Monitoring Neutrophil Elastase and Cathepsin G Activity in Human Sputum Samples
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.
Our FRET probe and protocols enable the rapid and sensitive quantification of free and surface-bound elastase and cathespin G activity in sputum from patients with neutrophilic airway diseases. These methods, make it possible to explore different aspects of proteases pathophysiology. Plate reader measurements permit for large screenings.
Confocal microscopy visualizes enzyme activity with subcellular solution. While flow cytometry, enables single cell phenotyping and protease activity quantification in a personalized fashion. This technique can be expanded to different airway diseases such as cystic fibrosis, bronchiectasis or COPD.
And it can be adapted to different BioSamples such as blood or bronchial alveola lavash or mice samples. Before starting the sputum induction procedure, inhale 200 micrograms of the beta 2 receptor antagonists salbutamol. Afterwards, inhale hypertonic 6%saline solution for 15 minutes using a nebulizer.
Collect the expectorated sputum in a Petri dish. Separate mucus clumps from the saliva into a Petri dish with the help of a pipette tip. Weigh the mucus, then add four volume parts per weight of 10%Sputolysin and PBS to the sputum.
Incubate the mixture at room temperature on a rocking shaker for 15 minutes to dissolve the mucus. Quench the reaction by adding one milliliter of cold PBS for each milliliter of 10%Sputolysin. Pipette the mixture to obtain a homogeneous solution.
Filter the mixture through a 100 micron nylon cell strainer into a 50 milliliter tube. Repeat the filtration step through a 40 micron strainer, then centrifuge for 10 minutes at 300 times G at four degrees Celsius. Transfer the supernatant into a fresh tube and store it on ice.
Gently resuspend the cell pellet in 500 microliters of cold PBS and place it on ice. Thaw enzymes on ice and set up an enzyme standard curve as described in the text manuscript. In parallel to the standard preparation, dilute sputum samples in activation buffer.
On the plate reader, set the excitation wavelength for the NE FRET probe NEmo-1 to 354 nanometers and the emission wavelength to 400 nanometers for donor and 490 nanometers for acceptor. For the CG FRET probe sSam set the excitation wavelength to 405 nanometers and the emission to 485 nanometers for donor, and 580 nanometers for acceptor. Add 40 microliters of samples, standards, or blanks into the wells of a black 96 well half area plate and add the master mix.
Start the plate reader and record the donor to acceptor ratio increase after every 60 to 90 seconds for at least 20 minutes, or until the increase in the signal reaches a plateau. Export the data and calculate the donor to acceptor ratio by dividing the donor RFU by the acceptor RFU for each time point and sample. Then calculate the donor to acceptor ratio mean and standard deviation.
Determine the slope within the linear growth of the donor to acceptor ratio change. For each measurement, resuspend 30, 000 sputum cells in a volume of 50 microliters of PBS in a 1.5 milliliter tube. Incubate the sputum cells with a specific inhibitor as a negative control and an appropriate enzyme as a positive control for 10 minutes at room temperature.
Add 50 microliters of PBS containing FRET reporter and a nuclear stain to each tube to get a final concentration of 2 micromolar. Incubate the mixture at room temperature for 10 to 20 minutes. Quench the reaction by adding 100 microliters of ice cold PBS and place the samples on ice.
Cytospin the mixture on microscopy slides. Then air dry and fix the cells with ice cold 10%methanol for 10 minutes. After fixation, air dry and mount the sample with an appropriate mounting medium.
Capture the images using a confocal microscope. Image at least 100 cells per condition for conclusive statistics. Resuspend 1 million cells in 100 microliters of PBS in a five milliliter FACS polystyrene round bottom tube, and place the tube on ice.
Add two microliters of FC block to each sample. Then incubate the samples for five minutes at room temperature. Add antibody each tube and incubate on ice in the dark for 30 minutes.
Wash the cells with two milliliters of cold PBS and centrifuge at 300 times G and four degrees Celsius. Finally, resuspend in 200 microliters of PBS. Divide the suspension into two tubes, 100 microliters each and add five microliters of cell viability stain.
Add appropriate specific NSP inhibitor to the negative control tube and incubate the sample for 10 minutes at room temperature in the dark. Add PBS to the sample and filter it through a 40 micron filter into a clean FACS tube. Add the reporter to the negative control sample and gently vortex.
Start acquiring cells incubated with this specific inhibitor and if necessary, slightly adjust the gates as well as the reporter PMTs voltages. To record changes in the donor to accept a ratio due to membrane bound protease activity, record 1000 neutrophils from each tube, every five to 10 minutes. Calculate the FRET ratio by dividing the donor by the acceptor channel values for the samples measured on the gated viable single neutrophils.
Images of neutrophils pre incubated with 100 micromolar Sivilestat or left untreated before reporter NEmo-2 addition are shown here. Nuclear signal was used to identify neutrophils by their characteristics segmented nuclei and the ROI was selected manually. The donor to accept a ratio of sputum neutrophils was plotted.
Each dot represents the mean of one ROI. Flow cytometry gating makes it possible to discriminate and study sputum neutrophils. The MFI distribution at zero and 10 minutes was observed after reporter addition.
The mean donor and acceptor MFI values for 1000 sputum neutrophils were calculated. The D/A ratio reached a plateau after an initial increase. The healthy donors sputum induction takes some practice before perfection.
Remember to relax and to breathe the aerosol for 10 to 15 minutes before expectoration. In addition, always keeps sputum cells on ice and process them as soon as possible after expectoration. We found that surface bound protease activity correlates with early lung damage in children and with severity of lung disease in adult CF patients.
Therefore together, these methods enable the exploration of proteases as early inflammation biomarkers in neutrophilic airway diseases.
Overview
This article presents protocols for visualizing and quantifying neutrophil protease activity in human sputum. The methods described are applicable for evaluating anti-inflammatory treatments and conducting biomarker validation.
Key Study Components
Area of Science
- Neuroscience
- Biology
- Clinical Research
Background
- Neutrophil proteases play a significant role in airway diseases.
- Understanding their activity can aid in the development of therapeutic strategies.
- Current methods allow for detailed analysis of protease function in clinical samples.
- Techniques such as FRET and flow cytometry enhance the sensitivity of measurements.
Purpose of Study
- To provide a detailed methodology for quantifying protease activity in sputum.
- To facilitate research on the pathophysiology of neutrophilic airway diseases.
- To enable large-scale screenings and personalized medicine approaches.
Methods Used
- FRET probes for measuring elastase and cathepsin G activity.
- Confocal microscopy for visualizing enzyme activity.
- Flow cytometry for single-cell analysis and phenotyping.
- Standardized sputum induction and processing protocols.
Main Results
- Successful quantification of protease activity in sputum samples.
- Demonstrated the feasibility of the methods for various airway diseases.
- Provided a comprehensive analysis of neutrophil activity.
- Identified key parameters for optimizing experimental conditions.
Conclusions
- The protocols enable effective study of neutrophil proteases in clinical samples.
- These methods can advance research in airway disease treatments.
- Further validation of biomarkers and therapeutic targets is encouraged.
Frequently Asked Questions
Executive Industry Relevance
Quantifying neutrophil elastase and cathepsin G activities in human sputum enables mechanistic de-risking of protease-driven inflammatory pathways in cystic fibrosis and COPD. These FRET-based methods provide predictive confidence for target validation by distinguishing free versus membrane-bound protease pools, supporting biomarker qualification and therapeutic response monitoring. The standardized workflow facilitates cross-functional translation from discovery to preclinical evaluation.
Strategic Applications in Biopharma R&D
Early Discovery & Target Validation
- Scientific Value: Interrogates therapeutic hypotheses by quantifying neutrophil serine protease activity in disease-relevant sputum matrices.
- Operational Value: Enables functional target validation through discrimination of free and surface-bound protease pools using FRET reporters.
- Predictive Confidence: Supports portfolio triage by correlating protease activity levels with lung disease severity in neutrophilic airway diseases.
Screening & Assay Development
- Assay Readiness: Prepares validated biological systems for high-throughput plate reader screening of protease inhibitors.
- Quantitative Outputs: Generates donor-to-acceptor ratio measurements enabling reproducible compound evaluation across laboratories.
- Platform Reuse: Adapts to blood, BAL, and murine samples, supporting scalable implementation in discovery and diagnostic workflows.
Translational & Preclinical Research
- Disease Relevance: Links neutrophil protease activity to pathophysiological mechanisms in cystic fibrosis and COPD, aligning with translational biomarker strategies.
- Preclinical Continuity: Enables risk-adjusted advancement decisions by quantifying protease inhibition in primary human sputum neutrophils.
- Mechanistic De-risking: Clarifies protease contribution to tissue remodeling and immune regulation, reducing ambiguity in target selection.
Pipeline & Workflow Integration
The method integrates into the discovery continuum from hypothesis testing in early discovery to lead identification and preclinical validation, supported by its ability to quantify protease activity in clinically relevant matrices.
- Discovery Biology: Supports hypothesis testing and pathway clarification by measuring neutrophil elastase and cathepsin G activities in sputum from neutrophilic airway diseases.
- Screening: Enables assay readiness and reproducibility through standardized FRET reporter protocols compatible with plate reader, confocal microscopy, and flow cytometry platforms.
- Analytics: Provides quantitative donor-to-acceptor ratio readouts that allow comparison of protease inhibition across conditions and time points.
- Translational Research: Connects to preclinical continuity by correlating protease activity with lung disease severity in cystic fibrosis and COPD patient samples.
- Enterprise Reuse: Establishes a reusable capability for protease activity quantification across sample types, diseases, and therapeutic modalities.
Operational & Enterprise Impact
- Scientific Value: Predictive confidence in target validation, reduction of mechanistic ambiguity in neutrophil protease pathophysiology.
- Operational Value: Standardization, reproducibility, and scalability across plate reader, microscopy, and flow cytometry readouts.
- Strategic Value: Better go/no-go decisions, capital efficiency, and reduced late-stage biological risk in inflammatory disease programs.
- Portfolio Impact: Risk-adjusted prioritization and advancement decisions based on protease activity biomarkers in cystic fibrosis and COPD.
Implementation Considerations
- Requires expertise in protease biochemistry, FRET assay design, and sputum processing protocols.
- Needs plate reader, confocal microscope, and flow cytometry instrumentation with appropriate filter sets for NEmo-1 and sSam probes.
- Demands cross-team standardization of sputum induction, processing, and FRET ratio calculation methods.
- Requires adaptation considerations for varying sample types such as blood, BAL, and murine models while maintaining protease activity stability.
- Practical limitations include sample processing time sensitivity and the need for immediate processing post-expectoration to preserve neutrophil viability and protease activity.
Biopharma FAQs
Chapters in this video
0:05
Introduction
1:07
Cell Isolation and Supernatant Separation
2:36
Quantification of Soluble Neutrophil Serine Protease (NSP) Activity via Fluorimeter or Plate Reader Assay
4:13
Membrane-bound NSPs Activity Measurement via Fluorescence Microscopy
5:30
Membrane-bound NSPs Activity Measurement via Flow Cytometry
7:25
Results: Representative Images and Quantification of Membrane-Bound NE Activity on Neutrophils Isolated from CF Patient Sputum
8:26
Conclusion
