Immunology and Infection
Published: May 21st, 2021
The protocols herein described provide a guide to visualize and quantify the activity of neutrophil proteases in human sputum. The applications of such analysis span from the evaluation of anti-inflammatory treatments, to biomarker validation, drug screening and large cohort clinical studies.
Proteases are regulators of countless physiological processes and the precise investigation of their activities remains an intriguing biomedical challenge. Among the ~600 proteases encoded by the human genome, neutrophil serine proteases (NSPs) are thoroughly investigated for their involvement in the onset and progression of inflammatory conditions including respiratory diseases. Uniquely, secreted NSPs not only diffuse within extracellular fluids but also localize to plasma membranes. During neutrophil extracellular trap (NETs) formation, NSPs become an integral part of the secreted chromatin. Such complex behavior renders the understanding of NSPs pathophysiology a challenging task. Here, detailed protocols are shown to visualize, quantify and discriminate free and membrane-bound neutrophil elastase (NE) and cathepsin G (CG) activities in sputum samples. NE and CG are NSPs whose activities have pleiotropic roles in the pathogenesis of cystic fibrosis (CF) and chronic obstructive pulmonary disease (COPD): they promote tissue remodeling, regulate downstream immune responses and correlate with lung disease severity. The protocols show how to separate fluid and cellular fraction, as well as the isolation of neutrophils from human sputum for enzymatic activity quantification via small-molecule Förster resonance energy transfer-based (FRET) reporters. To gather specific insights into the relative role of NE and CG activities, a FRET readout can be measured by different technologies: i) in vitro plate reader measurements allow for high-throughput and bulk detection of protease activity; ii) confocal microscopy spatiotemporally resolves membrane-bound activity at the cell surface; iii) small-molecule FRET flow cytometry enables for the rapid evaluation of anti-inflammatory treatments via single-cell protease activity quantification and phenotyping. The implementation of such methods opens the doors to explore NSPs pathobiology and their potential as biomarkers of disease severity for CF and COPD. Given their standardization potential, their robust readout and simplicity of transfer, the described techniques are immediately shareable for implementation across research and diagnostic laboratories.
Neutrophil elastase (NE), cathepsin G (CG), proteinase 3 (PR3) and neutrophil serine protease 4 (NSP4) are the four neutrophil serine proteases (NSPs)1. They are stored, together with myeloperoxidase, within neutrophil primary or azurophilic granules. Due to their elevated proteolytic content, the secretion of primary granules is tightly regulated and neutrophils have to be sequentially challenged with priming and activating stimuli2.
Inside the phagolysosome, NSPs function as intracellular bactericidal agents3. When secreted, NSPs become strong mediators of inflammation: they cleave cytokines and surface receptors, activating parallel pro-inflammatory pathways3. Importantly, inflammatory conditions feature an uncontrolled NSPs secretion. For example, within inflamed airways, excessive NE activity causes mucus hypersecretion, goblet cells metaplasia, CFTR inactivation and extracellular matrix remodeling4,5. Cathepsin G participates in inflammation as well: it specifically cleaves and activates two components of the IL-1 family, IL-36α and IL-36β6. In concert with NE, CG cleaves protease-activated receptors on the airway epithelium and also activates TNF-α and IL-1β.
Endogenous anti-proteases such as alpha-1-antitrypsin, alpha-1-antichymotrypsin and the secretory leukocyte protease inhibitor regulate neutrophil elastase and cathepsin G activity5. However, over the course of lung disease progression, the continuous secretion of proteases exceeds stoichiometrically the anti-protease shield, leading to non-resolving neutrophilia in the airways, inflammation worsening and tissue damage5,7. Although NE concentration and activity in soluble fractions of patient airways has been shown to be a promising biomarker of disease severity8, NE and CG also associate to the neutrophil plasma membrane and to extracellular DNA via electrostatic interactions9,10 where they become less accessible to anti-proteases. Importantly, preclinical studies defined a scenario where cell surface-associated protease activity appears earlier and/or independently of its soluble counterpart4,11. In fact, to become detectable, free protease activity first needs to overwhelm the anti-protease shield. Instead, at the cell surface, membrane-bound protease activity remains at least partially intact due to the inaccessibility of large inhibitors to the cell plasma membrane12. Such complex protease behavior has important consequences on neutrophil-mediated inflammation onset and propagation, and therefore needs to be investigated with precise and informative tools.
Over the years, Förster resonance energy transfer (FRET)-based probes found numerous biomedical applications as tools that efficiently and rapidly assess a specific protease activity in human samples13. To function, protease reporters are composed of a recognition motif (i.e., a peptide), which is recognized by the target enzyme and rely on FRET, a physical process where, upon excitation, a donor fluorophore transfers energy to an acceptor molecule. The processing operated by the enzyme on the reporter, namely the cleavage of the recognition part, results in the acceptor to diffuse away from the donor: the enzyme activity is therefore measured as a time-dependent change in the donor over the acceptor fluorescence. Such read-out is self-normalizing and ratiometric, hence only marginally affected by environmental conditions such as pH and local probe concentration. NEmo-114 and sSAM15 are FRET probes that report specifically on NE and CG activity, respectively. However, such reporters do not localize specifically to any cellular compartment, therefore they are employed to monitor the protease activity present in human fluids. In order to monitor protease activity in a spatially localized fashion, we and others developed FRET probes that associate to subcellular components via molecular tags14,15,16,17,18,19. Such a synthetic strategy allowed the development of NEmo-2 and mSAM, two FRET probes equipped with lipid anchors that localize to the plasma membrane. These reporters fueled a deeper understanding of NE and CG proteases in cystic fibrosis and chronic obstructive pulmonary diseases14,15.
Here, detailed protocols are provided for the visualization and quantification of soluble and membrane-bound NE and CG activities in human sputum by means of NEmo and SAM series of FRET probes. To address diverse aspects of NSPs pathophysiology and provide an array of methods that can be employed according to the user-specific need, the analysis via fluorescence spectroscopy, fluorescence microscopy and flow cytometry are shown.
The following protocols describe analysis performed on human sputum. Human sample handling was approved by the ethics committee of the University of Heidelberg and written informed consent was obtained from all patients or their parents/legal guardians (S-370/2011) and healthy controls (S-046/2009).
NOTE: The following protocols describe the sample preparation and the quantification of neutrophil serine proteases (NSPs) activity. The experimental procedures presented herein focus on human sputum and neutrophil elastase14,20,21 (NE) or cathepsin G15 (CG) activity measurement. However, slight adaptations in the sample preparation protocol render the analysis of blood-derived cells and tumor homogenates possible. In addition, matrix metalloproteinase 12 and cathepsin S activities can be investigated similarly by means of dedicated FRET probes22,23,24,25,26.
1. Sample preparation: cell isolation and supernatant separation
NOTE: If possible, the treatment of sputum should be carried out within 120 min after expectoration and the sputum should be stored on ice until further processing.
2. Neutrophil serine protease activity measurement
NOTE: Here, different methods are introduced to quantify NSPs activity by means of FRET reporters. The choice of the technology is dictated by the specific biomedical question and purpose of the experiment. The probes presented were extensively tested for their specificity against a set of lung relevant enzymes14,15. Although the probes are specific toward their target enzyme, always check the probe specificity on the clinical sample of interest. This can be achieved by incubating the sample with a specific protease inhibitor prior to probe addition, which should abolish any increase in the D/A ratio.
Figure 1: Representative images and quantification of membrane-bound NE activity on neutrophils isolated from CF patient sputum. a) Representative confocal microscopy images of neutrophils pre-incubated (top panel) for 10 min with 100 µM of Sivelestat (w/) or left untreated (w/o) (bottom panel) before reporter NEmo-2 (2 µM) addition. The first column from the left shows the nuclear stain, the second the donor channel, the third the acceptor channel and the last the calculated D/A ratio obtained by dividing donor and acceptor channels on a pixel-by-pixel basis. The borders of the region of interest (single neutrophil) are depicted as dashed line. Scale (10 µm) and calibration bars (D/A ratio) are indicated. b) Box- and dot-plots showing the D/A ratio of sputum neutrophils from a representative CF patient. Cells incubated with inhibitor and untreated cells are shown in grey and blue, respectively. Each dot represents one cell (N: w/ inhibitor = 113 and w/o inhibitor = 96). Please click here to view a larger version of this figure.
Figure 2: Gating strategy and representative plots of membrane-bound NE activity measured on neutrophils isolated from CF patient sputum. a) To gate sputum neutrophils the following antibodies are used: CD14 (1:50), CD16 (1:50), CD45 (1:33) and CD66b (1:50). The neutrophils are gated as 7-AAD-CD45+CD14-CD16+CD66b+ events. The gated events are analyzed for their donor (λexc= 405 nm, λem= 450/50 nm) and acceptor (λexc= 405 nm, λem= 585/42 nm) mean fluorescence intensities (MFIs). b) Representative histograms of CF sputum neutrophils analyzed for their membrane-bound NE activity. The left column shows the donor signal, the right column shows the acceptor signal. The top row shows mean fluorescence intensities of cells treated with Sivelestat (w/) for 10 min before addition of the reporter. The bottom row shows untreated (w/o) cells whose reporter fluorescence is measured immediately (0 min, grey) and 10 min (blue) after reporter addition. Neutrophils are gated according to the strategy shown in panel a. c) The data table shows a representative dataset consisting of raw MFIs for the donor and acceptor signal on neutrophils measured over several time points (0-3-5-10-15-20 min) as well as the calculated D/A ratio. The D/A ratio can be normalized, i.e., to the 0 min time point (white font). 0 min indicates a recording done as soon as possible after reporter addition to the flow tube with stained sputum cells. MFIs data are shown as mean ± standard deviation for 1000 neutrophils. Please click here to view a larger version of this figure.
The results shown in Figure 1a illustrate a representative microscopy dataset. The nuclear signal is used to identify neutrophils by their characteristic segmented nuclei. The region of interest (ROI) is selected manually (dashed line in Figure 1a). The D/A ratio image is calculated by dividing the intensities of the donor channel by the intensities of the acceptor channel on a pixel-by-pixel basis. In the last step the average D/A ratio per cell (ROI) is calculated. In Figure 1b each dot represents the mean of one ROI (neutrophil). It is recommended to image and evaluate about 100 cells per condition.
A representative flow cytometry gating strategy is shown in Figure 2a. Such gating allows to discriminate and study sputum neutrophils. To avoid fluorescence spillover or compensation artifacts, it is recommended to dedicate a laser line (e.g., blue laser) to the FRET probe fluorescence detection. Flow cytometry fluorescence compensation should be performed for antibodies and not for the FRET probe. Figure 2b depicts the MFI distribution at 0 and 10 min after reporter addition. Each row in Figure 2c indicates the mean donor and acceptor MFIs values for 1000 sputum neutrophils. The D/A ratio is calculated by dividing the donor and acceptor MFIs. The time course in Figure 2c on the right side shows the progression of the measurement: after a rapid initial increase, the D/A ratio reaches a plateau, according to the activity of the membrane-bound enzyme.
The reported protocols explain different approaches to quantify the activity of neutrophil elastase and cathepsin G in human sputum samples. Critical points for a successful enzyme activity measurement are the i) precise timing and standardization of the operative procedure and ii) the use of reliable negative and positive controls. If these conditions are met, the described methods are not limited to sputum but can also be easily adapted to the analysis of protease activity in blood, bronchoalveolar lavage fluids and tissue sections or homogenates.
Each of the three techniques has its strengths and limitations, which often complement each other. For example, flow cytometry allows for the rapid analysis of rare cell populations as well as cell phenotyping but lacks spatial resolution information, which can be achieved by microscopy. Instead, plate reader measurements permit for the parallel assessment of several samples or conditions in a high-throughput fashion. Since fresh sputum cells cannot be frozen and stored, the three methods require that samples must be processed rapidly after expectoration. This limits the flexibility or the throughput of the membrane-bound activity measurements. The development of a flow cytometry protocol that allows to fix cells after probe addition and enzymatic cleavage would open to the parallel measurement of a higher number of tubes. Moreover, particular attention should be paid to the handling and storage of the FRET probes. In fact, some aminoacids present in the peptide substrate, such as methionine, undergo oxidation which leads to decreased reporter sensitivity. To increase the reporter's shelf life (estimated of about three months at 20 °C), they can be stored in small volume aliquots (1-2 µL) under inert gas such as Nitrogen or Argon.
In CF and other chronic inflammatory lung diseases it is important to detect the inflammation as early as possible, and reliable biomarkers have the potential to achieve such a goal. The possibility to detect surface-bound NSPs activity, which has been shown to be harmful for the surrounding tissue, also in conditions when there is no or little free NE activity, adds another level of valuable information, which can be hardly achieved by means of other existing methods4,11.
The reporters can be used to study the link of membrane-bound associated NSP activity with severity and progression of lung disease, especially at its early-onset. The methods can be utilized to monitor treatment efficacy (e.g., anti-inflammatory treatments or highly effective CFTR modulators and potentiators28) and investigate the resulting dampening of neutrophil-driven inflammation. In addition, the protocols are based on non-invasive sample procedures which carry very low risk for the patient and, therefore, can be used on a very broad scale and open the doors to numerous exciting applications.
The authors declare no conflict of interests.
This project was supported by grants from the German Ministry for Education and Research (FKZ 82DZL004A1 to M.A.M) and the German Research Foundation (SFB-TR84TP B08 to M.A.M). Work described in this manuscript was supported by the German Center of Lung Research (DZL) and the EMBL Heidelberg through a PhD fellowship for M.G. We thank J. Schatterny, S. Butz and H. Scheuermann for expert technical assistance.
|100 µm Nylon cell strainer
|2300 EnSpire (Multilabel Plate Reader)
|35x10mm Dish, Nunclon Delta
|Thermo Fisher Scientific
|40 µm Nylon cell strainer
|50 mL tubes
|7-AAD, viability dye
|5 µL/100 µL
|OHAUS Instruments (Shanghai) Co., Ltd.
|BD Falcon Round-Bottom Tubes 5 mL
|BD LSRFortessa cell analyzer
|black flat bottom 96 well half area plate
|Corning Life Science
|Elastin Products Company
|Cathepsin G Inhibitor I
|Combitips advanced 1.0 mL
|0030 089 430
|cOmplete proteinase inhibitor
|Countig chambers improved Neubauer
|Glaswarenfabrik Karl Hecht GmbH & Co KG
|coverslips Ø 25mm
|Thermo Fisher Scientific
|Thermo Fisher Scientific
|DRAQ5 (nuclear stain)
|FACSDiva software, v8.0.1
|Fiji (Fiji Is Just ImageJ)
|Flow Jo software, v10
|FluoQ Plugin, v3-97
|Heraeus Megafuge 16R
|Thermo Fisher Scientific
|Human Sputum Leucocyte Elastase
|Elastin Products Company
|Leica SP8 confocal microscope
|PEQLAB Biotechnologie GmbH
|mouse anti-human CD14, Pe-Cy7, clone M5E2
|mouse anti-human CD16, AF700, clone 3G8
|mouse anti-human CD45, APC-Cy7, clone 2D1
|mouse anti-human CD66b, PE/Dazzel 594, clone G10F5
|Pari Boy SX with an LC Sprint jet nebulizer
|phosphate buffered saline
|ROTI Histokitt (mounting medium)
|Carl Roth GmbH + Co.KG
|SuperFrost Plus Adhesion slides
|Thermo Fisher Scientific
|Trypan Blue solution
Copyright © 2024 MyJoVE Corporation. All rights reserved