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In This Article

  • Summary
  • Abstract
  • Introduction
  • Protocol
  • Results
  • Discussion
  • Disclosures
  • Acknowledgements
  • Materials
  • References
  • Reprints and Permissions

Summary

This protocol describes a flow cytometry-based, high-throughput screening method to identify small-molecule drugs that inhibit β2 integrin activation on human neutrophils.

Abstract

This protocol aims to establish a method for identifying small molecular antagonists of β2 integrin activation, utilizing conformational-change-reporting antibodies and high-throughput flow cytometry. The method can also serve as a guide for other antibody-based high-throughput screening methods. β2 integrins are leukocyte-specific adhesion molecules that are crucial in immune responses. Neutrophils rely on integrin activation to exit the bloodstream, not only to fight infections but also to be involved in multiple inflammatory diseases. Controlling β2 integrin activation presents a viable approach for treating neutrophil-associated inflammatory diseases. In this protocol, a monoclonal antibody, mAb24, which specifically binds to the high-affinity headpiece of β2 integrins, is utilized to quantify β2 integrin activation on isolated primary human neutrophils. N-formylmethionyl-leucyl-phenylalanine (fMLP) is used as a stimulus to activate neutrophil β2 integrins. A high-throughput flow cytometer capable of automatically running 384-well plate samples was used in this study. The effects of 320 chemicals on β2 integrin inhibition are assessed within 3 h. Molecules that directly target β2 integrins or target molecules in the G protein-coupled receptor-initiated integrin inside-out activation signaling pathway can be identified through this approach.

Introduction

Many inflammatory diseases are characterized by the infiltration of neutrophils at the site of swelling or injury1. To infiltrate these tissues, neutrophils must complete the neutrophil recruitment cascade, which involves arrest to the endothelium, extravasation across the vessel wall, and recruitment into the tissue2. Circulating neutrophils need β2 integrin activation to complete this cascade, especially for the arrest phase. Thus, integrin-inhibiting drugs that reduce neutrophil adhesion, extravasation, and recruitment may effectively treat inflammatory diseases3,4.

β2 integrins have been targeted for inflammatory diseases before. Efalizumab, a monoclonal antibody directly targeting integrin αLβ2, was developed to treat psoriasis5. However, efalizumab was withdrawn due to its lethal side effect - progressive multifocal leukoencephalopathy resulting from JC virus reactivation6,7. New anti-inflammatory integrin-based therapies should consider maintaining the anti-infection functions of leukocytes to minimize side effects. The side effects of efalizumab might be due to the prolonged circulation of monoclonal antibodies in the bloodstream, which could inhibit immune functions in the long term8. A recent study shows that efalizumab mediates αLβ2 crosslinking and the unwanted internalization of α4 integrins, providing an alternative explanation for the side effects9. Thus, short-lived, small-molecule antagonists might avoid this problem.

A high-throughput method to screen small-molecule β2 integrin antagonists using human neutrophils is presented here. β2 integrin activation requires conformational changes of the integrin ectodomain to gain access to and increase its binding affinity to its ligand. In the canonical switchblade model, the bent-closed integrin ectodomain first extends to an extended-closed conformation and then opens its headpiece to a fully activated extended-open conformation10,11,12,13. There is also an alternative pathway that starts from the bent-closed to bent-open and extended-open, eventually14,15,16,17,18,19. The conformation-specific antibody mAb24 binds to an epitope in the human β2-I-like domain when the headpiece of the ectodomain is open20,21,22,23.

Here, mAb24-APC is used to determine whether the β2 integrins are activated. To activate neutrophils and integrin, N-formylmethionyl-leucyl-phenylalanine (fMLP), a bacterial-derived short chemotactic peptide that can activate neutrophil β2 integrins24, is used as a stimulus in this protocol. When fMLP binds to the Fpr1 on neutrophils, downstream signaling cascades involving G-proteins, phospholipase Cβ, and phosphoinositide 3-kinase γ are activated. These signaling events ultimately result in integrin activation via the inside-out signaling pathway18,25. Besides small molecule antagonists that directly bind to β2 integrins and prevent conformational changes of integrin activation26, compounds that can inhibit components in the β2 integrin inside-out activation signaling pathway would also be detected with this method. Automated flow cytometers enable high-throughput screening. Identifying new antagonists may not only deepen our understanding of integrin physiology but also provide translational insight into integrin-based anti-inflammation therapy.

Protocol

Heparinized whole-blood samples were obtained from de-identified healthy human donors after obtaining informed consent, as approved by the Institutional Review Board of UConn Health, following the principles of the Declaration of Helsinki. Informed consent was obtained from all donors. The inclusion/exclusion criteria for this study were carefully developed to ensure the suitability of participants and to minimize potential risks. Eligible participants were aged between 18 and 65 years, of any ethnicity, fluent in English, and capable of providing informed consent. Excluded participants included those unable to provide informed consent for themselves, such as those requiring a legally authorized representative, individuals under 18 or over 65 years, incarcerated individuals, and pregnant women. Additionally, participants had to be free from anti-inflammatory medication usage and inflammatory conditions. Current infections or ongoing chronic or acute inflammatory conditions were also exclusion criteria. Finally, individuals with a current or recent history of COVID-19 infection were ineligible for the study. These criteria were designed to ensure participant safety and suitability while minimizing potential confounding factors that could impact the study results.

1. Preparation of reagents

  1. Neutrophil medium: Prepare the neutrophil medium by adding 2% human serum albumin to RPMI-1640 without phenol red (see Table of Materials).
  2. fMLP solution: Prepare the fMLP solution by diluting it 100 times from the 10 mM fMLP dimethyl sulfoxide (DMSO) storage solution (see Table of Materials) with RPMI 1640 without phenol red, resulting in a 100 µM fMLP solution.
  3. Antibody solution: Dilute 120 µL of allophycocyanin (APC)-conjugated monoclonal antibody mAb24 (mAb24-APC) (see Table of Materials), which reports the high-affinity conformation of β2 integrin, in 10 mL of neutrophil medium, creating a 1.2 µg/mL mAb24-APC solution.
  4. Compound library: Dilute the initial compound library with a concentration of 10 mM to 1 mM by adding 5 µL of the 10 mM library (see Table of Materials) to 45 µL of DMSO in 384-well plates using the liquid handler. Subsequently, transfer 5 µL per well of the 1 mM compound library to an empty 384-well plate using the liquid handler.
  5. As illustrated in Figure 1, four columns (1, 2, 23, 24) contained only DMSO but no compounds, serving as positive and negative controls in the screening. Further dilute each well by adding 45 µL of RPMI-1640 without phenol red, resulting in a final concentration of 100 µM for all compounds.

2. Neutrophil isolation from human blood

  1. Using a serological pipette, carefully layer 4 mL of blood over 8 mL of a commercially available density gradient medium (see Table of Materials) in a 15 mL centrifuge tube (Figure 2A).
  2. Centrifuge the blood at 550 × g for 30-50 min at 20 °C. Decelerate the rotor gradually (deceleration factor of 1).
    NOTE: The successful separation of neutrophils (band 2 in Figure 2B) from red blood cells may vary among donors. For most donors, a 30 min centrifugation is sufficient. However, for some donors, an additional 10-30 min of centrifugation may be required if separation is not successful (Figure 2C).
  3. Carefully remove the plasma (the yellow liquid on top) and mononuclear cells (the upper cloudy band; PBMC band in Figure 2B) using a 1 mL pipette.
  4. Collect neutrophils from the lower cloudy band (neutrophil band in Figure 2B) and approximately 3-4 mL below the clear liquid into a 15 mL centrifuge tube containing 10 mL of phosphate-buffered saline (PBS). Gently mix the neutrophil suspension by inverting it 2-3 times.
  5. Centrifuge the suspension at 400 × g for 10 min at 20 °C, and then carefully remove the supernatant by decanting.
  6. Gently resuspend the pellet with 5 mL of PBS and centrifuge it at 300 × g for 5 min at 20 °C.
  7. Remove the supernatant by decanting, and carefully eliminate any residual supernatant around the tube mouth and on the tube wall using vacuum suction. Resuspend the pellet in 1 mL of neutrophil medium.
  8. Count the cell numbers using a hemocytometer. Typically, 1 to 4 × 107 neutrophils were obtained from 8 mL of human blood.
    NOTE: There may be red blood cell contamination in the neutrophil suspension. Red blood cells do not significantly affect most assays and can prevent neutrophil activation/priming27. Red blood cells need to be lysed before counting to obtain an accurate neutrophil concentration. Add 10 µL of the cell suspension to 891 µL of deionized water for 10-30 s to lyse the red blood cells, then add 99 µL of 10× PBS to balance the osmotic pressure, preventing lysis of the neutrophils.
  9. Adjust the cell density to 6.25 × 105 cells/mL by adding neutrophil medium.

3. Preparation of the 384-well plate

  1. In a 1.5 mL centrifuge tube, combine 1 mL of the antibody solution (1.2 µg/mL mAb24-APC) with 0.2 mL of neutrophil medium to create a 1 µg/mL mAb24-APC solution. Then, add 25 µL of this mixture to the negative control wells in a 384-well plate (the blue wells in Figure 1).
  2. In a 15 mL centrifuge tube, mix 9 mL of the antibody solution with 21.6 µL of the fMLP solution (100 µM) and 1.7784 mL of neutrophil medium to create a solution containing 1 µg/mL mAb24-APC and 200 nM fMLP. Next, add 25 µL of this mixture to the positive control and testing wells in a 384-well plate (the red and blue wells in Figure 1).
  3. Transfer 5 µL of the 100 µM compound solutions from the compound library plate to the 384-well plate using a multi-channel pipette.
  4. Centrifuge the liquid for 1 min at 500 × g, at room temperature.
    ​NOTE: To centrifuge plates, a swing-bucket rotor and plate buckets are required.

4. Treatment of cells

  1. Add 20 µL of the neutrophil suspension to each well using a 16-channel pipette (5-50 µL range, for each column of the 384-well plate). Gently mix 5-10 times using the pipette, maintaining a consistent time interval between each pipetting.
    NOTE: The final concentrations in the wells are as follows: neutrophils 2.5 × 105 cells/mL (all wells); mAb24-APC 0.5 µg/mL (all wells); fMLP 100 nM (positive control and testing wells); compounds 10 µM (testing wells).
  2. Incubate the plate on a shaker at 300 rpm, at room temperature (RT), for 10 min.
  3. Fix the cells by adding 3 µL of 16% paraformaldehyde (PFA) to each well using a 16-channel pipette (final PFA concentration ~0.91%). Then, incubate on ice for 10 min.
    ​NOTE: It is recommended to maintain a consistent time interval between different columns when adding neutrophils and PFA. This ensures that neutrophils in each well have the same incubation time with fMLP and mAb24-APC.

5. Flow cytometry

  1. Turn on the flow cytometer (see Table of Materials), and ensure that the computer connected to the instrument is also turned on.
  2. Ensure that the sheath fluid container is filled, and the waste container is empty and properly connected to the instrument.
  3. Load the 384-well plate onto the sample loader of the flow cytometer. Ensure that the A1 well of the plate aligns with the A1 mark on the loader.
  4. Open the software and create a new experiment. Select 384 well and choose the desired fluorophores. Next, click on Plate setup, drag to select all wells, and then click on Sample. Turn on the "High throughput" switch, and select High under the rate panel.
    1. In the volume box, enter 50. Select samples in odd-numbered columns and then activate the "Agitation" switch. Finally, name the samples in the right panel.
  5. Click on Plot and gate. Then, click on the Apply button (two arrows on a green-filled circle), and subsequently, click on the play button (triangle shape). Wait for a few seconds to observe some cells from the first well, and then click on the pause button.
    1. Adjust the FSC and SSC voltages to ensure proper visualization of cells on the plot. Click on Acquisition, and then click on the play button (triangle shape) to start the assay.
  6. The flow cytometer will sequentially sample ~50 µL of 1000-3000 neutrophils from each well. It will take approximately 3 h to complete the entire 384-well plate.
  7. After completing all samples, export the .fcs files for the next step.

6. Data analysis

  1. Open the flow cytometry data analysis software (see Table of Materials). Select and drag the folder containing all .fcs files into the software window, then release to import the data into the software.
  2. Double-click on one sample, gate single neutrophils based on FSC and SSC28,29,30 in the popped-up window, as shown in Figure 3. Select the gated rows and drag them to the folder, then release to apply the gating to all samples in that folder.
  3. Calculate and export the APC median fluorescence intensity (MFI) of neutrophils from each well using the software's table editor function. Click on Add Column under the Edit tab. Select Median in the Statistic tab, choose the gated population neutrophils, and select APC as the parameter. Click on the OK button.
  4. Return to the "Table editor" tab, select All Samples in the "Group" tab, and press the Create table button. A new window will appear displaying the created table. Save it as a text, CSV, or Excel file.
  5. Open the exported table, calculate the mean value and standard deviation of the APC MFI for the positive and negative control samples (Figure 4).
  6. Calculate the Z'-factor (Z') of the plate using the equation:
    Z' = 1 - 3(σp + σn)/(µp - µn),
    where σp and σn are the standard deviations of the positive and negative controls, respectively, and µp and µn are the means of the positive and negative controls, respectively31.
    NOTE: The Z' factor indicates the separation of the positive and negative control distributions. A Z' of 0 means no separation, while a Z' of 0.5 indicates equal separation. As mentioned in a previous study31, Z' > 0.5 indicates an excellent assay for compound screening. A Z' in the range of 0.5 to 0 is acceptable, but it may only identify strong hits in the assay, which will require further validation in secondary assays.
  7. Consider compounds as "hits" for screening when the APC MFI of compound-treated neutrophils is lower than three times the standard deviation of the positive control MFI subtracted from the positive control MFI (P = 0.0013, represented by the dotted line in Figure 4).

Results

Data from a representative 384-well plate screening (Figure 4) revealed that negative controls had an MFI of mAb24-APC of 3236 ± 110, while positive controls had an MFI of mAb24-APC of 7588 ± 858. The Z' factor for this plate is approximately 0.33, which is within an acceptable range31. However, Z' requires further validation in secondary assays.

To normalize the data, all values were scaled to assign a maximum value of 1 ...

Discussion

The initiation and termination of neutrophil stimulation and staining are determined by the addition of neutrophils and the fixative PFA. Therefore, ensuring the same time interval between pipetting neutrophils or PFA into each column is critical. This ensures that the stimulation and staining time of neutrophils from each well remains consistent. Due to the short lifespan of neutrophils, the entire experiment, from collecting blood from donors to completing flow cytometry, must be carried out on the same day. Neutrophil...

Disclosures

The authors declare no competing financial interest.

Acknowledgements

We thank Dr. Evan Jellison and Ms. Li Zhu in the flow cytometry core at UConn Health for their assistance with flow cytometry, Dr. Lynn Puddington in the Department of Immunology at UConn Health for her support of the instruments, Ms. Slawa Gajewska and Dr. Paul Appleton in the clinical research core at UConn Health for their help in obtaining blood samples. We acknowledge Dr. Christopher "Kit" Bonin and Dr. Geneva Hargis from UConn School of Medicine for their help with scientific writing and editing of this manuscript. This research was supported by grants from the National Institutes of Health, National Heart, Lung, and Blood Institute (R01HL145454), National Institute of General Medical Sciences (P20GM121176), USA, a Career Development Award from the American Heart Association (18CDA34110426), and a startup fund from UConn Health. Figure 1 was created with BioRender.com.

Materials

NameCompanyCatalog NumberComments
16-channel pipettesThermo4661090NInstrument
384-well plateGreiner784201Materials
APC anti-human CD11a/CD18 (LFA-1) Antibody Clone: m24BioLegend363410Reagents
Bravo Automated Liquid Handling Platform Agilent16050-102384 multi-channel liquid handler
CentrifugeEppendorfModel 5810RInstrument
FlowJoBecton, Dickinson & CompanyNASoftware
Human Serum Albumin Solution (25%)GeminiBio800-120Reagents
LifitegrastThermofisher 50-208-2121Reagents
Nexinhib20Tocris6089Reagents
N-Formyl-Met-Leu-Phe (fMLP)SigmaF3506Reagents
Paraformaldehyde 16% solutionElectron Microscopy Sciences15710Reagents
Plate bucketsEppendorfUL155Accessory
Plate shaker Fisher88-861-023Instrument
PolymorphPrepPROGEN1895 (previous 1114683)Reagents
Prestwick Chemical Library Compound Plates (10 mM)Prestwick Chemical LibrariesVer19_3841520 small molecules, 98% marketed approved drugs (FDA, EMA, JAN, and other agencies approved)
RPMI 1640 Medium, no phenol redGibco11-835-030Reagents
Swing-bucket rotor EppendorfA-4-62Rotor
ZE5 Cell AnalyzerBio-Rad LaboratoriesModel ZE5Instrument

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