Eosinophil‘s Role Remains Uncertain as Anti–Interleukin-5 only Partially Depletes Numbers in Asthmatic Airway

Department of Allergy and Clinical Immunology, National Heart and Lung Institute, Faculty of Medicine, Imperial College, London, United Kingdom

	ABSTRACT

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The role of eosinophils as effector cells in asthma pathogenesis has been questioned since an anti–interleukin (IL)-5 monoclonal antibody (mepolizumab), which depleted blood and sputum eosinophils, failed to inhibit allergen-induced bronchoconstriction and airway hyperresponsiveness. However, the effect of IL-5 blockade on tissue eosinophils was not examined. We sought to determine whether mepolizumab depletes airway tissue eosinophils and their products. Twenty-four patients with mild asthma received three intravenous doses of either 750 mg of mepolizumab or placebo in a randomized, double-blind, parallel-group fashion over 20 weeks. Mepolizumab produced a median decrease from baseline of 55% for airway eosinophils (interquartile range, 29–89%; p = 0.009 versus placebo), 52% for bone marrow eosinophils (45–76%, p = 0.003), and 100% for blood eosinophils (range, 67–100%, p = 0.02). Mepolizumab had no appreciable effect on bronchial mucosal staining of eosinophil major basic protein. There were no significant changes in clinical measures of asthma (airway hyperresponsiveness, FEV₁, and peak flow recordings) between the mepolizumab and placebo-treated groups. Anti–IL-5 treatment reduces but does not deplete airway or bone marrow eosinophils. The role of the eosinophil remains uncertain. Further clinical studies in asthma with more effective antieosinophil strategies are required.

　

Key Words: eosinophils • asthma • anti–interleukin-5

	INTRODUCTION

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Asthma is a chronic inflammatory condition of the airways that is characterized by a prominent eosinophilic inflammatory infiltrate in the bronchial mucosa (1). Activated eosinophils secrete granular basic proteins that damage the bronchial epithelium and membrane-derived lipid mediators, which contract smooth muscle, increase mucous secretion, and cause vasodilation (2). The correlation between eosinophil numbers and disease severity (3) supports the hypothesis that the eosinophil is the central effector cell in ongoing airway inflammation in asthma.

Interleukin (IL)-5 is a key cytokine in eosinophil differentiation and maturation in the bone marrow as well as in recruitment and activation at sites of allergic inflammation (4). IL-5 stimulates the expansion and differentiation of eosinophil precursors (5), upregulates expression of its own specific receptor  chain during human eosinophil development (6), primes eosinophils for enhanced chemotaxis (7) and hyperadherence (8), and delays apoptosis (9). Clinical studies have shown an increase in IL-5 in bronchoalveolar lavage fluid (BALF) and bronchial biopsies in asthma at baseline (10). Concentrations of IL-5 correlated with clinical features (11), and IL-5 mRNA was upregulated in the bronchial mucosa after allergen-provoked asthma (12) and decreased after successful treatment with corticosteroids (13). In an ascaris model of allergen challenge in monkeys, anti–IL-5 almost completely abrogated eosinophilia and airway hyperresponsiveness (14).

For the previously mentioned reasons, IL-5 blockade was expected to deplete eosinophils and improve symptoms in subjects with asthma. Surprisingly, a humanized monoclonal antibody against IL-5 (mepolizumab), which effectively depleted eosinophils from blood and induced sputum in mild atopic subjects with asthma, had no effect on airway hyperresponsiveness or the late asthmatic reaction to inhaled allergen challenge (15). However, a critical, unanswered question was whether mepolizumab depleted bronchial mucosal eosinophils and their granule products. In this study, we have administered three doses of mepolizumab to subjects with mild asthma over a 20-week period to determine its effect on baseline bronchial mucosal eosinophils (and other cell types, including basophils and tissue deposition of major basic protein). Similar measurements were performed in the bone marrow and blood, and results were compared with clinical manifestations of asthma.

	METHODS

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Volunteers
Twenty-four volunteers with mild asthma, with a FEV₁ of 70% or more of predicted within an 18- to 55-year-old age range were recruited. All volunteers were atopic, as defined by a positive skin prick test to one or more aeroallergen. All were well controlled with short-acting ß₂-agonists, with no use of corticosteroids or other antiinflammatory drugs in the preceding 8 weeks. All volunteers gave a clear history of asthma, demonstrated airway hyperresponsiveness with a PC₂₀ to histamine of 4.0 mg/ml of less and were nonsmokers. The study was approved by the ethics committees of the Royal Brompton and Harefield NHS Trust and the London Chest Hospital, and all volunteers gave informed consent before participation in the study.

Study Design
This was a two-center, double-blind, placebo-controlled, parallel-group study based at the Royal Brompton and London Chest Hospitals. At an initial screening visit, baseline FEV₁ and histamine PC₂₀ were measured. From the first visit, volunteers kept a diary of their morning and evening peak expiratory flow rate (PEFR) for the duration of the study. Two weeks later, venous blood sampling, bone marrow aspiration, and fiberoptic bronchoscopy with endobronchial biopsy and bronchoalveolar lavage were performed. Two days later, the study medication (750 mg of mepolizumab or an equal volume of placebo) was administered as an intravenous infusion over 30 minutes in a double-blind fashion. The second and third infusions of study drug were given 4 and 8 weeks later, respectively. The blood sample, bone marrow aspiration, and bronchoscopy were repeated 2 weeks after the final infusion. Volunteers were then followed 4 and 8 weeks later with histamine PC₂₀ measurements  .

fig.ommitted Figure 1. Study design.

 
Tissue Processing and Immunohistochemistry
Bronchial mucosal biopsies were fixed, and immunohistochemistry was performed as described previously (10). The BALF was processed as described previously (12). Additional data on tissue and BALF processing and immunohistochemistry are provided in the online supplement. Measurement of major basic protein (MBP) deposition within the bronchial mucosal biopsies was performed as described previously (16). Additional data on measurement of MBP deposition are provided in the online supplement.

Blood eosinophils were measured within the department of hematology at the Royal Brompton Hospital using an Advia 120 cell counter (Bayer, Berkshire, UK).

After bone marrow aspiration, cytospins were prepared as described previously (17). Additional data on cytospin preparation and counting are provided in the online supplement.

Clinical Parameters
FEV₁ was measured according to standard operating procedure on a bellows spirometer (Vitalograph, Bucks, UK). The FEV₁ measured at Weeks 0 and 10 were used for the before and after comparisons. The histamine PC₂₀ was measured as described previously (18), and the before and after measurements were taken from the screening visit and at Week 12. PEFR measurements were recorded for the study duration. The before and after measurements for PEFR are the median values of the morning PEFR for the weeks preceding Weeks 0 and 10, respectively.

Statistical Analysis
The Wilcoxon signed rank test was used for within group paired comparisons. For between-group comparisons, the delta values of the effect of mepolizumab and placebo were compared (Mann-Whitney test). A p value of  0.05 was considered to be significant.

	RESULTS

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Eleven volunteers were randomized to receive mepolizumab and 13 to receive placebo. All 24 volunteers completed the study without reporting adverse events or asthma exacerbations. Both groups were well matched for baseline values  .

fig.ommitted TABLE 1. Baseline characteristics of mepolizumab- and placebo-treated groups

 
Peripheral Blood Eosinophils and Basophils
At 4 weeks after the first dose of mepolizumab, there was a significant decrease in peripheral blood eosinophil counts in the actively treated group when compared with placebo (p = 0.002). This decrease was maintained throughout the dosing period and was still evident at the time of the repeat bronchoscopy and bone marrow aspirate, Week 10 (p = 0.02)  . There was a median reduction of 100% from baseline of eosinophils in the actively treated group at Weeks 4 and 10 (interquartile range, 67–100%;  and ) . A return of blood eosinophil counts toward baseline was observed at a mean of 9 weeks after the last dose (range 4–20 weeks, data not shown).

fig.ommitted   Figure 2. Peripheral blood eosinophil count over the study duration in mepolizumab- and placebo-treated groups. Results are expressed as mean ± SEM.

 

fig.ommitted Figure 3. Effect of mepolizumab or placebo treatment on eosinophils within the bronchial mucosa, bone marrow, and blood. Eosinophils were measured using anti-MBP (BMK-13), modified Wright's stain, and Advia 120 cell counter, respectively.

 
BALF Eosinophils
Mepolizumab produced a 79% median reduction in BALF eosinophils (interquartile range, 43–99%) (p = 0.4 when compared with placebo)  .

fig.ommitted TABLE 2. Inflammatory cells in the airways and bone marrow before and after treatment with mepolizumab or placebo

 
Bronchial Mucosal Inflammation
Mepolizumab induced a significant decrease in the number of bronchial mucosal MBP-positive eosinophils compared with placebo treatment (p = 0.009) ( and  and ). The median reduction in airway mucosal MBP+ eosinophil numbers was 55.0% in those actively treated with mepolizumab (interquartile range, 29–89%). When bronchial biopsies were stained with a high-affinity antibody to detect both intracellular and released eosinophil major basic protein, there was no significant reduction in staining intensity after mepolizumab treatment, despite a 55% reduction in intact eosinophils, and no difference when compared with placebo ( and ). Compared with placebo, there were no significant changes in airway basophils, neutrophils, macrophages, mast cells, or CD3+ cells after treatment with mepolizumab .

fig.ommitted Figure 4. (A) Bronchial mucosal biopsies stained with a monoclonal antibody against MBP before and after mepolizumab with evidence of residual tissue eosinophils after treatment (positive staining is developed with Fast Red). (B) Immunofluorescent staining with a polyclonal anti-MBP antibody of bronchial biopsies before and after mepolizumab, demonstrating no significant change in the levels of extracellular MBP after anti–IL-5 treatment, despite a 55% decrease in intact tissue eosinophils.

 
Bone Marrow Cells
Treatment with mepolizumab induced a significant decrease in the percentage of eosinophils within the bone marrow when compared with placebo treatment (p = 0.003) with a median reduction in the actively treated group of 52.0% (interquartile range, 45–76%;  and ). There was a 26% reduction in the percentage of bone marrow basophils when mepolizumab treatment was compared with placebo (p = 0.09) .

Clinical Measures of Asthma
Within the mepolizumab-treated group, there was a significant increase in median morning peak flow between the run-in week and the week after the last dose of treatment (p < 0.05), although the treatment effect was not significant when compared with placebo (peak flow diaries were available from nine subjects in each group; ) . There were no changes in clinic FEV₁ or airway hyperresponsiveness measured at screening and Week 12 in the mepolizumab-treated group and no differences from placebo .

fig.ommitted TABLE 3. Clinical measurements of asthma before and after treatment with mepolizumab or placebo

 

	DISCUSSION

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A previous single-dose study of mepolizumab treatment in asthma reported no effect on allergen challenge or baseline airway hyperresponsiveness despite a substantial reduction in blood and sputum eosinophilia at the highest dose used (15) and was interpreted as evidence against the eosinophil hypothesis of asthma. Although the power of the lung function data in this study has been questioned (19), it did exclude any major effect of mepolizumab on the late asthmatic reaction. We have extended the work of Leckie and colleagues by demonstrating that treatment of subjects with mild asthma with three intravenous doses of mepolizumab-reduced blood eosinophils by a similar extent (median 100% from baseline) and BALF eosinophils by a similar magnitude to that seen in sputum (median 79% from baseline). However, mepolizumab only reduced airway tissue and bone marrow eosinophils by 50%. Furthermore, tissue staining with a high-affinity polyclonal antibody to eosinophil major basic protein showed no significant reduction with anti–IL-5 treatment. Thus, despite high dose anti–IL-5 treatment, there were residual airway eosinophils, with evidence of ongoing degranulation in subjects with asthma. We found no significant difference between anti–IL-5 and placebo on PEFR, airway hyperresponsiveness, or FEV₁. The relatively modest depletion of airway eosinophils by mepolizumab and residual evidence of eosinophil degranulation may explain the minimal clinical effects of such treatment in subjects with asthma.

The significant increase in morning PEFR is interesting and may be indicative of a small effect; however, there is no statistical significance between the groups, and the FEV₁ was unchanged. Similarly, a different humanized monoclonal antibody to IL-5 was reported to have no clinical effect in severe asthma (20). In subjects with asthma of similar severity to those studied here, we have previously reported that oral corticosteroid therapy reduced mucosal eosinophil infiltration by a median of 81% (21). We interpret our current findings as showing that mepolizumab does not deplete airway eosinophils sufficiently to impact appreciably on the clinical features of asthma.

This study was powered to detect changes in inflammatory cells in the bronchial mucosa rather than on clinical outcome measures. Therefore, given the size of this study, it is not possible to exclude a differential effect of mepolizumab on subgroups of subjects with asthma. Larger studies are required to allow subgroup analysis on the effects of mepolizumab on subjects with asthma with different levels of airflow obstruction and/or tissue eosinophilia.

The different levels of depletion of blood compared with bone marrow and bronchial mucosal eosinophils by anti–IL-5 treatment could be explained in several ways. First, the monoclonal antibody may simply not penetrate tissues or act systemically to reach bone marrow and bronchial mucosa. Previous data from other monoclonal antibody treatments and pharmacodynamic studies of distribution of anti–IL-5 in animal studies make this explanation unlikely (22). A second possibility is that although IL-5 is important in eosinophil development, activation, and survival, other cytokines can sustain tissue eosinophilia in its absence. Obvious candidates are IL-3 and granulocyte monocyte colony stimulating factor, both of which have been shown to be expressed at increased concentrations in asthmatic airways compared with control subjects (23). Third, our own recent observations indicate that incubation of blood eosinophils with IL-5 in vitro profoundly downregulated expression of the IL-5 receptor  chain with a corresponding reduction in IL-5 responsiveness and that this effect was long lasting (B. Gregory, manuscript in preparation). This raises the possibility that bone marrow and tissue eosinophils recently exposed to IL-5 during development and mobilization are less dependent on this cytokine for survival. This may explain persistence and activation of airway eosinophils despite effective depletion of IL-5.

The inability of mepolizumab to decrease tissue MBP, despite a 55% reduction in eosinophils, can be interpreted in two ways. It may simply be that MBP has a long tissue half-life, or more likely, that despite an apparent decrease in intact eosinophils, the level of eosinophilic degranulation within the bronchial mucosa remains unchanged. Evidence to support this comes from a study demonstrating significant MBP deposition within the skin of patients with atopic dermatitis in the absence of intact eosinophils (16).

Basophils are less numerous in the airway infiltrate in asthma (24), but through interaction of allergen with surface high-affinity immunoglobulin E, receptor-bound immunoglobulin E may also be important contributors to allergen-induced asthma symptoms (25). The lack of effect of anti–IL-5 treatment on airway basophil infiltration and modest effect on bone marrow basophils are perhaps surprising given previous reports of IL-5 responsiveness of these cells (26, 27). However, we have previously shown predominant expression of IL-3 receptors by these cells, with much more modest surface expression of receptors for IL-5 and granulocyte monocyte colony stimulating factor so that basophil infiltration and survival in the tissues may depend more on other cytokines (28).

The previous report of lack of effect of mepolizumab in allergen challenge, despite a dramatic reduction in blood and sputum eosinophils, has been widely interpreted as questioning the role of the eosinophil in asthma. This is despite considerable evidence linking eosinophils with airway pathology and clinical severity of asthma. Our findings demonstrate that even after three doses of mepolizumab there is residual airway eosinophilia, suggesting that this strategy fails to deplete this cell type in the target organ. Thus, the eosinophil cannot be excluded as a target for asthma therapy.

	Acknowledgments

 
The authors are grateful to Dr. Gerry Gleich for supplying the affinity chromatography-purified rabbit anti-human MBP antibody. Dr. Neil Barnes provided consultant cover for all volunteers at the London Chest Hospital.

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