Mepolizumab Attenuates Airway Eosinophil Numbers, but Not Their Functional Phenotype, in Asthma

Abstract

Rationale: Mepolizumab, an IL-5-blocking antibody, reduces exacerbations in patients with severe eosinophilic asthma. Mepolizumab arrests eosinophil maturation; however, the functional phenotype of eosinophils that persist in the blood and airway after administration of IL-5 neutralizing antibodies has not been reported.

Objectives: To determine the effect of anti-IL-5 antibody on the numbers and phenotypes of allergen-induced circulating and airway eosinophils.

Methods: Airway inflammation was elicited in participants with mild allergic asthma by segmental allergen challenge before and 1 month after a single intravenous 750-mg dose of mepolizumab. Eosinophils were examined in blood, bronchoalveolar lavage, and endobronchial biopsies 48 hours after challenge.

Measurements and main results: Segmental challenge without mepolizumab induced a rise in circulating eosinophils, bronchoalveolar lavage eosinophilia, and eosinophil peroxidase deposition in bronchial mucosa. IL-5 neutralization before allergen challenge abolished the allergen-induced rise in circulating eosinophils and expression of IL-3 receptors, whereas airway eosinophilia and eosinophil peroxidase deposition were blunted but not eliminated. Before mepolizumab treatment, bronchoalveolar lavage eosinophils had more surface IL-3 and granulocyte-monocyte colony-stimulating factor receptors, CD69, CD44, and CD23 and decreased IL-5 and eotaxin receptors than blood eosinophils. This activation phenotype indicated by bronchoalveolar lavage eosinophil surface markers, as well as the release of eosinophil peroxidase by eosinophils in the bronchial mucosa, was maintained after mepolizumab.

Conclusions: Mepolizumab reduced airway eosinophil numbers but had a limited effect on airway eosinophil activation markers, suggesting that these cells retain functionality. This observation may explain why IL-5 neutralization reduces but does not completely eradicate asthma exacerbations. Clinical trial registered with www.clinicaltrials.gov (NCT00802438).

Keywords: IL-3 receptor; IL-5; allergen challenge.

Figure 1.

Study design. This was an experimental, nonclinical study rather than a traditional pharmaceutical industry–sponsored “clinical trial.” Participants with mild allergic asthma underwent whole-lung allergen inhalation challenge (WLAC) to determine the allergen provocation dose resulting in a 20% reduction in FEV₁ (AgPD₂₀). An early allergic reaction (EAR), defined as a drop in FEV₁ ≥ 20% within 1 hour of challenge, was required for continuation. One month later, bronchoscopy with bronchoalveolar lavage (BAL) was performed in two subsegments followed by segmental bronchoprovocation with allergen (SBP-Ag) at a dose of 1% (low dose) or 20% (high dose) of the subject’s AgPD₂₀. Forty-eight hours later, bronchoscopy with BAL was performed in each subsegment and biopsies (Bx) were taken from an antigen-naive site and from the high-dose segment. BAL eosinophilia greater than 50% in the high-dose segment was required for continuation. One month after SBP-Ag, spirometry was performed to confirm continued asthma stability, and then a single 750-mg intravenous dose of mepolizumab was administered. One month after dosing, circulating eosinophils were required to be decreased by at least 80% of the baseline value, or fewer than 100 eosinophils/μl. If the threshold was not met, mepolizumab dosing was repeated. If the threshold was met, participants underwent bronchoscopy with BAL in two subsegments followed by SBP-Ag at the same 1 and 20% AgPD₂₀ doses administered for the baseline challenge before mepolizumab. The postmepolizumab challenge was performed in the contralateral lung. Forty-eight hours after SBP-Ag, bronchoscopy with BAL was performed in each subsegment and Bx were taken from an antigen-naive site and from the high-dose segment. D0, D2 = Day 0, Day 2; EOS = eosinophils; mepo = mepolizumab; PC₂₀ = provocative concentration of methacholine causing a 20% fall in FEV₁; PD₂₀ = provocative dose resulting in a 20% reduction in FEV₁; PreAg = before antigen administration; PostAg = after antigen administration; seg/s = segment/s.

Figure 2.

Mepolizumab prevents an allergen-induced rise in circulating eosinophils. (A) Before administration of mepolizumab, there was a significant rise in circulating eosinophil counts 48 hours (D2) after airway allergen challenge. The allergen-induced rise did not occur when challenge was given 1 month postmepolizumab. (B) WBC counts were not changed by allergen challenge or mepolizumab. Data represent medians with first and third quartiles; whisker lines represent the 10th and 90th percentiles (n = 10). Pre- versus postallergen P values are indicated above the solid lines; pre- versus postmepolizumab P values are indicated above the dashed lines. D0, D2 = Day 0, Day 2; ns = not significant; preAg = before segmental allergen challenge; WBC = white blood cells.

Figure 3.

Mepolizumab decreases, but does not eliminate, allergen-induced bronchoalveolar lavage (BAL) eosinophils. Both before and after mepolizumab, there was an antigen dose–dependent increase in the percentage of BAL eosinophils (A) and total numbers of BAL cells per milliliter of BAL fluid (B) 48 hours (D2) after segmental bronchoprovocation with allergen. The magnitude of the antigen-induced eosinophilia was significantly lower after mepolizumab. Data represent medians with first and third quartiles; whisker lines represent the 10th and 90th percentiles (n = 10). P values for pre- versus post-1% and post-1% versus 20% segmental bronchoprovocation with allergen are indicated above the solid lines; pre- versus postmepolizumab P values are indicated above the dashed lines. AgPD₂₀ = allergen provocation dose resulting in a 20% reduction in FEV₁; D0, D2 = Day 0, Day 2; EOS = eosinophils; preAg = before antigen administration.

Figure 4.

Mepolizumab decreases expression of the IL-3 receptor on circulating eosinophils but has no effect on IL-5 family cytokine receptor expression on BAL eosinophils. Forty-eight hours after segmental bronchoprovocation with allergen (SBP-Ag), the cytokine receptor expression pattern was assessed on blood eosinophils and on airway eosinophils obtained from the segment challenged with 20% of the participant’s AgPD₂₀ (allergen provocation dose resulting in a 20% reduction in FEV₁) (P values indicated above the solid lines). After mepolizumab administration, IL-3 receptor α chain (IL-3Rα/CD123/IL3RA) and β chain (βc/CD131/CSF2RB) were significantly diminished on blood eosinophils. Overall, however, mepolizumab did not alter the pattern of receptor expression on blood versus BAL eosinophils (P values indicated above the dashed lines). (A) Cell surface protein. Data represent medians with first and third quartiles (n = 9). (B) mRNA fold change normalized to each individual’s premepolizumab circulating eosinophils. Symbols represent values for individual subjects (n = 6 for blood eosinophils; n = 3 for BAL eosinophils). BAL = bronchoalveolar lavage; D2 = Day 2; EOS = eosinophils; GM-CSFRα = granulocyte–macrophage colony–stimulating factor receptor α chain; IL-5Rα = IL-5 receptor α chain; LAD = below the level of accurate detection; ns = not significant.

Figure 5.

Mepolizumab has no significant effect on IL-5–responsive cell surface activation markers on blood or BAL eosinophils. Forty-eight hours after segmental bronchoprovocation with allergen, the expression pattern of activation markers was assessed on blood eosinophils and on airway eosinophils obtained from the segment challenged with 20% of the participant’s AgPD₂₀ (allergen provocation dose resulting in a 20% reduction in FEV₁) (P values indicated above the solid lines). The differences in activation marker expression did not change after mepolizumab administration (P value indicated above the dashed lines). Data represent medians with first and third quartiles (CD44 and CD23, n = 8; CCR3, n = 7; CD69, n = 6). BAL = bronchoalveolar lavage; D2 = Day 2; FcεRII = low-affinity receptor for IgE; ns = not significant.

Figure 6.

Eosinophil release of eosinophil peroxidase (EPX) and EPX-containing granules is reduced, but still present, in bronchial mucosa after mepolizumab administration. (A) Representative photomicrographs, taken using a ×10 objective (rear photographs) and a ×40 objective (forward photographs), demonstrating EPX staining patterns. (B) After mepolizumab administration, the EPX staining score (medians with first and third quartiles) was significantly less for the antigen-naive site and tended to decrease for the site that had been challenged with 20% of the participant’s AgPD₂₀ (allergen provocation dose resulting in a 20% reduction in FEV₁) (n = 8). (C) EDN mRNA fold change normalized to each individual’s premepolizumab circulating eosinophils. Symbols represent values for individual subjects (n = 6 for blood eosinophils; n = 3 for BAL eosinophils). Pre– versus post–SBP-Ag P values are indicated above the solid lines; pre- versus postmepolizumab P values are indicated above the dashed lines. Ag = antigen; BAL = bronchoalveolar lavage; EDN = eosinophil-derived neurotoxin; EOS = eosinophils; ns = not significant; SBP-Ag = segmental bronchoprovocation with allergen.