringency condition (hybridization at 50°C and posthybridization washing at 60°C, 0.1x SSC) to minimize nonspecific hybridization. Negative controls employed hybridization with the sense probe and pretreatment of slides with RNase A (Promega, Southampton, U.K.) before hybridization with the antisense probe. For autoradiography, slides were dipped into K-5 emulsion (Ilford, Basildon, U.K.) and exposed at 4°C for 2 wk in absolute darkness in a desiccated environment. The slides were developed (D-19 developing solution, Eastman Kodak, Rochester, NY), rinsed, and counterstained with Harris hematoxylin. Dense deposits of silver grains on autoradiographs were present over cells expressing chemokine mRNA. Slides were counted in duplicate, blind to the patients’ clinical status, using an eyepiece graticule, as previously described (6, 7, 12). The results were expressed as the total number of positive cells per field (per 0.202 mm2) of submucosa. The coefficient of variability of the duplicate counts obtained from all slides was <5%.
Immunohistochemistry
Sections (6 µm) were freshly cut from frozen bronchial biopsies. For identification of eosinophils, basophils, eotaxin, and CCR3 protein product, IHC of bronchial biopsies was performed using alkaline phosphatase-antialkaline phosphatase (APAAP) method as described previously (6, 7, 12). Rabbit anti-mouse Ig, APAAP, and control IgG1 were purchased from Dako (High Wycombe, U.K.). EG2, a mAb against human activated eosinophils, was purchased from Pharmacia (Uppsala, Sweden). A mouse anti-human eotaxin mAb (2G6) and anti-human CCR3 mAb (7B11) were provided by Drs. C. Mackay, P. Ponath, and W. Newman (LeukoSite) (12, 16). BB1, a mAb recognizing a human basophil granular protein, was a gift from Dr. A. F. Walls (Southampton General Hospital, Southampton, U.K.). This mAb did not react with lymphocytes, monocytes, platelets, neutrophils, eosinophils, mast cells, or any other cell type or tissue structure (17). The optimal concentrations of all Abs used were determined in pilot experiments. Briefly, the slides were incubated with 20% human serum in PBS for 20 min, then incubated with monoclonal anti-human eotaxin (2G6, 1/50), or monoclonal mouse anti-CCR3 (7B11, 1/50) in 20% human serum/PBS overnight at room temperature. Slides then were treated with rabbit anti-mouse IgG (1/30, 30 min) and APAAP (1/30, 30 min), respectively. For immunostaining of BB1+ basophils, the slides were pretreated with 0.1% saponin (Sigma)/PBS for 30 min, incubated with BB1 Ab (1/10) overnight at room temperature, then processed as described above. Positive cells stained red after development with Fast Red (Sigma). Omission or substitution of the primary Ab with an irrelevant Ab of the same species was used as a negative control. All the slides were counted in duplicate under blind conditions to the patients’ clinical status. Results were expressed as the total number of positive cells per field (per 0.202 mm2) of submucosa. The coefficient of variability of the duplicate counts obtained from all slides was <5%. No immunoreactivity was observed in sections stained with omission of the primary Ab or substitution of this Ab with an irrelevant Ab of the same species.
Sequential IHC and ISH
To identify the cell sources of eotaxin, eotaxin-2, RANTES, MCP-3, and MCP-4 mRNA, sequential IHC/ISH was employed as previously described (6, 12). Briefly, cell phenotypes were first identified by IHC using the APAAP technique and phenotype-specific murine mAb. The mAb used were directed against human epithelial cells (cytokeratin, clone MNF 116), endothelial cells (CD31, clone JC/70A), macrophages (CD68, clone EBM11), mast cell tryptase (clone AA1), neutrophil elastase (clone NP57; Dako), T cells (CD3; Becton Dickinson, Oxford, U.K.), and eosinophil cationic protein (EG2). Sections from five atopic asthmatics and five nonatopic asthmatics with high expression of eotaxin, eotaxin-2, RANTES, MCP-3, and MCP-4 RNA were chosen. After developing with Fast Red for cell phenotypes, ISH was performed using digoxygenin-labeled riboprobes (6, 12) specific for these C-C chemokines. The mRNA-positive signals were visualized using nitro blue tetrazolium/bicinchononic acid (Sigma) as the chromogen (6, 12). The numbers of positive cells expressing phenotypic markers, chemokine mRNA, or both were counted in the epithelial area and submucosa in whole sections. The results were expressed as the percent distribution of cells expressing eotaxin, eotaxin-2, RANTES, MCP-3, and MCP-4 mRNA and the percentage coexpressed by each cell type within the epithelium and sumucosa of biopsies.
Statistical analysis
Data were analyzed with the aid of a commercially available statistical package (Minitab for Windows, Minitab release 9.2, Minitab, State College, PA). The Mann-Whitney U test was then used for intergroup comparison (with Bonferroni’s correction). Correlation coefficients were obtained by Spearman’s rank-order method. A p value <0.05 was accepted as statistically significant.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Details of the study population are shown in Table I
. As expected, atopic asthmatics had
significantly elevated total serum IgE compared with NAA and NC
(p < 0.01). Both AA and NAA had significantly
lower FEV1 and histamine
PC20 than those of AC and NC. Unlike NC, AA, NAA,
and AC had relatively high blood eosinophil accounts (Table I).
Compared with AA, AC, and NC, the nonatopic asthmatics were
significantly older. The total serum IgE in NAA was slightly higher
than in NC, although it was significantly lower than in AA and AC.
These observations are consistent with previous findings
(5, 6, 7).
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The numbers of EG2+ eosinophils in the
bronchial mucosa from both atopic and nonatopic asthmatic patients were
significantly elevated compared with those in both nonasthmatic control
groups (Fig. 1). Although the number of
BB1+ basophils was 
10-fold less than that of
EG2+ eosinophils, the numbers of
BB1+ basophils in bronchial biopsies from
asthmatics (AA and NAA) were significantly elevated compared with
control values (AC+ NC; p < 0.05; Fig. 1). There were
no statistically significant differences between AA and NAA, although
the mean numbers of BB1+ basophils in AA were
slightly higher than those in NAA.
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Bronchial biopsies from asthmatic patients had elevated numbers of
cells encoding mRNA for eotaxin, which significantly increased compared
with those from both the atopic and nonatopic control subjects without
asthma (p < 0.001; Fig. 2). No significant differences between
either AA and NAA or AC and NC were observed. Immunohistochemistry
revealed increases in the numbers of eotaxin-immunoreactive cells in
the bronchial mucosa of asthmatics compared with nonasthmatic controls
(p < 0.05; Fig. 2). By ISH and IHC
eotaxin-positive cells were identified in the epithelium and throughout
the submucosa, either as isolated cells or as aggregates. Cells
encoding mRNA for eotaxin-2 were also significantly increased in the
bronchial mucosa of asthmatic patients regardless of their atopic
status compared with those in nonasthmatic controls (Fig. 2) Taking the
atopic and nonatopic subjects with asthma as a group, significant
inverse correlations were detected between the bronchial mucosal
expression of eotaxin (mRNA and protein) and the histamine
PC20 (r = -0.5;
p < 0.05; Fig. 3). The
numbers of eotaxin+ (mRNA and protein) cells in
asthmatics (AA+NAA) were also significantly correlated to the numbers
of EG2+ cells (r = 0.75;
p < 0.001). Eotaxin-2 mRNA+
cells were also significantly correlated with numbers of
EG2+ eosinophils (r = 0.5;
p < 0.05), but not with FEV1 or
PC20.
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Bronchial biopsy sections from the atopic and nonatopic asthmatics
had significantly elevated numbers of MCP-4 mRNA+
cells per unit area of bronchial biopsies compared with both atopic and
nonatopic control subjects without asthma (Fig. 4). A significant increase in the numbers
of cells expressing mRNA for both RANTES and MCP-3 was also detected in
the biopsies from asthmatics compared with nonasthmatic controls (Fig. 4). None of these C-C chemokines significantly correlated with
eosinophils or markers of asthma severity. MCP-4 and RANTES mRNA were
located throughout of epithelial area and submucosa. MCP-3 mRNA were
generally located in submucosa, and low numbers were detected within
the epithelial layer.
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The numbers of CCR3-expressing cells (mRNA and protein) were
significantly elevated in asthmatics compared with AC and NC (Fig. 5). However, nonsignificant differences
between either AA and NAA or AC and NC were observed. In the asthmatic
group (AA + NAA), numbers of CCR3 (mRNA and protein)-positive cells
were directly correlated with EG2+ eosinophil
numbers (r = 0.8; p < 0.001) and
inversely with histamine PC20 (r
= -0.51; p < 0.05).
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Cells expressing mRNA for C-C chemokines were determined by double
IHC/ISH. In the epithelium, up to 70% of mRNA-positive cells were
colocated to cytokeratin+ epithelial cells. The
remains of the mRNA+ cells were
CD68+ macrophages, CD3+ T
cells, or tryptase+ mast cells (Fig. 6). In the submucosa,
CD31+ endothelial cells,
CD68+ macrophages, and CD3+
T lymphocytes were the major cell sources expressing these C-C
chemokines (Fig. 7). No significant
differences in the various cell types expressing mRNA for these
chemokines in either epithelium or submucosa were observed between AA
and NAA (n = 5 for each group). The percentages of each
cell type coexpressing mRNA for eotaxin, eotaxin-2, RANTES, MCP-3, and
MCP-4 in the epithelium and the submucosa
are summarized in Tables II and III.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Whether these mediators are elaborated by the same mechanisms and through the same pathways in atopic and nonatopic asthma is presently unclear. Walker and colleagues have suggested that intrinsic and extrinsic asthma might be regarded as distinct immunopathological entities, since they found evidence that atopic asthma was characterized by a cytokine profile typical of Th2-type response in bronchoalveolar lavage (BAL) fluid, while intrinsic asthma was associated with isolated, elevated expression of IL-5 (21). They postulated that the absence of production of IL-4 explained the lack of IgE elevation in these patients. On the other hand, studies that have examined C-C chemokine as well as cytokine expression in atopic and nonatopic asthma have provided evidence for similarities, rather than differences, in the immunopathogenesis of these clinically distinct forms of the disease.
For example, elevated numbers, compared with controls, of activated T cells, eosinophils (22), high affinity IgE receptor-bearing cells (8), IL-5 and IL-4 mRNA, and protein product-positive cells have been shown to be features of the bronchial mucosa of both atopic and nonatopic asthma (5, 6). Furthermore, no differences in the number of mRNA-positive cells for IL-13 (23), RANTES, and MCP-3 were detected (7).
Using mAb EG2 and BB1, the numbers of eosinophils and basophils were
evaluated in the bronchial biopsies from asthmatics at baseline and
from controls. Immunohistochemistry showed that the numbers of
EG2+ eosinophils were significantly higher in
asthmatics (AA and NAA) than controls (AC and NC; Fig. 1
). Although the
number of EG2+ eosinophils was slightly higher in
NAA than in AA, there were no significant differences between AA and
NAA. This is consistent with previous findings (5, 6, 7).
Unlike eosinophils, BB1+ basophils were
undetectable in the biopsies from either AC or NC, suggesting that
BB1+ basophils are not infiltrated into tissue in
the normal situation. Compared with controls (AC + NC), small, but
statistically significant, numbers of BB1+
basophils were detected in bronchial mucosa from asthmatics (AA + NAA;
p < 0.05; Fig. 1). The number of
BB1+ basophils in AA was higher than that in NAA,
but the difference was not statistically significant. Interestingly,
the number of BB1+ basophils was much less than
that of EG2+ eosinophils in bronchial mucosa. The
reason is unclear. BB1 is an mAb directed against a basophil granule
constituent with a molecular mass of 124 kDa (17). Thus,
completely degranulated basophils, if present, may be undetected.
In general, chemokine expression was elevated in both asthmatic groups compared with nonasthmatic controls. There was a trend for more pronounced expression in nonatopic patients compared with atopic asthmatics of similar severity. This could be the result of elevated numbers of macrophages and other inflammatory cells detected in the bronchial mucosa of intrinsic asthmatics and could be responsible, in turn, for the relatively increased numbers of blood and airway eosinophils in intrinsic asthma. We and others had previously shown that eotaxin expression in bronchial biopsies from atopic asthmatics correlated with eosinophil infiltration and clinical features (12, 18, 19). In this study, eotaxin mRNA and protein product-positive cells significantly correlated with EG2+ eosinophils and histamine PC20 in asthmatics (AA + NAA). There was also a significant correlation between eotaxin-2 mRNA+ cells and EG2+ eosinophils (see Results). Both eotaxin and eotaxin-2 activate and attract eosinophils, basophils, and Th-2 cells (24), but not other leukocytes, and appear to act exclusively via CCR3 receptor (25). In terms of structure, however, eotaxin and eotaxin-2 are distant, sharing only 39% identical amino acids, while there is a 43% homology for MCP-4, 39% for MCP-3 and eotaxin, and 32% for RANTES (25). In addition, MCP-3, MCP-4, and RANTES have chemotactic activity for eosinophils and basophils (26, 27). Thus, it is likely that several chemokines contribute to eosinophil recruitment in allergic inflammation. Eotaxin-deficient mice had reduced eosinophils in BAL fluid early, but not late, after allergen challenge (28). Using the allergen-induced human skin late-phase reaction as a model, we found that that expression of eotaxin and MCP-3 was associated with the early 6-h recruitment of eosinophils, while eotaxin-2, RANTES, and MCP-4 appear to be involved in later 24-h infiltration of eosinophils (29). These observations suggest that different mechanisms may guide the early vs late eosinophilia in allergic inflammation. Taha et al. (20) showed that eotaxin and MCP-4 mRNA increased in bronchial mucosa of atopic asthmatics and that eotaxin mRNA+ cells significantly correlated with eosinophils. BAL fluid from asthmatic patients contained chemotactic activity for eosinophils that was attributable in part to both eotaxin and MCP-4 (18). However, a combination of eotaxin, MCP-4, and RANTES accounted for only about 50% of the eosinophil chemotactic activity in the BAL, suggesting the existence of other eosinophil chemotactic factors (18). In view of this multiplicity of potential influences on eosinophils, it is perhaps surprising that we found a direct correlation between eotaxin (but not RANTES, MCP-3, or MCP-4) mRNA expression and eosinophil numbers, clinical features, and the degree of bronchial hyper-responsiveness. The specificity of eotaxin for eosinophils, as opposed to the multiple cell targets of RANTES, MCP-3, and MCP-4, may explain these positive clinical correlations.
Previous reports indicated that the various C-C chemokines are
expressed by many cell types, including epithelial cells, endothelial
cells, macrophages, T lymphocytes, muscle cells, fibroblasts,
platelets, Langerhans cells, dendritic cells, and eosinophils
themselves (30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41). Here we showed by double IHC/ISH that
cytokeratin+ epithelial cells,
CD31+ endothelial cells,
CD68+ macrophages, and CD3+
T lymphocytes were the majority of cells expressing these C-C
chemokines (Figs. 6 and 7 and Tables II and III). We also noted that
some MCP-3 mRNA+ cells (35%) were not
colocalized to any of the cell types examined. In vitro, it had been
reported that fibroblasts and platelets also express MCP-3 (42, 43). Thus, fibroblasts and platelets (for which phenotypic
markers are unavailable) and other unknown cell types may contribute to
the expression of MCP-3 in bronchial mucosa.
The mechanisms of multi-C-C chemokine gene overexpression in the
bronchial mucosa from asthmatics are incompletely understood. A number
of proinflammatory cytokines and mediators are probably involved. For
example, IL-1 and TNF- might up-regulate the expression of eotaxin
(30, 32), MCP-4 (31), and other C-C
chemokines in epithelial and endothelial cells in vitro. On the other
hand, Th2 cell-derived cytokines probably contribute to
eotaxin-mediated tissue eosinophilia, because adoptive transfer of Th2
cells into mice induced Ag-dependent lung eotaxin expression and
eosinophilia (44). Also, IL-4, the prototypic Th2
cytokine, enhanced eotaxin expression by epithelial and endothelial
cells and dermal fibroblasts in vitro (30, 45), and
injection of IL-4 into rats induced eosinophil accumulation in skin
that was partially mediated by endogenous production of eotaxin
(46). Again, intratracheal mouse recombinant eotaxin into
IL-5 transgenic mice induced 4
integrin-dependent bronchial hyper-responsiveness and eosinophil
migration (47). IL-13, another Th2 cytokine, induced
eotaxin expression on epithelium (48). In addition, the
Th2 cytokines IL-4 and IL-13 both induce up-regulation of VCAM-1 on
endothelium, which is probably involved in eotaxin-induced eosinophil
accumulation. Finally, peptido-lipid mediators (i.e., leukotrienes
C4, D4, and
E4) as well as histamine may also regulate the
expression of C-C chemokines. We have shown in preliminary observations
that these agents can increase eotaxin expression on human endothelial
cells in vitro (49), indicating that these mediators may
contribute to the eosinophil influx by up-regulating eotaxin and other
C-C chemokines.
CCR3 mRNA is mainly associated with eosinophils, with limited expression by macrophages and mast cells (12), although a recent report identified CCR3 on blood basophils (50). Heath et al. (16) have shown that >95% of the response of eosinophils to eotaxin, RANTES, MCP-3, and MCP-4 is mediated through CCR3 receptor and could be blocked completely with an anti-CCR3 mAb. On the other hand, basophils express CCR1-CCR4 (24). In addition, it is known that RANTES interacts with two more receptors, CCR1 and CCR4 (24, 51), and that MCP-3 binds to CCR1 and CCR2 (24, 52), while MCP-4 binds to CCR2 and CCR3 (24, 31, 53). Thus, these C-C chemokines may contribute to the infiltration of other inflammatory cells, including macrophages in the bronchial mucosa of asthmatics.
It is worth noting that the elevated airway expression of all the chemokines studied was demonstrably not a feature of atopy per se, but instead a feature specific to asthma. In addition, it is noteworthy that C-C chemokine mRNA expression was detectable in a proportion of the control subjects. Similar observations were found in animal model. For instance, eotaxin expression and eosinophil infiltrates were detectable in lamina propria of the jejunum from normal wild-type mice, while eosinophils were reduced in the jejunum in eotaxin-deficient mice (54). Thus, a basal physiological degree of chemokine expression in the airway may be required for the trafficking of patrolling leukocytes involved in host defense.
Thus, we present evidence that combined production of various eosinophil-active chemokines within the bronchial mucosa is a characteristic feature of bronchial asthma regardless of its atopic status. The mechanisms controlling the transcriptional regulation of C-C chemokines are still poorly understood. CCR3 seems to be the principal receptor for eosinophil responses to C-C chemokines, and blocking this receptor could be a promising therapeutic tool in the management of asthma.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Prof. B. Kay, Department of Allergy and Clinical Immunology, National Heart and Lung Institute, Imperial College School of Medicine, Dovehouse Street, London, U.K. SW3 6LY. E-mail address:
3 Abbreviations used in this paper: ISH, in situ hybridization; FEV1, forced expiratory volume in 1 s; BAL, bronchoalveolar lavage; IHC, immunohistochemistry; MCP, monocyte chemoattractant protein; AA, atopic asthmatics; NAA, nonatopic asthmatics; AC, nonasthmatic atopic controls; NC, nonatopic nonasthmatic controls; RAST, radioallergosorbent; APAAP, alkaline phosphatase-antialkaline phosphatase.
Received for publication June 28, 1999. Accepted for publication September 17, 1999.
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 E. J. Cheadle, K. Riyad, D. Subar, D. G. Rothwell, G. Ashton, H. Batha, D. J. Sherlock, R. E. Hawkins, and D. E. Gilham Eotaxin-2 and Colorectal Cancer: A Potential Target for Immune Therapy Clin. Cancer Res., October 1, 2007; 13(19): 5719 - 5728. [Abstract] [Full Text] [PDF]
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 A. Mori, K. Ogawa, K. Someya, Y. Kunori, D. Nagakubo, O. Yoshie, F. Kitamura, T. Hiroi, and O. Kaminuma Selective suppression of Th2-mediated airway eosinophil infiltration by low-molecular weight CCR3 antagonists Int. Immunol., August 1, 2007; 19(8): 913 - 921. [Abstract] [Full Text] [PDF]
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M. E. Scheicher, M. M. Teixeira, F. Q. Cunha, A. L. Teixeira Jr, J. T. Filho, and E. O. Vianna Eotaxin-2 in sputum cell culture to evaluate asthma inflammation Eur. Respir. J., March 1, 2007; 29(3): 489 - 495. [Abstract] [Full Text] [PDF]
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M. S. Rahman, A. Yamasaki, J. Yang, L. Shan, A. J. Halayko, and A. S. Gounni IL-17A Induces Eotaxin-1/CC Chemokine Ligand 11 Expression in Human Airway Smooth Muscle Cells: Role of MAPK (Erk1/2, JNK, and p38) Pathways J. Immunol., September 15, 2006; 177(6): 4064 - 4071. [Abstract] [Full Text] [PDF]
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 C. A. Reichel, A. Khandoga, H.-J. Anders, D. Schlondorff, B. Luckow, and F. Krombach Chemokine receptors Ccr1, Ccr2, and Ccr5 mediate neutrophil migration to postischemic tissue J. Leukoc. Biol., January 1, 2006; 79(1): 114 - 122. [Abstract] [Full Text] [PDF]
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J. Fan, N. M. Heller, M. Gorospe, U. Atasoy, and C. Stellato The role of post-transcriptional regulation in chemokine gene expression in inflammation and allergy Eur. Respir. J., November 1, 2005; 26(5): 933 - 947. [Abstract] [Full Text] [PDF]
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P. Joubert, S. Lajoie-Kadoch, I. Labonte, A. S. Gounni, K. Maghni, V. Wellemans, J. Chakir, M. Laviolette, Q. Hamid, and B. Lamkhioued CCR3 Expression and Function in Asthmatic Airway Smooth Muscle Cells J. Immunol., August 15, 2005; 175(4): 2702 - 2708. [Abstract] [Full Text] [PDF]
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S. Ying, B. O'Connor, J. Ratoff, Q. Meng, K. Mallett, D. Cousins, D. Robinson, G. Zhang, J. Zhao, T. H. Lee, et al. Thymic Stromal Lymphopoietin Expression Is Increased in Asthmatic Airways and Correlates with Expression of Th2-Attracting Chemokines and Disease Severity J. Immunol., June 15, 2005; 174(12): 8183 - 8190. [Abstract] [Full Text] [PDF]
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S. Phipps, F. Benyahia, T.-T. Ou, J. Barkans, D. S. Robinson, and A. B. Kay Acute Allergen-Induced Airway Remodeling in Atopic Asthma Am. J. Respir. Cell Mol. Biol., December 1, 2004; 31(6): 626 - 632. [Abstract] [Full Text] [PDF]
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N. M. Heller, S. Matsukura, S. N. Georas, M. R. Boothby, P. B. Rothman, C. Stellato, and R. P. Schleimer Interferon-{gamma} Inhibits STAT6 Signal Transduction and Gene Expression in Human Airway Epithelial Cells Am. J. Respir. Cell Mol. Biol., November 1, 2004; 31(5): 573 - 582. [Abstract] [Full Text] [PDF]
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U. Forssmann, I. Hartung, R. Balder, B. Fuchs, S. E. Escher, N. Spodsberg, Y. Dulkys, M. Walden, A. Heitland, A. Braun, et al. n-Nonanoyl-CC Chemokine Ligand 14, a Potent CC Chemokine Ligand 14 Analogue That Prevents the Recruitment of Eosinophils in Allergic Airway Inflammation J. Immunol., September 1, 2004; 173(5): 3456 - 3466. [Abstract] [Full Text] [PDF]
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A. D. Schecter, A. B. Berman, L. Yi, H. Ma, C. M. Daly, K. Soejima, B. J. Rollins, I. F. Charo, and M. B. Taubman MCP-1-dependent signaling in CCR2-/- aortic smooth muscle cells J. Leukoc. Biol., June 1, 2004; 75(6): 1079 - 1085. [Abstract] [Full Text] [PDF]
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 D Bumbacea, J Scheerens, B S Mann, R G Stirling, and K F Chung Failure of sputum eosinophilia after eotaxin inhalation in asthma Thorax, May 1, 2004; 59(5): 372 - 375. [Abstract] [Full Text] [PDF]
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O. Kalayci, E. Birben, L. Wu, T. Oguma, K. Storm van's Gravesande, V. Subramaniam, H. K. Sheldon, E. S. Silverman, and C. M. Lilly Monocyte Chemoattractant Protein-4 Core Promoter Genetic Variants: Influence on YY-1 Affinity and Plasma Levels Am. J. Respir. Cell Mol. Biol., December 1, 2003; 29(6): 750 - 756. [Abstract] [Full Text] [PDF]
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U. Atasoy, S. L. Curry, I. Lopez de Silanes, A.-B. Shyu, V. Casolaro, M. Gorospe, and C. Stellato Regulation of Eotaxin Gene Expression by TNF-{alpha} and IL-4 Through mRNA Stabilization: Involvement of the RNA-Binding Protein HuR J. Immunol., October 15, 2003; 171(8): 4369 - 4378. [Abstract] [Full Text] [PDF]
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D. M. Conroy, L. A. Jopling, C. M. Lloyd, M. R. Hodge, D. P. Andrew, T. J. Williams, J. E. Pease, and I. Sabroe CCR4 blockade does not inhibit allergic airways inflammation J. Leukoc. Biol., October 1, 2003; 74(4): 558 - 563. [Abstract] [Full Text] [PDF]
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E. Rojas-Ramos, A.F. Avalos, L. Perez-Fernandez, F. Cuevas-Schacht, E. Valencia-Maqueda, and L.M. Teran Role of the chemokines RANTES, monocyte chemotactic proteins-3 and -4, and eotaxins-1 and -2 in childhood asthma Eur. Respir. J., August 1, 2003; 22(2): 310 - 316. [Abstract] [Full Text] [PDF]
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P. Forsythe and A. D. Befus CCR3: A Key to Mast Cell Phenotypic and Functional Diversity? Am. J. Respir. Cell Mol. Biol., April 1, 2003; 28(4): 405 - 409. [Full Text] [PDF]
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C.-H. Cui, T. Adachi, H. Oyamada, Y. Kamada, T. Kuwasaki, Y. Yamada, N. Saito, H. Kayaba, and J. Chihara The Role of Mitogen-Activated Protein Kinases in Eotaxin-Induced Cytokine Production from Bronchial Epithelial Cells Am. J. Respir. Cell Mol. Biol., September 1, 2002; 27(3): 329 - 335. [Abstract] [Full Text] [PDF]
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 N. Hizawa, E. Yamaguchi, S. Konno, Y. Tanino, E. Jinushi, and M. Nishimura A Functional Polymorphism in the RANTES Gene Promoter Is Associated with the Development of Late-Onset Asthma Am. J. Respir. Crit. Care Med., September 1, 2002; 166(5): 686 - 690. [Abstract] [Full Text] [PDF]
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 X.-Z. Shang, B.-C. Chiu, V. Stolberg, N. W. Lukacs, S. L. Kunkel, H. S. Murphy, and S. W. Chensue Eosinophil Recruitment in Type-2 Hypersensitivity Pulmonary Granulomas : Source and Contribution of Monocyte Chemotactic Protein-3 (CCL7) Am. J. Pathol., July 1, 2002; 161(1): 257 - 266. [Abstract] [Full Text] [PDF]
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J. M. Schuh, K. Blease, S. L. Kunkel, and C. M. Hogaboam Eotaxin/CCL11 is involved in acute, but not chronic, allergic airway responses to Aspergillus fumigatus Am J Physiol Lung Cell Mol Physiol, July 1, 2002; 283(1): L198 - L204. [Abstract] [Full Text] [PDF]
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S. A. Bryan, P. J. Jose, J. R. Topping, R. Wilhelm, C. Soderberg, D. Kertesz, P. J. Barnes, T. J. Williams, T. T. Hansel, and I. Sabroe Responses of Leukocytes to Chemokines in Whole Blood and Their Antagonism by Novel CC-Chemokine Receptor 3 Antagonists Am. J. Respir. Crit. Care Med., June 15, 2002; 165(12): 1602 - 1609. [Abstract] [Full Text] [PDF]
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S. H. P. Oliveira, S. Lira, C. Martinez-A, M. Wiekowski, L. Sullivan, and N. W. Lukacs Increased responsiveness of murine eosinophils to MIP-1{beta} (CCL4) and TCA-3 (CCL1) is mediated by their specific receptors, CCR5 and CCR8 J. Leukoc. Biol., June 1, 2002; 71(6): 1019 - 1025. [Abstract] [Full Text] [PDF]
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Z. Zhu, B. Ma, T. Zheng, R. J. Homer, C. G. Lee, I. F. Charo, P. Noble, and J. A. Elias IL-13-Induced Chemokine Responses in the Lung: Role of CCR2 in the Pathogenesis of IL-13-Induced Inflammation and Remodeling J. Immunol., March 15, 2002; 168(6): 2953 - 2962. [Abstract] [Full Text] [PDF]
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K. Watanabe, P. J. Jose, and S. M. Rankin Eotaxin-2 Generation Is Differentially Regulated by Lipopolysaccharide and IL-4 in Monocytes and Macrophages J. Immunol., February 15, 2002; 168(4): 1911 - 1918. [Abstract] [Full Text] [PDF]
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I. Sabroe, C.M. Lloyd, M.K.B. Whyte, S.K. Dower, T.J. Williams, and J.E. Pease Chemokines, innate and adaptive immunity, and respiratory disease Eur. Respir. J., February 1, 2002; 19(2): 350 - 355. [Abstract] [Full Text] [PDF]
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S. K. Banerjee, H. W. J. Young, J. B. Volmer, and M. R. Blackburn Gene expression profiling in inflammatory airway disease associated with elevated adenosine Am J Physiol Lung Cell Mol Physiol, February 1, 2002; 282(2): L169 - L182. [Abstract] [Full Text] [PDF]
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Y. Fujitani, Y. Kanaoka, K. Aritake, N. Uodome, K. Okazaki-Hatake, and Y. Urade Pronounced Eosinophilic Lung Inflammation and Th2 Cytokine Release in Human Lipocalin-Type Prostaglandin D Synthase Transgenic Mice J. Immunol., January 1, 2002; 168(1): 443 - 449. [Abstract] [Full Text] [PDF]
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 R. Martinelli, I. Sabroe, G. LaRosa, T. J. Williams, and J. E. Pease The CC Chemokine Eotaxin (CCL11) Is a Partial Agonist of CC Chemokine Receptor 2b J. Biol. Chem., November 9, 2001; 276(46): 42957 - 42964. [Abstract] [Full Text] [PDF]
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J. L. Chunn, H. W. J. Young, S. K. Banerjee, G. N. Colasurdo, and M. R. Blackburn Adenosine-Dependent Airway Inflammation and Hyperresponsiveness in Partially Adenosine Deaminase-Deficient Mice J. Immunol., October 15, 2001; 167(8): 4676 - 4685. [Abstract] [Full Text] [PDF]
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P.J. Barnes Cytokine modulators as novel therapies for airway disease Eur. Respir. J., July 2, 2001; 18(34_suppl): 67S - 77s. [Abstract] [Full Text] [PDF]
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N. Berkman, S. Ohnona, F. K. Chung, and R. Breuer Eotaxin-3 but Not Eotaxin Gene Expression Is Upregulated in Asthmatics 24 Hours after Allergen Challenge Am. J. Respir. Cell Mol. Biol., June 1, 2001; 24(6): 682 - 687. [Abstract] [Full Text] [PDF]
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M. Cecilia Berin, L. Eckmann, D. H. Broide, and M. F. Kagnoff Regulated Production of the T Helper 2-Type T-Cell Chemoattractant TARC by Human Bronchial Epithelial Cells In Vitro and in Human Lung Xenografts Am. J. Respir. Cell Mol. Biol., April 1, 2001; 24(4): 382 - 389. [Abstract] [Full Text]
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C. LAMBLIN, F. BOLARD, P. GOSSET, A. TSICOPOULOS, T. PEREZ, J. DARRAS, A. JANIN, A. B. TONNEL, Q. HAMID, and B. WALLAERT Bronchial Interleukin-5 and Eotaxin Expression in Nasal Polyposis . Relationship with (A)symptomatic Bronchial Hyperresponsiveness Am. J. Respir. Crit. Care Med., April 1, 2001; 163(5): 1226 - 1232. [Abstract] [Full Text]
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 W. W. Busse and R. F. Lemanske Asthma N. Engl. J. Med., February 1, 2001; 344(5): 350 - 362. [Full Text] [PDF]
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C. Stellato, M. E. Brummet, J. R. Plitt, S. Shahabuddin, F. M. Baroody, M. C. Liu, P. D. Ponath, and L. A. Beck Cutting Edge: Expression of the C-C Chemokine Receptor CCR3 in Human Airway Epithelial Cells J. Immunol., February 1, 2001; 166(3): 1457 - 1461. [Abstract] [Full Text] [PDF]
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A. Heinemann, A. Hartnell, V. E. L. Stubbs, K. Murakami, D. Soler, G. LaRosa, P. W. Askenase, T. J. Williams, and I. Sabroe Basophil Responses to Chemokines Are Regulated by Both Sequential and Cooperative Receptor Signaling J. Immunol., December 15, 2000; 165(12): 7224 - 7233. [Abstract] [Full Text] [PDF]
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N. Zimmermann, S. P. Hogan, A. Mishra, E. B. Brandt, T. R. Bodette, S. M. Pope, F. D. Finkelman, and M. E. Rothenberg Murine Eotaxin-2: A Constitutive Eosinophil Chemokine Induced by Allergen Challenge and IL-4 Overexpression J. Immunol., November 15, 2000; 165(10): 5839 - 5846. [Abstract] [Full Text] [PDF]
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L. N. KHAN, O. M. KON, A. J. MACFARLANE, Q. MENG, S. YING, N. C. BARNES, and A. BARRY KAY Attenuation of the Allergen-induced Late Asthmatic Reaction by Cyclosporin A Is Associated with Inhibition of Bronchial Eosinophils, Interleukin-5, Granulocyte Macrophage Colony-Stimulating Factor, and Eotaxin Am. J. Respir. Crit. Care Med., October 1, 2000; 162(4): 1377 - 1382. [Abstract] [Full Text] [PDF]
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B. LAMKHIOUED, E. A. GARCIA-ZEPEDA, S. ABI-YOUNES, H. NAKAMURA, S. JEDRZKIEWICZ, L. WAGNER, P. M. RENZI, Z. ALLAKHVERDI, C. LILLY, Q. HAMID, et al. Monocyte Chemoattractant Protein (MCP)-4 Expression in the Airways of Patients with Asthma . Induction in Epithelial Cells and Mononuclear Cells by Proinflammatory Cytokines Am. J. Respir. Crit. Care Med., August 1, 2000; 162(2): 723 - 732. [Abstract] [Full Text] [PDF]
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 U HELWIG, K M LAMMERS, P GIONCHETTI, F RIZZELLO, M CAMPIERI, M UGUCCIONI;, J WEDEMEYER, A LORENTZ, M P MANNS, and S C BISCHOFF MCP-3 in inflammatory bowel disease Reply Gut, July 1, 2000; 47(1): 155 - 155. [Full Text] [PDF]
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 S. P. Hogan, A. Mishra, E. B. Brandt, P. S. Foster, and M. E. Rothenberg A critical role for eotaxin in experimental oral antigen-induced eosinophilic gastrointestinal allergy PNAS, June 6, 2000; 97(12): 6681 - 6686. [Abstract] [Full Text] [PDF]
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J. R. White, J. M. Lee, K. Dede, C. S. Imburgia, A. J. Jurewicz, G. Chan, J. A. Fornwald, D. Dhanak, L. T. Christmann, M. G. Darcy, et al. Identification of Potent, Selective Non-peptide CC Chemokine Receptor-3 Antagonist That Inhibits Eotaxin-, Eotaxin-2-, and Monocyte Chemotactic Protein-4-induced Eosinophil Migration J. Biol. Chem., November 17, 2000; 275(47): 36626 - 36631. [Abstract] [Full Text] [PDF]
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