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. 2016 Aug:435:77-84.
doi: 10.1016/j.jim.2016.06.002. Epub 2016 Jun 7.

Colocalization of neutrophils, extracellular DNA and coagulation factors during NETosis: Development and utility of an immunofluorescence-based microscopy platform

Laura D Healy  1 , Cristina Puy  2 , Asako Itakura  3 , Tiffany Chu  2 , David K Robinson  2 , Alan Bylund  2 , Kevin G Phillips  2 , Elizabeth E Gardiner  4 , Owen J T McCarty  5
Affiliations

Colocalization of neutrophils, extracellular DNA and coagulation factors during NETosis: Development and utility of an immunofluorescence-based microscopy platform

Laura D Healy et al. J Immunol Methods. 2016 Aug.

Abstract

Background: Neutrophils, the most populous innate immune cell type, are the first responders to sites of infection and inflammation. Neutrophils can release their DNA to form extracellular traps (NETs), webs of DNA and granular proteases that contribute to pathogen clearance and promote thrombus formation. At present, the study of NETs is in part limited to the qualitative analysis of fluorescence microscopy-based images, thus quantification of the interactions between NETs and coagulation factors remains ill-defined.

Aim: Develop a quantitative method to measure the spatial distribution of DNA and colocalization of coagulation factor binding to neutrophils and NETs utilizing fluorescence-based microscopy.

Approach: Human neutrophils were purified from peripheral blood, bound to fibronectin and treated with the PKC-activator phorbol myristate acetate (PMA) to induce neutrophil activation and NETs formation. Samples were incubated with purified coagulation factors or plasma before staining with a DNA-binding dye and coagulation factor-specific antibodies. The spatial distribution of DNA and coagulation factors was imaged via fluorescence microscopy and quantified via a custom-built MATLAB-based image analysis algorithm. The algorithm first established global thresholding parameters on a training set of fluorescence image data and then systematically quantified intensity profiles across treatment conditions. Quantitative comparison of treatment conditions was enabled through the normalization of fluorescent intensities using the number of cells per image to determine the percent and area of DNA and coagulation factor binding per cell.

Results: Upon stimulation with PMA, NETs formation resulted in an increase in the area of DNA per cell. The coagulation factor fibrinogen bound to both the neutrophil cell body as well as NETs, while prothrombin, FX and FVIIa binding was restricted to the neutrophil cell body. The Gla domain of FX was required to mediate FX-neutrophil binding. Activated protein C (APC), but not Gla-less APC, bound to neutrophil cell bodies and NETs in a punctate manner. Neither FXIIa nor FXIa were found to bind either neutrophil cell bodies or NETs. Fibrinogen binding was dependent on extracellular DNA, while FX and APC required phosphatidylserine exposure for binding to activated neutrophils.

Conclusions: We have developed a quantitative measurement platform to define the spatial localization of fluorescently-labeled coagulation factor binding to neutrophils and extracellular DNA during NETosis.

Keywords: Biophysical measurement; Coagulation factors; Fluorescence microscopy; Neutrophil extracellular traps.

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Conflict of interest statement

Disclosures

The authors have no conflicts of interest to declare.

Figures

Figure 1
Figure 1. NETs promote binding of select purified coagulation factors
Acid-washed glass coverslips were coated with 20 µg/mL fibronectin and then blocked with denatured BSA (5 mg/mL). Purified human neutrophils (2×106/mL) were plated on the coverslips, and were treated with HBSS or PMA (10 nM) for 3 hours at 37°C. Cell samples were washed and treated with vehicle (HBSS buffer), fibrinogen (2.6 mg/mL), prothrombin (FII, 100 µg/mL), FX (10 µg/mL), FVIIa (300nM), APC (300 nM), protein C (300 nM) was then incubated for 15 minutes with the cell samples at 37°C. Samples were then fixed with 4% PFA. (A) Samples were incubated overnight with primary antibodies. Samples were then incubated with Hoechst 33342 (1:1000) and secondary antibodies Alexa Fluor 488 goat anti-rabbit and Alexa Fluor 546 goat anti-mouse (Invitrogen, 1:500). Images were normalized to secondary antibody alone images and quantified in a custom MATLAB program to quantify each pixel positive signal, shown above are representative images of coagulation factors with positive cell staining.
Figure 2
Figure 2. DNA was quantified as area per image and per cell
Images were normalized to secondary antibody alone images and quantified in a custom MATLAB program to quantify each pixel positive signal as (A) the area of DNA per image and (B) area DNA per cell. Data are mean±SEM n=3.
Figure 3
Figure 3. Purified coagulation factor proteins quantified as area per image and per cell
Acid-washed glass coverslips were coated with 20 µg/mL fibronectin and then blocked with denatured BSA (5 mg/mL). Purified human neutrophils (2×106/mL) were plated on the coverslips, and were treated with HBSS or PMA (10 nM) for 3 hours at 37°C. Cell samples were washed and treated with vehicle (HBSS buffer), fibrinogen (2.6 mg/mL), prothrombin (FII, 100 µg/mL), FX (10 µg/mL), FX-GD (10 µg/mL), protein C (300 nM), APC (300 nM), APC-GD (300 nM), FVIIa (300nM), FXIa (20 µg/mL), FXIIa (20 µg/mL) in the presence of HK (20 µg/mL) and ZnCl2 (25 µM) was then incubated for 15 minutes with the cell samples at 37°C. Samples were then fixed with 4% PFA. (A) Samples were incubated overnight with primary antibodies. Samples were then incubated with Hoechst 33342 (1:1000) and secondary antibodies Alexa Fluor 488 goat anti-rabbit and Alexa Fluor 546 goat anti-mouse (Invitrogen, 1:500). Images were normalized to secondary antibody alone images and quantified in a custom MATLAB program to quantify each pixel positive signal as (A) the area of factor per image and (B) area of signal per cell. Data are mean±SEM n=3.
Figure 4
Figure 4. NETs promotes localization of plasma prothrombin, FX and fibrinogen
Representative images of neutrophils and NETs treated with buffer or plasma and stained for DNA, fibrinogen and either (A) prothrombin or (B) FX.
Figure 5
Figure 5. Coagulation factors in platelet poor plasma colocalize to NETs, quantified as area per image and per cell
Acid-washed glass coverslips were coated with 20 µg/mL fibronectin and then blocked with denatured BSA (5 mg/mL). Purified human neutrophils (2×106/mL) were plated on the coverslips, and were treated with HBSS or PMA (10 nM) for 3 hours at 37°C. Cell samples were washed and incubated with platelet-poor plasma or vehicle (HEPES containing 2mM CaCl2, 2mM MgCl2 and 0.1% BSA) (1:1) for 15 min at 37°C. Samples were incubated overnight with primary antibodies. Samples were then incubated with Hoechst 33342 (1:1000) and secondary antibodies Alexa Fluor 488 goat anti-rabbit and Alexa Fluor 546 goat anti-mouse (Invitrogen, 1:500). Images were normalized to secondary antibody alone images and quantified in a custom MATLAB program to quantify each pixel positive signal as (A) the area per image and (B) area per cell. Data are mean±SEM n=3.
Figure 6
Figure 6. Coagulation factors bind neutrophils and neutrophil extracellular traps in a DNA- and phospholipid-dependent manner quantified as area per image and per cell
Acid-washed glass coverslips were coated with 20 µg/mL fibronectin and then blocked with denatured BSA (5 mg/mL). Purified human neutrophils (2×106/mL) were plated on the coverslips, and were treated with HBSS or PMA (10 nM) for 3 hours at 37°C. Cell samples were washed and treated with vehicle (HBSS buffer), DNase I (10,000U/mL), RGDS (20 µM), Annexin V (10 µg/mL) for 10 minutes at 37°C. Cell samples were then washed and treated with vehicle (HBSS buffer), fibrinogen (2.6 mg/mL), FX (10 µg/mL), and APC (300 nM) was then incubated for 15 minutes with the cell samples at 37°C. Samples were then fixed with 4% PFA. (A) Samples were incubated overnight with primary antibodies. Samples were then incubated with Hoechst 33342 (1:1000) and secondary antibodies Alexa Fluor 488 goat anti-rabbit and Alexa Fluor 546 goat anti-mouse (Invitrogen, 1:500). Images were normalized to secondary antibody alone images and quantified in a custom MATLAB program to quantify each pixel positive signal as (A) the area of factor per image and (B) area of signal per cell. Data are mean±SEM n=3.
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