The effect of eosinophils on collagen gel contraction and implications for tissue remodelling

Abstract

Asthma is characterized by an eosinophilic inflammation and a subepithelial fibrosis in the airways. Eosinophils contain several cytotoxic substances, such as eosinophil cationic protein (ECP), which can promote inflammation and cause tissue damage. This has generated the hypothesis that eosinophils may drive remodelling of extracellular matrix (ECM). To investigate the role of eosinophils we used an in vitro model for remodelling, the three-dimensional collagen gel contraction assay. Two sources of eosinophils were used in this study, isolated human peripheral eosinophils (purity > 95%) and stimulated [interleukin (IL)-5, IL-3 and granulocyte macrophage-colony stimulating factor (GM-CSF)] HL-60 clone 15 cells. Human eosinophils or HL-60 cells were cast together with human lung fibroblasts (HFL1) in type I collagen gels. Both types of eosinophils augmented fibroblast-mediated collagen gel contraction in a time and concentration-dependent manner. At 48 h, the gel area in HFL1/eosinophil co-culture was 46.5% +/- 0.5 (mean +/- s.e.m.) of initial area and in HFL1 culture 52.3% +/- 0.1 (P < 0.001). Respective figures for HFL1/stimulated HL-60 co-culture and HFL1 culture only were 44.1% +/- 0.5 and 52.4% +/- 0.4 (P < 0.001). The release of ECP was increased when fibroblasts were cultured with eosinophils compared to eosinophils cultured alone. In addition, native ECP added to fibroblast gel cultures also augmented contraction. Our results suggest that eosinophils may interact with mesenchymal cells, promoting remodelling of ECM and that ECP constitutes one potential eosinophil-derived mediator driving this process. We conclude that this may be one important mechanism by which eosinophil-ECM interactions will lead to airway tissue remodelling in asthma.

Fig. 1

Time-dependent augmentation of fibroblast-mediated collagen gel contraction by eosinophils (Eos). Fibroblasts (HFL1: 3 × 10⁵/ml gel) and Eos (10⁶/ml gel) or the two cell types together (HFL1/Eos) were cast into a 24-well tissue culture plate. The assay was performed by releasing the gels into medium immediately after gelation. The area of each gel was measured by an image analyser on 4 consecutive days. Vertical axis: gel area expressed as percentage of original gel size. Data presented as mean of triplicate and standard error of mean (± s.e.m.) from one representative experiment. P < 0·001: HFL1 versus HFL1/Eos at all data points.

Fig. 2

Eosinophil concentration-dependent augmentation of fibroblast-mediated collagen gel contraction. Increasing numbers of eosinophils were mixed together with fibroblasts in a three-dimensional collagen gel. After gelation, the gels were released and the area of the floating gels was measured after 48 h of culture. Vertical axis: gel area expressed as percentage of original gel size. Horizontal axis: number of eosinophils (Eos) added to gels. Data shown as mean of triplicate and ± s.e.m. ***P < 0·001, **P < 0·01, n.s. = P > 0·05.

Fig. 3

Differentiated clone 15 HL-60 cells (diff HL-60) augment fibroblast-mediated collagen gel contraction. Clone 15 HL-60 cells were differentiated (IL-5, IL-3, GM-CSF) and cast together with fibroblasts in collagen gels. Undifferentiated clone 15 HL-60 cells (HL-60) were used as control cells. Vertical axis: gel area expressed as percentage of original gel size. Data presented as mean of triplicate and standard error of mean (± s.e.m.). P < 0·001: HFL1/HL-60 versus HFL1/diff HL-60 on days 2, 3 and 4.

Fig. 4

Cell numbers in collagen gels estimated by DNA content. Floating collagen gels containing fibroblasts (HFL1), co-culture of fibroblasts and eosinophils (HFL1/Eos) or eosinophils (Eos) alone were cultured for 2 days. The gels were then dissolved in collagenase and DNA contents in pellets were determined by a fluorometric assay. Vertical axis: optical density (OD value, arbitrary units) measured at wavelength 355/460 nm. Data shown as means ± s.e.m., n = 6.

Fig. 5

ECP is released in co-culture. Fibroblasts together with eosinophils (HFL1/Eos) or eosinophils (Eos) alone were cast into collagen gels. After 2 days of culture, the gels and surrounding medium were pooled and analysed for ECP (ECP-CAP-FEIA). Vertical axis: concentration of ECP (ng/ml). Horizontal axis: data are given as individual results from two independent experiments. □ = Experiment 1, ▪ = experiment 2.

Fig. 6

Native ECP augment fibroblast-mediated collagen gel contraction in a concentration-dependent manner. ECP in various concentrations were added to fibroblast/collagen gels. After gelation, the gels were released and the area of the floating gels was measured after 2 days of culture. Vertical axis: gel area expressed as percentage of original gel size. Horizontal axis: ECP concentration added to gels. Data shown as mean of triplicate ± s.e.m. ***P < 0·001, **P < 0·01, n.s. = P > 0·05.