COPD exacerbation severity and frequency is associated with impaired macrophage efferocytosis of eosinophils

Abstract

Background: Eosinophilic airway inflammation is observed in 10-30% of COPD subjects. Whether increased eosinophils or impairment in their clearance by macrophages is associated with the severity and frequency of exacerbations is unknown.

Methods: We categorised 103 COPD subjects into 4 groups determined by the upper limit of normal for their cytoplasmic macrophage red hue (<6%), an indirect measure of macrophage efferocytosis of eosinophils, and area under the curve sputum eosinophil count (≥ 3%/year). Eosinophil efferocytosis by monocyte-derived macrophages was studied in 17 COPD subjects and 8 normal controls.

Results: There were no differences in baseline lung function, health status or exacerbation frequency between the groups: A-low red hue, high sputum eosinophils (n=10), B-high red hue, high sputum eosinophils (n=16), C-low red hue, low sputum eosinophils (n=19) and D- high red hue, low sputum eosinophils (n=58). Positive bacterial culture was lower in groups A (10%) and B (6%) compared to C (44%) and D (21%) (p=0.01). The fall in FEV1 from stable to exacerbation was greatest in group A (ΔFEV1 [95 % CI] -0.41 L [-0.65 to -0.17]) versus group B (-0.16 L [-0.32 to -0.011]), C (-0.11 L [-0.23 to -0.002]) and D (-0.16 L [-0.22 to -0.10]; p=0.02). Macrophage efferocytosis of eosinophils was impaired in COPD versus controls (86 [75 to 92]% versus 93 [88 to 96]%; p=0.028); was most marked in group A (71 [70 to 84]%; p=0.0295) and was inversely correlated with exacerbation frequency (r=-0.63; p=0.006).

Conclusions: Macrophage efferocytosis of eosinophils is impaired in COPD and is related to the severity and frequency of COPD exacerbations.

Figure 1

Consort diagram showing the design of the study.

Figure 2

Cytoplasmic macrophage red hue as an indirect measure of macrophage efferocytosis of eosinophils. a) Percentage area of sputum macrophage cytoplasmic red hue in COPD subjects against sputum eosinophil area under the curve (AUC) %/year. The cut-off points for the upper limit of the normal ranges are as shown on the horizontal and vertical axes, 6 (16) and 3% respectively. b) Representative images of sputum macrophages: A: Subjects with high eosinophils ≥ 3% and low red hue <6%; B: high eosinophils ≥3% and high red hue ≥6%; C: low eosinophils <3% and low red hue <6%; D: low eosinophils <3% and high red hue >6%. Group B and D subjects have purple coloured cytoplasm in their macrophages. Group A and C have light blue cytoplasm. c) Change of FEV₁ between stable and first exacerbation visits for the 4 groups. The lines represent mean (SEM).

Figure 3

Monocyte–derived macrophages efferocytosis of eosinophils. a) Representative immunofluorescence staining of macrophages cultured with apoptotic eosinophils. Macrophages were stained with CD68-FITC (green), ECP-PE (red) and DAPI (blue). Left panel shows efferocytosis of eosinophils by a macrophage positively stained with ECP and the right panel shows ECP positive eosinophils and a macrophage with no evidence of efferocytosis. b) Comparison of macrophage efferocytosis of eosinophils in the 4 COPD groups and normal controls. The horizontal bars represent the median. c) Dot-plot of macrophage efferocytosis of eosinophils in those subjects with or without frequent exacerbations (≥2 exacerbations/year). Horizontal bars are medians. d) Scatter blot demonstrating the inverse correlation between the efferocytosis of eosinophils by macrophages and the frequency of exacerbations. Group A: triangles pointed down, B: triangles pointed up, C: closed circles, D: open circles. e) MDM % red hue from stained cytospins before and after eosinophil feeding for both COPD and normal subjects. Arrows represent medians of percentage area of red hue for each subject.