Activated producing HOCL neutrophils revealed by flow cytometry and confocal microscopy with celestine blue B

Hypochlorous acid (HOCl) is produced with myeloperoxidase (MPO) in activated neutrophils. To assay MPO activity we chose a HOCl-selective dye i.e. celestine blue B (CB) that changes into pink glycol after oxidation. Our aim was to elaborate fluorescent methods of measuring HOCl production with activated neutrophils. Scanning CB fluorescence before and after its reaction with HOCl revealed activation and maximum emission, at 487 and 578 nm respectively, which are specific for oxidized product. Activated with Phorbol 12-myristate 13-acetate (PMA), the neutrophils were incubated with MPO inhibitor, 4-aminobenzoic acid hydrazide, which decreased fluorescence intensity (activation 487 nm, emission 578 nm) as compared with inhibitor-free samples. By confocal microscopy method we obtained images of CB- and DAPI-stained neutrophils. Neutrophil extracellular traps (NET) are clearly visible: DAP1 staining of multiple DNA bands co-localizes with fluorescence of oxidized CB. Flow cytometry showed that intensity of neutrophils activated by 50 nM PMA was 5 times higher (p < 0.05) than in PMA-free cells.

HOCl, HOCl, hypochlorous acid, myeloperoxidase, celestine blue B, neutrophil, confocal microscopy, flow cytometry, fluorescence

1. Панасенко О.М., Горудко И.В., Соколов А.В. Хлорноватистая кислота как предшественник свободных радикалов в живых системах // Успехи биологической химии. - 2013. - Т. 53. - С. 195-244

2. Соколов А.В., Голенкина Е.А., Костевич В.А., Васильев В.Б., Судьина Г.Ф. Взаимодействие церулоплазмина и 5-липоксигеназы // Биохимия. - 2010. - Т. 75, № 12. -С. 1687-1694

3. Dypbukt JM, Bishop C, Brooks WM, Thong B, Eriksson H, Kettle AJ (2005) A sensitive and selective assay for chloramine production by myeloperoxidase. Free Radical Biology and Medicine, (39), 1468-1477.

4. Flemmig J, Zschaler J, Remmler J, Arnhold J (2012). The fluorescein-derived dye aminophenyl fluorescein is a suitable tool to detect hypobromous acid (HOBr)-producing activity in eosinophils. Journal of Biological Chemistry, (287), 27913-27923.

5. Franck T, Minguet G, Delporte C, Derochette S, Zouaoui Boudjeltia K, van Antwerpen P, Gach O, Deby-Dupont G, Mouithys-Mickalad A, Serteyn D (2015). An immunological method to combine the measurement of active and total myeloperoxidase on the same biological fluid, and its application in finding inhibitors which interact directly with the enzyme. Free Radical Research, (49), 790-799.

6. Huang J, Milton A, Arnold RD, Huang H, Smith F, Panizzi JR, Panizzi P (2016). Methods for measuring myeloperoxidase activity toward assessing inhibitor efficacy in living systems. Journal of Leukocyte Biology, (99), 541-548.

7. Malle E, Furtmüller PG, Sattler W, Obinger C (2007). Myeloperoxidase: a target for new drug development? British Journal of Pharmacology, (152), 838-854.

8. Sokolov AV, Kostevich VA, Kozlov SO, Donskyi IS, Vlasova II, Rudenko AO, Zakharova ET, Vasilyev VB, Panasenko OM (2015). Kinetic method for assaying the halogenating activity of myeloperoxidase based on reaction of celestine blue B with taurine halogenamines. Free Radical Research, (46), 777-789.

9. Weiss SJ, Klein R, Slivka A, Wei M (1982). Chlorination of taurine by human neutrophils. Evidence for hypochlorous acid generation. Journal of Clinical Investigation, (70), 598-607.

10. Urban CF, Ermert D, Schmid M, Abu-Abed U, Goosmann C, Nacken W, Brinkmann V, Jungblut PR, Zychlinsky A (2009). Neutrophil extracellular traps contain calprotectin, a cytosolic protein complex involved in host defense against Candida albicans. PLoS Pathogens, (5), e1000639.