Anti–IL-5 (mepolizumab) therapy reduces eosinophil activation ex vivo and increases IL-5 and IL-5 receptor levels

Effect of anti–IL-5 on circulating leukocytes and peripheral blood eosinophil counts. Study protocol (A) showing eosinophil responses represented by individual lines (B) and in paired individual changes before (week 8) and after (week 20) anti–IL-5 therapy (C). The percentage of eosinophils and lymphocytes (mean ± SD, n = 25) and the numbers of total white blood cell (WBC) counts expressed as × 10³ cells (k) per cubic millimeter are shown (D). A dot diagram of peripheral blood eosinophil levels is shown (E).

Plasma IL-5 levels before and after anti–IL-5 therapy. Plasma IL-5 levels represented for patients and healthy control subjects (NL; A) with paired individual changes (B) before (week 8) and after (week 20) anti–IL-5 therapy are shown. Dots under detection limit (dashed line) are extrapolated data in a log × log scale curve analysis and do not represent true values. The horizontal bars represent mean values.

Molecular analysis of plasma IL-5. The average of subject and control samples are presented as the original IL-5 level detected at week 20 and after filtration in the upper and lower chambers (see the Methods section for details). Recombinant IL-5 at 100 pg/mL was used as a control to demonstrate that IL-5 is normally found in the lower chamber (A). The upper chamber fraction precipitation with nonsaturating quantities of protein A/G (50 μL) in representative samples and a control sample of T-cell supernatant after PHA stimulation is shown (B). A representative protein A/G dose-response curve is shown (C). The combined results of subjects' plasma (week 20) samples before and after protein A/G precipitation with saturating amounts (200 μL) is shown (D). The dashed line represents the detection limit value. Horizontal bars represent mean (SD) values (n = 12). ∗∗P < .01, ∗∗∗P < .001.

Effect of anti–IL-5 on peripheral blood CCR3⁺ cell levels and the expression of IL-5Rα mcf in peripheral blood eosinophils. The percentage of CCR3⁺ cells in peripheral blood leukocytes (A and B) and the expression of IL-5Rα mcf (C and D) are represented as group distribution (Fig 4, A and C) and paired analysis (Fig 4, B and D) for all study subjects before (week 8) and after (week 20) anti–IL-5 therapy. The horizontal bars represent mean values.

Effect of anti–IL-5 on intracellular IL-5 production by T cells. Anti–IL-5 therapy increased the percentage of peripheral blood CD4⁺ and CD8⁺ cells producing intracellular IL-5. The percentage of CD4⁺ (A and B) and CD8⁺ (C and D) cells producing intracellular IL-5 is shown before (week 8) and after (week 20) anti–IL-5 therapy for all study subjects. The unpaired (Fig 5, A and C) and paired (Fig 5, B and D) data are shown. The horizontal bars represent mean values.

Effect of anti–IL-5 therapy on peripheral blood eosinophil activation in response to eotaxins. Peripheral blood eosinophil activation was assessed based on eotaxin-induced eosinophil shape change ex vivo in response to eotaxin-1 (A), eotaxin-2 (B), and eotaxin-3 (C) in patients before (week 8) and after (week 20) anti–IL-5 therapy. Eosinophil shape changes in response to eotaxin-1 and eotaxin-2 are shown in a paired analysis; eosinophil shape changes in response to eotaxin-3 are shown in a nonpaired analysis. ^∗P < .05, ^∗∗P < .01.

Lymphocyte and subpopulation analysis before and after anti–IL-5 therapy. The absolute lymphocyte levels and percentage of various subpopulations (percentage of CD3⁺, CD4⁺, CD8⁺, CD19⁺, and CD16⁺CD56⁺ cells) are shown for the combined cohorts before (week 8, solid bars) and after (week 20, open bars) anti–IL-5 therapy. ns, Not significant; NK, natural killer. The horizontal bars represent mean (SD) values.

Effect of anti–IL-5 on intracellular IL-4, IL-13, IFN-γ, and TNF-α production. The percentage of peripheral blood CD4⁺ and CD8⁺ cells producing intracellular IL-4, IL-13, IFN-γ, and TNF-α after anti–IL-5 therapy is shown for the combined cohorts before (week 8) and after (week 20) anti–IL-5 therapy. ns, Not significant.

Effect of anti–IL-5 on IL-4, IL-5, IL-10, IL-13, GM-CSF, and IFN-γ secretion by PHA-stimulated PBMCs. The secreted levels of IL-4 (A), IL-5 (B), IL-10 (C), GM-CSF (D), and IFN-γ (E) by PHA-stimulated PBMCs are shown for all cohorts before (week 8) and after (week 20) anti–IL-5 therapy. The different responses of mepolizumab on IL-13 secretion by PHA-stimulated PBMCs (F) are shown for all subjects (G) and only paired patients in cohort A (H) before (week 8) and after (week 20) anti–IL-5 therapy.

Effect of IL-5 on IL-5Rα expression on eosinophils in vitro. IL-5Rα mcf expression on eosinophils preincubated with a wide concentration range of IL-5 concentrations in vitro: the histogram with isotype control is shown in A. The mean value of IL-5Rα expression is shown in B (mean ± SD, n = 3).