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Contribution of Eotaxin-1 to Eosinophil Chemotactic Activity of Moderate and Severe Asthmatic Sputum

Respiratory Cell and Molecular Biology, Division of Infection, Inflammation, and Repair, University of Southampton School of Medicine, Southampton; Cambridge Antibody Technology, Cambridge, United Kingdom; and Pneumology and Allergology, Faculty of Medicine, University of Liège, Liège, Belgium

Correspondence and requests for reprints should be addressed to Gordon Dent, Ph.D., School of Life Sciences, Huxley Building, Keele University, Keele, Staffordshire ST5 5BG, UK. E-mail: g.dent{at}hfac.keele.ac.uk

	ABSTRACT

TOP ABSTRACT METHODS RESULTS DISCUSSION REFERENCES

 
The CC chemokine eotaxin-1 (CCL11) is chemotactic for eosinophils, basophils, and type 2 helper T cells and may play a role in allergic inflammation. We investigated its contribution as an eosinophil chemoattractant in asthmatic airway secretions (sampled as induced sputum), which possess chemotactic activity for eosinophils and T cells. Sputum samples collected from healthy subjects and subjects with mild, stable-moderate, unstable-moderate, and severe asthma were processed with phosphate-buffered saline and assayed for eotaxin by ELISA and for eosinophil chemotactic activity by fluorescence-based chemotaxis assay. The contribution of eotaxin to chemotactic activity was studied by using a high-affinity neutralizing human anti-eotaxin antibody, CAT-213. Sputum eotaxin concentration was significantly raised in moderate and severe asthma (p < 0.05 versus healthy control subjects) but not in mild asthma. Chemotactic activity was significantly increased in all asthmatic groups relative to healthy subjects (p < 0.05) and was significantly inhibited by CAT-213 (100 nM) in subjects with moderate and severe asthma, with median inhibition of 52% (p < 0.05), 78% (p < 0.0001), and 86% (p < 0.0001), respectively, in samples representing stable-moderate, unstable-moderate, and severe asthma. Eotaxin contributed to the eosinophil chemotactic activity of sputum from subjects with more severe forms of asthma but not mild asthma, suggesting that its contribution is more important in more severe disease. This activity is inhibited significantly by CAT-213.

Key Words: antibodies • asthma • chemokines • chemotaxis

The chemokine eotaxin (CC chemokine ligand 11, CCL11), initially purified from bronchoalveolar lavage (BAL) fluid of allergen-sensitized guinea pigs (1), has been implicated in many manifestations of human allergic inflammation involving eosinophil infiltration (2), including bronchial asthma (3). It is a potent chemotactic factor for eosinophils (4) that binds with high affinity to CC chemokine receptor 3 (CCR3), which is expressed on cells that are central to asthma pathogenesis, such as eosinophils, basophils, and a subset of T lymphocytes (the helper T cell type 2 subpopulation of clonal T cells) (5–7). Eotaxin levels have been shown to be elevated in BAL fluid, induced sputum, and plasma of subjects with asthma, and its expression is also increased in the bronchial mucosa in asthma (8, 9). Sputum levels of eotaxin have been shown to correlate with eosinophil counts, whereas plasma levels contribute to the odds of a diagnosis of asthma and impaired lung function but correlate poorly with severity indices of asthma and atopy such as forced expiratory volume in 1 second (FEV₁), blood eosinophilia, and serum IgE (9, 10). Several cellular sources of eotaxin in the lungs have been identified, including T lymphocytes, macrophages, eosinophils, smooth muscle, fibroblasts, and bronchial epithelial cells (8, 11–15), but the relative contribution of each of these to the overall quantities of eotaxin produced is unknown.

Airway eosinophilia is a characteristic feature of asthma that persists despite treatment with inhaled and/or oral corticosteroids. In some studies it has been shown to correlate broadly with the degree of airway hyperresponsiveness and disease severity, but more importantly it has been shown to be related to asthma exacerbation, with studies showing that treatment targeted at eosinophil reduction in sputum reduces rates of exacerbation (16–20). Identification of the major chemoattractants for eosinophils and other inflammatory cells may, therefore, provide opportunities for the development of putative asthma therapies, and chemoattractants that evade the regulatory effects of corticosteroids might present novel targets for therapeutic intervention. Eotaxin is one candidate, being elevated in induced sputum of patients with asthma who are treated with inhaled corticosteroids (8, 9), although it is not known to what extent this accounts for any chemotactic activity in the airways. We have previously shown clearly elevated eosinophil chemotactic activity in induced sputum from patients with asthma (18), but have not related this activity either to disease severity or to any particular chemoattractant.

The aim of this study was, therefore, to relate airway eotaxin levels and its contribution as a chemoattractant for airway eosinophils to asthma severity. Our hypothesis was that both increase in relation to asthma severity despite the use of corticosteroids, identifying eotaxin and its chemotactic activity as a target for treatment in poorly controlled asthma.

CAT-213 is a human anti-eotaxin IgG4 antibody that was isolated by phage display technology (S. Main, R. Handy, J. Wilton, S. Smith, L. Williams, L. Du Fou, J. Andrews, L. Conroy, R. May, I. Anderson, and T. Vaughan, unpublished data). It neutralizes the ability of human eotaxin to cause chemotaxis and increase intracellular Ca²⁺ levels in human CCR3-transfected cells and inhibits human eotaxin-induced shape change of human eosinophils in vitro (21). CAT-213 is highly specific for human eotaxin and does not bind to a number of other chemokines and cytokines including eotaxin-2 (CCL24) and eotaxin-3 (CCL26), monocyte chemotactic proteins 1–4 (CCL2, CCL8, CCL7, and CCL13), RANTES (regulated upon activation, normal T cell expressed and secreted; CCL5), and tumor necrosis factor- (S. Main, R. Handy, J. Wilton, S. Smith, L. Williams, L. Du Fou, J. Andrews, L. Conroy, R. May, I. Anderson, and T. Vaughan, unpublished observations). This antibody is, therefore, a useful tool for elucidation of the contribution of eotaxin to the chemotactic activity of airway secretions.

To address the objectives of our study we have used induced sputum as a means of sampling the airway lining fluid as a source of chemotactic activity for eosinophils. We have used CAT-213 to inhibit in vitro the eosinophil chemotactic activity that is present in sputum (18). Some of the results of these studies have been previously reported in the form of abstracts (22, 23).

	METHODS

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Materials
Synthetic human eotaxin was purchased from Albachem (Edinburgh, UK). Human eotaxin ELISA kits were purchased from R&D Systems (Abingdon, UK). Forty-eight-well acrylic microchemotaxis chambers and polycarbonate filter membranes were supplied by NeuroProbe (Gaithersburg, MD). Calcein (3,3'-bis[N,N-bis-(carboxymethyl)-aminomethyl]-fluorescein) acetoxymethyl ester was purchased from Molecular Probes (Leiden, The Netherlands). Hanks' balanced salt solution and N-2-hydroxyethylpiperazine-N'-ethane sulfonic acid solution were from Invitrogen (Paisley, UK); sterile bovine serum albumin solution and general-use protease inhibitor cocktail [containing 4-(2-aminoethyl)-benzenesulfonyl fluoride, aprotinin, bestatin, E-64, and leupeptin] were from Sigma (Poole, UK).

Anti-human eotaxin-1 human monoclonal antibody IgG4, CAT-213, and human null control antibody IgG4, CAT-001, were produced by Cambridge Antibody Technology (Cambridge, UK).

Subjects
Sixty-seven volunteers were recruited as sputum donors. Twelve nonsmoking subjects with no positive skin-prick response to common aeroallergens or history of respiratory disease acted as a control group. Fifty-five subjects with a physician diagnosis of asthma and positive skin-prick responses to at least one common aeroallergen were allocated to subgroups according to disease severity, as defined by symptoms and requirement for drug therapy (Table 1) . Subject characteristics are summarized in Table 2 .

Sputum Induction and Processing
Sputum induction was performed with hypertonic saline, as previously described (18).

In an extensive series of validation experiments, eotaxin was found to be undetectable by ELISA in dithioerythritol (DTE)-processed sputum samples, owing to a loss of immunoreactivity probably resulting from thiol reduction of disulfide bridges (24). Therefore, expectorated samples were divided and processed by two parallel procedures. A small quantity of sputum was processed with DTE (1 volume of DTE per volume of sputum) and used for total and differential cell counts, as described (18). The remainder of the whole, unselected sample was mixed with phosphate-buffered saline (PBS) containing protease inhibitors (4 volumes of PBS per volume of sputum) and vortex-mixed vigorously. The PBS-treated samples were filtered through gauze and the supernatants were centrifuged at 400 x g for 10 minutes to precipitate cells and mucus. Supernatants were decanted and stored at –80°C until required for assay: no reduction in detectable eotaxin concentration was observed during storage of up to 6 months. The PBS-processed samples were used for immunoassay and chemotaxis assay. Supernatants were subjected to sonication and high-speed centrifugation (12,000 x g for 2 minutes) immediately before use to clarify the samples and remove any residual insoluble mucus.

Eosinophil Isolation
Eosinophils were isolated from heparin-anticoagulated peripheral blood of volunteers with mild asthma by differential density centrifugation and negative immunomagnetic selection, as described (25). The blood donors were different from those who donated sputum.

Eosinophils were labeled with calcein, as described (26). Calcein does not affect the migratory responses of cells (27), and this was confirmed in preliminary experiments comparing chemotactic responses assessed by microscopic cell counting of unlabeled and calcein-labeled granulocytes (not shown).

Chemotaxis Assay
Eosinophil chemotaxis was measured in a fluorescence-based modified Boyden chamber assay, essentially as described (18) but using cell-associated fluorescence to quantify the magnitude of responses. Briefly, medium (Hanks' balanced salt solution supplemented with 10 mM N-2-hydroxyethylpiperazine-N'-ethane sulfonic acid and 0.35% [wt/vol] bovine serum albumin), eotaxin, or sputum supernatants were mixed in a 1:1 ratio with medium or antibodies and preincubated for 30 minutes at 37°C in the lower wells of 48-well microchemotaxis chambers. The lower wells were covered with uncoated 8-µm pore-size polycarbonate membrane filters before assembly of the chambers. Eosinophils (1–2 x 10⁵/well) were added to the upper wells and the chambers were incubated for a further 1 hour at 37°C. Chambers were dismantled and nonmigrated cells were scraped from the upper surface of the filters. Filters were allowed to dry and cell-associated fluorescence within and on the lower surface of the filters was quantified with a Syngene bioimager (Gene Genius; Synoptics, Cambridge, UK).

Data are expressed as a chemotactic index, calculated as the number of cells migrating in response to stimulus divided by the number of cells migrating in response to medium.

Eotaxin Immunoassay
Eotaxin was measured in sputum supernatants by ELISA (R&D Systems). The detection limit of the assay corresponded to a sputum eotaxin concentration of 2 pM. To prevent the exclusion of low values, which would distort the group statistics, samples with an eotaxin concentration below 2 pM were assigned a value of zero for statistical analyses.

Statistical Analysis
Estimates of eotaxin median effective concentration (EC₅₀) and CAT-213 median inhibitory concentration (IC₅₀) values were made by nonlinear regression on a four-parameter logistic model (SigmaPlot 8.0 for Windows; SPSS, Chicago, IL) and are expressed as geometric mean with 95% confidence intervals from the indicated numbers of experiments.

All statistical analyses were performed with InStat for Windows 3.0 (GraphPad Software, San Diego, CA). Chemotactic indices for each group of samples were evaluated by one-sample t test versus a hypothetical mean of 1.0 (representing zero net chemotactic activity). Sputum eosinophil counts and eotaxin concentrations were not normally distributed (p < 0.0001 by Kolmogorov–Smirnov test for normality) and chemotactic indices exhibited significantly different standard deviations among subject groups; therefore, nonparametric tests were used for all comparisons. Data for individual variables from the five subject groups were analyzed by the Kruskal–Wallis test, with post hoc pairwise comparisons of groups performed by the Dunn multiple comparisons test. Comparison of pairs of variables was performed by rank correlation. For evaluation of the effects of CAT-213 and CAT-001, chemotactic responses to medium-, CAT-001-, and CAT-213-pretreated sputum samples were compared within each subject group by the Friedmann test with post hoc Dunn multiple comparisons test. In all cases, a probability (p) less than 0.05 was defined as significant.

	RESULTS

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Neutralization of Chemotactic Activity of Human Eotaxin by CAT-213
Synthetic human eotaxin induced chemotactic responses in asthmatic human eosinophils with an EC₅₀ of 15 nM (5.0–43 nM) (Figure 1a) . The chemotactic activity of a submaximally effective concentration of eotaxin (30 nM) was neutralized concentration dependently by a 30-minute preincubation with CAT-213 (IC₅₀ of 0.95 nM [0.03–33 nM]) but not by the isotype control antibody, CAT-001 (Figure 1b). Neutralization by CAT-213 was essentially complete at 100 nM (95 ± 0.31% inhibition).

View larger version (21K): [in this window] [in a new window]   Figure 1. Neutralization of eotaxin chemotactic activity by CAT-213. (a) Chemotactic response of asthmatic human eosinophils to synthetic human eotaxin. Data represent means ± SEM of five experiments conducted in triplicate. (b) Response to 30 nM eotaxin preincubated for 30 minutes with CAT-213 (10 pM–100 nM) or isotype control antibody CAT-001 (100 nM). Data represent means ± SEM of three experiments conducted in triplicate.

View larger version (30K): [in this window] [in a new window]   Figure 2. Eotaxin concentrations in induced sputum supernatants from healthy subjects and from subjects with asthma. For group classifications, see Table 1. Concentrations were corrected for dilution through sputum processing and indicate original concentrations in expectorated sputum. The dashed line indicates the lower detection limit of the assay: samples with lower concentrations were assigned a value of zero (see text). Horizontal lines indicate the median; boxes indicate the interquartile range; whiskers indicate 10th and 90th percentiles. *p < 0.05 compared with healthy control group.

View larger version (23K): [in this window] [in a new window]   Figure 3. Eosinophil chemotactic activity of induced sputum supernatants from healthy subjects and from subjects with asthma. For group classifications, see Table 1. The dashed line indicates baseline cell migration. Data are expressed as chemotactic index (see text) and are shown as median, interquartile range, and 10th and 90th percentiles, as described in Figure 2. **p < 0.01, ***p < 0.001 compared with healthy control group.

Strikingly, both sputum eosinophil count and sputum eotaxin concentration exhibited significant negative rank correlation with FEV₁ for all subjects with asthma (Figures 4a and 4b) , indicating that more severe airflow limitation is related to higher levels of sputum eosinophilia and eotaxin. No significant correlation was observed between FEV₁ and sputum eosinophil chemotactic activity (r_s = –0.030, p = 0.829).

View larger version (19K): [in this window] [in a new window]   Figure 4. Correlation between FEV1 and (a) sputum eosinophil count (n = 55), (b) sputum eotaxin concentration (n = 57), and (c) percent inhibition of sputum eosinophil chemotactic activity by the anti-eotaxin antibody CAT-213 (n = 48). Spearman rank correlation coefficients are shown for all subjects with asthma in whose sputum samples the indices were measured or evaluated. For clarity, eotaxin concentrations are plotted on a logarithmic scale and zero values are shown as 1; however, correlation was performed with raw data.

Preincubation of sputum supernatants with 100 nM CAT-001 for 30 minutes at 37°C had no effect on the eosinophil chemotactic activity of samples from any group. Preincubation of healthy control or mild asthmatic samples with 100 nM CAT-213 also had no effect on chemotactic activity. However, samples from both moderate asthmatic groups and the severe asthmatic group exhibited significant inhibition of their eosinophil chemotactic activity after preincubation with CAT-213 (Figure 5) , with median inhibition of 51.9% (interquartile range, 20.0–65.8%), 77.5% (58.1–97.1%), and 86.1% (60.3–110%) for stable-moderate, unstable-moderate, and severe asthmatic groups, respectively.

View larger version (23K): [in this window] [in a new window]   Figure 5. Effects of CAT-213 on eosinophil chemotactic activity of sputum supernatants. Supernatants were preincubated with medium, isotype control antibody (CAT-001, 100 nM), or CAT-213 (100 nM) for 30 minutes before addition of eosinophils to chemotaxis chambers. Dashed lines indicate baseline cell migration. Responses are expressed as chemotactic index (see text) and are shown as median, interquartile range, and 10th and 90th percentiles from 12 samples per group, as described in Figure 2. *p < 0.05, ****p < 0.0001 compared with medium.

	DISCUSSION

TOP ABSTRACT METHODS RESULTS DISCUSSION REFERENCES

 
Using a high-affinity human anti-eotaxin antibody, we have demonstrated that eotaxin is a major chemoattractant for eosinophils in asthma. The expression of eotaxin and its contribution to chemotaxis of eosinophils is related to the severity of asthma, with more than 80% of the chemotactic activity in moderate to severe disease being attributable to this chemokine. This identifies eotaxin in the airway lining fluid as a valid target for this novel antibody and suggests that the greatest value of its application to the treatment of asthma may be in moderate to severe disease.

This study extends the observations made in past studies (8–10, 28). In one of these, only partial (about 28%) inhibition of eosinophil chemotactic activity in asthmatic BAL fluid was demonstrated (8). Because this study used a polyclonal neutralizing antibody with undefined affinity, it is possible that a greater degree of inhibition might have been achieved if a high-affinity antibody, such as CAT-213, had been used. In contrast to our study, the patients in the study of Lamkhioued and coworkers had mild steroid-naive disease (8), which, as our study suggests, might have led to an underestimation of the contribution of eotaxin to more severe forms of asthma. The subject population was small and incompletely characterized with respect to severity, making it difficult to fully appreciate the clinical relevance. Furthermore, it is likely that there are differences between sputum and BAL fluid, which sample different compartments of the lungs. It should be noted that the chemotactic indices achieved with BAL fluid were substantially higher than those reported here for sputum supernatants, suggesting that the activity of chemoattractants is affected by the origin, nature, and/or processing of the sample. The procedure used for processing of sputum in our study was found to be optimal for eotaxin determination by ELISA (24) and gave reproducible activity in a range of chemotaxis assays (our unpublished observations). The fact that these were optimal conditions for recovery of eotaxin may render the samples processed in this way exceptionally rich in this chemokine, leading to the inhibitory effects of the anti-eotaxin antibody being apparent to an extent that would not have been observed in samples processed in different ways.

In addition to associating eosinophil chemotaxis with disease severity, our study is novel with respect to quantifying the contribution of eotaxin to this process. The ability of CAT-213 to neutralize the chemotactic activity of moderate and severe asthmatic sputum was striking, with the median chemotactic index in the patients with severe asthma being reduced to 1.2, indicating that migration induced by factors present in the airway lining fluid in the presence of CAT-213 was barely different from spontaneous migration (migration in medium alone). No significant inhibition of the chemotactic activity was observed when using sputum from patients with mild asthma, but all the remaining asthmatic groups showed significant inhibition of their activity by the antibody.

The contribution of eotaxin, as assessed by the magnitude of inhibition, was surprising in view of the low concentrations of eotaxin present in the samples: the median concentration of 30 pmol/L in samples from the unstable-moderate group is below the range of concentrations of synthetic human recombinant eotaxin required to induce eosinophil chemotaxis (Figure 1a). This might reflect the presence in asthmatic sputum samples of other cytokines that prime eosinophils for chemotactic responses to eotaxin or synergy with other chemokines and lipid mediators. It should be noted that the concentrations of eotaxin measured in BAL fluid by Lamkhioued and coworkers were also about two orders of magnitude lower than the concentrations of recombinant eotaxin required to induce chemotactic responses, even in interleukin (IL)-5-primed eosinophils (8). The possibility must also be considered, however, that the procedures used for determining levels of eotaxin in sputum samples might systematically underestimate the absolute quantities. The use of PBS—as opposed to thiol-reducing agents such as DTE—for sample processing does not solubilize mucus as effectively as DTE and may leave a proportion of the analyte trapped in mucus, which is precipitated along with cells during centrifugation (24). This is an unlikely explanation given the excellent (100%) spiking recovery of recombinant eotaxin, although it is possible that recombinant and native eotaxin might interact differently with mucins. Furthermore, the ELISA uses antibodies raised against recombinant proteins for both trapping and labeling of analytes. This introduces the issue of differing immunoreactivity of recombinant and native proteins: if an extensively glycosylated native protein binds to the ELISA antibodies with lower affinity than does the recombinant standard, the assay will consistently give measurements that are lower than the actual concentration of the analyte (29). Standardized preparations of native proteins are needed to overcome this problem, and these are being developed and cataloged by the World Health Organization (29). Finally, it is conceivable that native eotaxin might possess greater potency as a CCR3 agonist than either the recombinant protein or the synthetic protein used in our study. In view of these issues, we would recommend that attention be focused on the relative levels of eotaxin in the various groups studied here, and their relationship to airflow obstruction and eotaxin dependence of sputum chemotactic activity, rather than the absolute quantities. We would also suggest that functional assays, as conducted in this study, provide more relevant information about the role of the mediator regardless of issues related to detection and quantification by immunoassay.

Bearing these caveats in mind, the significantly elevated eotaxin concentrations in unstable-moderate and severe asthmatic sputum samples (median values of 30 and 21 pmol/L, respectively) correspond to 250 and 175 pg/ml; these are approximately half the levels reported in patients with unstable asthma by Yamada and coworkers (9). This difference probably reflects the fact that the earlier study selected the viscous parts of sputum, whereas in this study whole sputum samples were used. In our study more stringent statistical tests were used to take into account multiple comparisons. These methodologic differences notwithstanding, the magnitude of the difference in sputum eotaxin levels between healthy control subjects (mean, 14 pg/ml or 1.7 pmol/L) and corticosteroid-treated patients with stable asthma (mean, 111 pg/ml or 13 pmol/L) in that study is sufficiently large to suggest that corticosteroid-treated patients with stable-moderate asthma might also have raised levels of eotaxin.

Although Yamada and coworkers demonstrated a significant positive correlation between sputum concentrations of eotaxin and eosinophil cationic protein (a marker of eosinophil degranulation), they found no correlation between eotaxin concentrations and sputum eosinophil numbers for three of four asthmatic patient groups, with only corticosteroid-free patients with stable asthma exhibiting such a correlation (9). We similarly failed to show any correlation between eotaxin levels and eosinophil numbers for any subject group; nor did we find a correlation between sputum eosinophil chemotactic activity and eosinophil numbers for four of our five subject groups, with only patients with stable-moderate asthma showing a significant correlation. Eotaxin production in the airways has been shown to precede eosinophil accumulation that results from allergen challenge and to peak simultaneously with bronchoalveolar eosinophil numbers (3, 30). This temporal association might suggest a cause–effect relationship. However, eosinophil numbers in the airway lumen are unlikely to be closely related to chemoattractant levels because they reflect the sum effect of chemotactic migration, cell survival within the airways, and cell clearance via the mucociliary apparatus. Furthermore, airway eosinophilia is a result of the combination of chemoattractant gradients that exist between the inflamed tissue and the circulation, adhesion mechanisms, and factors that regulate cell survival. It is possible that different factors account for varying proportions of the total sputum chemotactic activity in each individual and that the total sputum eosinophilia is accounted for by the local concentrations of eosinophil survival factors at least as much as by levels of chemoattractants.

We did, however, observe significant negative correlations between FEV₁ and three indices in the total set of asthmatic sputum samples, namely eosinophil count, eotaxin concentration, and percent inhibition of eosinophil chemotactic activity by CAT-213. The latter shows that subjects with asthma whose eosinophil chemotactic activity is dependent on eotaxin tend to have more severely impaired lung function. This provides further indication of the particular importance of eotaxin as a factor that determines eosinophil recruitment in more severe asthma, and also highlights the benefit of conducting functional assays when attempting to determine the contribution of a mediator to the disease process.

Our definition of disease severity was based on both symptoms and requirement for therapy (Table 1). Given that greater disease severity was associated with greater use of inhaled—and, in the severe asthmatic group, oral—glucocorticoids, the potential influence of corticosteroids on eotaxin production within the airways must be considered. Corticosteroid therapy was associated with significantly reduced eotaxin sputum concentrations, but not reduced eosinophil numbers, in subjects with stable asthma (9). This suggests that the pathologic significance of eotaxin in airway secretions might vary with disease status: patients whose disease is controlled by corticosteroid therapy have lower sputum concentrations of eotaxin than patients exhibiting a similar severity of symptoms despite their not having required treatment with corticosteroids. On the other hand, it may be the case that corticosteroids fail to affect other eosinophil chemotactic or priming factors, so that the reduced levels of eotaxin do not result in reduced airway eosinophilia or severity of disease compared with those patients not requiring corticosteroids. Strikingly, corticosteroid therapy was not associated with reduced sputum eotaxin concentrations in patients with severe asthma (9), possibly indicating a shift in emphasis toward steroid-insensitive cells as a source of eotaxin in severe asthmatic airways.

Antibodies are proving to be an important class of drugs and are also likely to be important in the treatment of asthma. Long elimination half-life (about 2–3 weeks) and good bioavailability after subcutaneous delivery make them suitable for chronic diseases such as rheumatoid arthritis and asthma. Adalimumab (anti–tumor necrosis factor- human IgG1) has been shown to provide disease-modifying treatment for patients with rheumatoid arthritis; this antibody can be distinguished from other immunoglobulin treatments because it is a human protein generated by phage display technology (31). The anti-IgE antibody omalizumab, the first antibody to be approved for the treatment of asthma, reduces exacerbations and corticosteroid use in patients with allergic asthma (32). Like adalimumab, CAT-213 is a human antibody generated by phage display technology. The pharmacology of CAT-213 has been characterized previously (21). In a Phase I single-dose clinical study CAT-213 was shown to be safe and well tolerated, remaining present in serum in an active form for more than 60 days after intravenous injection (33). In a more recent clinical study, CAT-213 reduced mucosal eosinophil infiltration induced by allergen challenge in subjects with rhinitis (34, 35). CAT-213 has been shown to inhibit eosinophil responses in vitro and in vivo in animals and humans.

Given the persistence of eosinophils in more severe forms of asthma and in particular the association with asthmatic exacerbations (16) it is possible that CAT-213 could reduce symptoms in asthmatic patients with more severe disease. Initial trials with an anti–IL-5 antibody, which reduced blood and sputum eosinophil numbers to negligible levels, were disappointing in that they failed to show efficacy in asthma (36). Even though this antibody caused a significant reduction in sputum eosinophils induced by allergen challenge, it had no effect on the associated airway responses. This led to a controversy about the role not only of IL-5 but also eosinophils as factors of importance in asthma. However, the persistence of airway tissue eosinophilia after neutralization of IL-5 has called these conclusions into question (37). As further evidence of the pathogenetic role of eosinophils, in an uncontrolled study, the anti–IL-5 antibody mepolizumab caused a marked reduction in eosinophil numbers in blood and affected skin of patients with hypereosinophilic syndrome and atopic dermatitis that was associated with clinical improvement (38). A potential added value of an anti-eotaxin antibody is that eotaxin is also a chemoattractant for basophils and a subset of helper T Type 2 cells, which also play important roles in asthma pathogenesis and pathophysiology. Future research should focus on elucidating whether CAT-123 is also able to inhibit those activities.

In conclusion, eotaxin accounts for a significant proportion of the eosinophil chemotactic activity of induced sputum from subjects with moderate and severe asthma and this proportion increases with increasing asthma severity. This suggests that neutralizing antibodies directed against eotaxin may have therapeutic potential in the control of airway eosinophilia in moderate and severe asthma.

	Acknowledgments

 
The authors gratefully acknowledge the valuable contributions of Gilbert Angco, Jon Ward, Monique Henket, and Dr. Rory O'Donnell to volunteer recruitment, clinical assessment, and sputum induction.

	FOOTNOTES

 
Supported by a grant from Cambridge Antibody Technology.

Conflict of Interest Statement: G.D. received $187,000 from Bayer in 2002–2003 as research support, was paid $1,000 by Merck as an honorarium for an article in Leukotriene Research & Clinical Review and has been reimbursed by Cambridge Antibody Technology for attendance at conferences and has a grant for this study funded by Cambridge Antibody Technology; C.H. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript; T.Y. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript; R.L.C.H. was employed full-time by Cambridge Antibody Technology from June 1998 to June 2003 and as an employee holds free and purchased shares and share options, currently valued <$10,000; J.P. has been employed full-time by Cambridge Antibody Technology since June 1999 and as an employee he holds free and purchased shares and share options, currently valued <$10,000; I.K.A. has been employed full-time by Cambridge Antibody Technology since March 1998 and as an employee he holds free and purchased shares and share options, currently valued <$10,000; R.L. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript; D.E.D. has a grant for this study funded by Cambridge Antibody Technology; R.D. has been on the advisory board of Aventis for the past 2 years and has received $1,500 for lectures sponsored by GlaxoSmithKline and has received sponsorship for basic research from AstraZeneca, Cambridge Antibody Technology (for the research reported in this paper), Immunex, GlaxoSmithKline and Bayer totaling $1,545,582 and has received $349,000 from GlaxoSmithKline for participating in two multi-center studies of the antiinflammatory effects of Advair and has a grant for this study funded by Cambridge Antibody Technology.

Received in original form June 27, 2003; accepted in final form March 2, 2004

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