Neutrophil Elastase Targets Select Proteins on Human Blood-Monocyte-Derived Macrophage Cell Surfaces

Abstract

Neutrophil elastase (NE) has been reported to be a pro-inflammatory stimulus for macrophages. The aim of the present study was to determine the impact of NE exposure on the human macrophage proteome and evaluate its impact on pro-inflammatory signals. Human blood monocytes from healthy volunteers were differentiated to macrophages and then exposed to either 500 nM of NE or control vehicle for 2 h in triplicate. Label-free quantitative proteomics analysis identified 41 differentially expressed proteins in the NE versus control vehicle datasets. A total of 26 proteins were downregulated and of those, 21 were cell surface proteins. Importantly, four of the cell surface proteins were proteoglycans: neuropilin 1 (NRP1), syndecan 2 (SDC2), glypican 4 (GPC4), and CD99 antigen-like protein 2 (CD99L2) along with neuropilin 2 (NRP2), CD99 antigen (CD99), and endoglin (ENG) which are known interactors. Additional NE-targeted proteins related to macrophage function were also measured including CD40, CD48, SPINT1, ST14, and MSR1. Collectively, this study provides a comprehensive unbiased view of selective NE-targeted cell surface proteins in chronically inflamed lungs.

Keywords: COPD; ELANE; cystic fibrosis; inflammation; macrophages; neutrophil elastase; polarization; proteoglycans.

Figure 1

Experimental workflow for investigating the effects of NE treatment on the human blood-monocyte-derived macrophage (BMDM) proteome. BMDMs from healthy donors were split equivalently into 6-well plates and then incubated without (control (Ctl)) or with 500 nM of neutrophil elastase (+NE) for 2 h in triplicate.

Figure 2

Summary of label-free quantification (LFQ) results. Results show the number of proteins quantified in each donor sample with NE treatment (black) and without NE treatment (gray) (A). Venn diagram shows the proteome overlap between donor samples (B). Venn diagram shows statistically significant (t-test; adjusted p-value < 0.05) differentially regulated proteins in NE vs. Ctl (C). Heatmap showing the log2 fold-change values of protein levels in NE vs. control that were detected in 3/3 donors and statistically significant in 3/3 or 2/3 donor (* indicates the expression level is not statistically significant) (D).

Figure 3

Down-regulated proteins. Enriched downregulated proteins from the GO cellular component annotation (A). Log10 of the median normalized LFQ abundances rank-plotted with 21 cell-membrane-annotated differentially downregulated proteins (yellow) labeled by gene. GPC4 is labeled in red as it is downregulated in only 2/3 donor samples yet is a member of the proteoglycan family (B).

Figure 4

Western blot and densitometry results for NRP1 (A,B), SDC2 (C,D), and GPC4 (E,F) from NE-targeted BMDM lysates. Three separate blood monocyte donations were obtained from healthy donors and cultured in RPMI with GM-CSF for 8–10 days to differentiate cells to blood monocyte derived macrophages. Cells from each donor were treated with control vehicle or NE (200 or 500 nM) for 1 or 2 h. Cell lysates were collected, protein quantified, and Western analyses were performed for NRP1 (A), SDC2 (C), and GPC4 (E). Left panels are representative Western blots for protein targets and as a control, β-actin. After densitometry of bands using ImageJ, relative expression corrected for β-actin was compared to control treated samples and summary data of relative expression for each protein shown in the panel on the right, NRP1 (B), SDC2 (D), and GPC4 (F). Significant differences in relative expression for N = 3 per protein was determined; *, p < 0.05; **, p < 0.01; ***, p < 0.001.