Micron 39 (2008) 367372 www.elsevier.

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Visualization of neutrophil extracellular traps in TEM

Wolf Dietrich Krautgartner a, Ljubomir Vitkov b,c,*
a

Department of Light & Electron Microscopy, Organismic Biology, University of Salzburg, Hellbrunnerstrae 34, A-5020 Salzburg, Austria b Department of Operative Dentistry & Periodontology, Medical University of Innsbruck, Anichstrae 35, A-6020 Innsbruck, Austria c Mayburgerplatz 7, A-5204 Strawalchen, Austria Received 25 February 2007; received in revised form 18 March 2007; accepted 19 March 2007

Abstract Neutrophil extracellular traps (NETs) have recently been described as an important innate defence mechanism in inammation. However, routine electron microscopic staining techniques faintly stain NETs and are therefore insufcient for enabling a distinction between these and the host cell debris as well as proteins regularly present at the site of inammation. In order to test suitable electron microscopic staining techniques, NETs induced ex vivo via phorbol myristate were absorbed on formvar. Four types of drop-on-grid positive staining were used: osmium tetroxide (Os), osmium tetroxideuranyl acetatelead citrate (OsUPb), ruthenium red-osmium tetroxide (RR-OsO4), and cuprolinic blue enhanced by sodium tungstate (CB-WO4). We observed no staining of NETs using Os, faint staining with OsUPb, better but still weak staining with CB-WO4 and outstanding staining with RR-OsO4. Furthermore, RR-OsO4 staining also enables the observation of bacterial mbriae-mediated adhesion, which is possibly responsible for the ability of NETs to bind bacteria. Thus, the offered RR-OsO4 staining technique may facilitate the study of the NETs-bacterial interactions. # 2007 Elsevier Ltd. All rights reserved.
Keywords: Cationic dyes; Cuprolinic blue; Ruthenium red; Deoxyribonuclease; Globular domains

1. Introduction Recently, a new mechanism for bacterial clearance by neutrophil extracellular traps (NETs) has been reported (Brinkmann et al., 2004). NETs are extracellular net-like bres generated by activated neutrophils and are able to disarm and kill bacteria extracellularly. They consist of a DNA backbone with embedded antimicrobial peptides and enzymes, e.g. histones and neutrophil elastase (Brinkmann et al., 2004; Fuchs et al., 2007). NETs bind Gram-positive as well as Gramnegative bacteria and are abundant in vivo in acute inammation. They appear to be a form of innate response that binds microorganisms, prevents them from spreading and ensures a high local concentration of antimicrobial agents to degrade virulence factors and kill bacteria (Brinkmann et al., 2004). Although NETs are of pronounced interest to the investigation of the host response to bacterial challenge, their visualization in inamed tissues by routine transmission electron microscopy techniques is not reliable, as there is

insufcient staining when applying routine techniques. Thus, the basic elements of NETs, the brils with a diameter of 15 17 nm have not been visualized by transmission electron microscopy (TEM) until now, but only by high resolution scanning electron microscopy (Brinkmann et al., 2004; Fuchs et al., 2007). Consequently, the question arises, whether a selective staining for chromatin could enable the visualization and differentiation of NETs by TEM in the presence of extracellular host proteins regularly present at the site of inammation. The aim of the present work was to offer a reliable method for visualization and differentiation of NETs by TEM. 2. Materials and methods 2.1. Neutrophil harvesting Mouse neutrophils were kindly supplied by Dr. J. Thalhammer and Dr. R. Weiss (Department of Molecular Biology, University of Salzburg, Austria). Peripheral blood mononuclear cells (PBMCs) were prepared from whole blood by erythrocyte lysis under gently hypotonic conditions. Two millilitre of heparin-treated whole mouse blood were mixed

* Corresponding author at: Mayburgerplatz 7, A-5204 Strawalchen, Austria. Tel.: +43 676 4041225; fax: +43 6215 20088. E-mail address: lvitkov@yahoo.com (L. Vitkov). 0968-4328/$ see front matter # 2007 Elsevier Ltd. All rights reserved. doi:10.1016/j.micron.2007.03.007

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with 5 ml of FACS Lysing Solution (Becton Dickinson, Schwechat, Austria) and incubated at room temperature for 10 min. Seven millilitre of the nutritive medium (MEM, PAA, Pasching, Austria) were added and the cells were centrifuged for 10 min at 1200 rpm. The supernatant was discarded and the pellet was washed two times in 10 ml MEM. Finally, PBMCs were resuspended in 1 ml MEM, supplemented with 1% fetal calf serum, 1% L-glutamine, 1% penicillin/streptomycin, 1 mM sodium pyruvate, 20 mM HEPES, 1% nonessential aminoacids and 2 mM beta-mercaptoethanol. The nal concentration of the freshly separated mouse PBMCs was 2 107 cells/ml, whereof 1060% were neutrophils. Twenty microlitre of the cell suspension were dropped on each grid (100 mesh gold grids covered by formvar). Subsequently, the grids were placed into a humid chamber at 37 8C for 90 min. The grids were blotted and dropped with 20 ml of the same nutritive solution supplemented by 25 nM PMA (phorbol 12-myristate 13-acetate, LC Laboratories, Woburn, MA 01801) at 37 8C for 60 min to induce the NET formation (Brinkmann et al., 2004). 2.2. DNA digestion Half of the samples were treated with 20 mM TrisHCl and 5 mM MgCl2 and the rest with 1 mg/ml deoxyribonuclease (DNase I, Roche Diagnostics GmbH, Mannheim, Germany) buffered with 20 mM TrisHCl and supplemented with 5 mM MgCl2 at 378 for 60 min. 2.3. Osmium tetroxide (Os) staining The grids were washed with 0.1 M sodium cacodylate buffered at pH 5.6. Fixation was performed by 1.2% glutaraldehyde buffered at pH 5.6 with 0.1 M sodium cacodylate for 15 min at room temperature. Subsequently, the grids were treated with 1.2% osmium tetroxide buffered at pH 5.6 with 0.1 M sodium cacodylate for 1 min at room temperature. 2.4. Osmium tetroxideuranyl acetatelead citrate (OsUPb) staining Half of the grids stained with osmium tetroxide were subsequently stained with 1% aqueous uranyl acetate (Ultrostain 1, Leica, Vienna, Austria) as well as 1% aqueous lead citrate (Ultrostain 2, Leica, Vienna, Austria) by LKB 2168 Ultrostainer (LKB Produkter AB, Bromma, Sweden). 2.5. Cuprolinic blue (CB) staining The grids were washed with 0.05 M sodium acetate buffered at pH 5.6. Subsequently, the grids were xed with 1.2% glutaraldehyde and 0.2% cuprolinic blue (Cuprolinic blue, Polysciences Inc., Warrington, PA 18976) (buffered at pH 5.6 with 0.05 M sodium acetate) in dark for 30 min at room temperature. Enhancement was performed with 1% aqueous sodium tungstate for 30 s and subsequent dehydration was performed with an ascending series of ethanol. The 30 and 50% ethanol drops contained also 1% sodium tungstate.

2.6. Ruthenium red-osmium tetroxide (RR-OsO4) staining The grids were washed with 0.1 M sodium cacodylate buffered at pH 5.6. Subsequently, the grids were xed with 1.2% glutaraldehyde and 0.05% ruthenium red (buffered at pH 5.6 with 0.1 M sodium cacodylate) for 15 min at room temperature. Postxation was performed with 1.2% osmium tetroxide (buffered at pH 5.6 with 0.1 M sodium cacodylate) and 0.05% ruthenium red for one min at room temperature. After staining, all of the grids were simply washed and airdried. The RR-OsO4 drop-on-grid specimens and particularly the drop-on-grid cuprolinic blue specimens were unstable and faded after a few days. 2.7. TEM processing All samples were examined with a transmission electron microscope LEO EM 910 (LEO Elektronenmikroskopie Ltd., Oberkochen, Germany) operating at 80 kV. 3. Results Os-stained samples revealed no structures. OsUPb staining revealed well formed, but faintly blackened networks consisting of a multitude of interwoven threads. They branched into thinner threads, but no brils with a diameter 1517 nm could be distinguished (Fig. 1). CB staining revealed a similar appearance of NETs, however, somewhat better staining was achieved (Figs. 24). RR-OsO4 staining showed at low magnication a similar appearance of NETs (Fig. 5). However, the higher magnication revealed that the thinner threads branched into ne bres (Fig. 6). A sharp demarcation of deeply blackened bres with diameters of circa 1517 nm and less frequently of circa 6 nm was evident (Fig. 7), however, no globular domains could be observed. Two predominant patterns of thread alignment were observed: a radiating one and a meshwork-like one. The radiating thread alignment pattern was characterized by the branching of a few thick threads into thinner ones, which on their part branched into bres (Figs. 57). The meshwork-like pattern covered areas extending to more than 1000 mm2 (Fig. 8) and was characterized by a relative uniformity of appearance of dense interwoven threads and bres with a diameter of circa 1517 nm (Fig. 9). In contrast, bres with a diameter of 6 nm were not observed within this pattern. The treatment by DNase prior to the xation completely disaggregated the NETs. In the RR-OsO4 stained samples, however, a multitude of tiny particles, very probably NET fragments, were observed (Fig. 10). The electron density of the particles is the same as that of the NETs (Fig. 11). 4. Discussion Morphologically, NETs have been characterized as DNaselabile net-like formations of bres with a diameter of 15 17 nm, with so-called globular domains on them with a diameter of approximately 25 nm (Brinkmann et al., 2004).

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Figs. 14. (1) OsUPb staining. NETs. Inset: The higher magnication shows lack of ne structural details. (2) CB staining. An overview of NETs. Some similarity with light microscopy micrographs (Brinkmann et al., 2004) is evident. (3) CB staining (a detail of Fig. 2). The deeply blackened spots (arrows) are probably phthalocyanine precipitates, as previously reported (Krautgartner et al., 2003, 2005). (4) CB staining (a detail of Fig. 3). The higher magnication shows lack of ne structural details.

Besides the bactericidal effects caused by neutrophil elastase and histones, the ability to trap bacteria is another key characteristic of NETs. The bacterial binding by NETs probably occurs by mbriae-mediated adhesion. Indeed, bacterial mbriae-mediated adhesion to histones (Zhu et al., 2005) and to DNA (van Schaik et al., 2005) has been demonstrated. Both DNA and bacterial mbriae are poorly depicted by routine TEM staining techniques. As DNA and microbial mbriae are polyanions, cationic stains have been used for their visualization by TEM. Thus, uranyl and lead ions (Pearse, 1985; Trendelenburg and Puvion-Dutilleul, 1987), in particular the combination of uranyl acetate and lead citrate, are still constantly employed for this purpose. However, these staining techniques have low specicity, feebly contrast the DNA and therefore disable a good resolution. Two cationic dyes, the phthalocyanine cuprolinic blue and ruthenium red have also been used for chromatin staining. CB has been used for visualizing DNA in light microscopy (Tas et al., 1983; Mendelson et al., 1983), however, enhancing the phthalocyanine staining by sodium tungstate enables the

attainment of a sufcient contrast in TEM (Scott et al., 1981). Despite the good staining specicity, the DNA contrast attained by this technique was insufcient to achieve high resolution. Nevertheless, the possibility to visualize the microbial adhesion via phthalocyanines at TEM level (Fassel et al., 1992; Krautgartner et al., 2003, 2005) makes this method, albeit restricted by its limited resolution, suitable to study the NETsbacterial interactions. Ruthenium red shows a strong afnity for chromatin (Stockert and Pelling, 1992; Engelhardt, 2000). However, as chromatin and also NETs consist of two main components, DNA and proteins, it remains unclear which component(s) really stain by RR-OsO4. Thus, it has been hypothesized by Engelhardt (Engelhardt, 2000) that the chromatin staining by RR-OsO4 could be caused by a reaction with the chromatin glycoproteins (Reeves et al., 1981; Turner et al., 1990). Although RR alone produces some degree of electron opacity (Stockert and Pelling, 1992), RR-OsO4 creates a much more intense contrasting. Indeed, the presently employed RR-OsO4 staining technique enabled the outstanding staining of the

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Figs. 57. (5) RR-OsO4 staining. An overview of NETs. Some similarity with light microscopic micrographs (Brinkmann et al., 2004) is evident. (6) RR-OsO4 staining (a detail of Fig. 5). The alignment of the threads is clearly distinguishable. (7) RR-OsO4 staining (a detail of Fig. 6). Fibres with diameter of 1517 nm form a meshwork. Arrows: some bres with a diameter of 6 nm are also evident.

NETs, despite the superimposition by cellular debris and proteins from the nutritive solution. The RR-positive NETs mainly consisted of bres with a diameter of circa 1517 nm, which were interwoven in net-like formations. RR-OsO4

staining revealed also bres with a diameter of circa 6 nm, which has not been observed with scanning electron microscopy. RR-OsO4 stains microbial mbriae (Fassel et al., 1992; Fassel and Edmiston, 1999; Vitkov et al., 2001, 2002, 2005a,b),

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Figs. 811. (8) RR-OsO4 staining. Huge areas are covered by meshwork of a relatively uniform density. (9) RR-OsO4 staining. The higher magnication reveals no basic differences to the loose networks. Inset: Interwoven bres with similar dimension as shown in Fig. 7. (10) RR-OsO4 staining. The sample is treated by deoxyribonuclease prior to the staining. No networks, but multitudes of tiny particles were observed. (11) RR-OsO4 staining (a detail from Fig. 10). Many deeply contrasted particles, probably NET fragments, are evident. The blackening of the particles is the same as that of the NETs.

proteoglycans (Chan and Wong, 1992; Rodgers et al., 1995) and chromatin. Consequently, the DNase digestion of the NET bres veried their DNA character. By contrast, no globular domains were visualized via RR-OsO4; consequently, they lack chromatin. Two modes of NET spreading were observed: radiating ones and web-like ones. In the radiating NETs, the threads were branched into bres with a diameter of 1517 nm. The threads have been suggested to consist of aggregated bres (Brinkmann et al., 2004). In the web-like NETs, the spread was more homogenous and fewer threads with smaller diameters were observed. In our opinion, the web-like appearance was a consequence of the superimposition of a multitude of better spread NETs. As the adhesion of NETs to the formvar causes a transformation of the three-dimensional NETs into twodimensional images, deductions concerning the three-dimensional relationship between the NET elements by virtue of the presented results are restricted. The ability of RR staining to visualize the microbial adhesion makes this method particularly appropriate for studying the NETs-bacterial interactions. The deeply blackened DNase-sensitive smooth stretches, with

their characteristic diameter, are the assertive criterion to identify NETs. 5. Conclusions The employed RR-OsO4 staining technique enables the visualization of both NETs and mbriae-mediated bacterial adhesion (Vitkov et al., 2001, 2002, 2005a,b). It thus facilitates the examination of NETs-bacterial interactions at TEM level. The combination of the following two characteristics guarantees the high specicity of this staining procedure: the blackening by RR-OsO4 and the disintegration of the RR-OsO4-positive netlike structures by DNase. Additionally, the combination of the specic bre thickness (1517 nm) and of the deep blackening of the bres is a very strong indication of NETs. Acknowledgements The authors thank Professor Josef Thalhammer and Dr. Robert Weiss for supplying the mouse neutrophils, Dr. Karin

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Oberascher, Mrs. Adda Maenhardt and Mrs. Michaela Klappacher for the technical assistance as well as Mr. Andreas Zankl for the image processing. References
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