Kinetic analysis of phagosomal production of reactive oxygen species

doi:10.1016/j.freeradbiomed.2010.11.024

Free Radical Biology and Medicine

Volume 50, Issue 3, 1 February 2011, Pages 438-447

https://doi.org/10.1016/j.freeradbiomed.2010.11.024 Get rights and content

Abstract

Phagocytes produce large quantities of reactive oxygen species for pathogen killing; however, the kinetics and amplitude of ROS production on the level of individual phagosomes are poorly understood. This is mainly due to the lack of appropriate methods for quantitative ROS detection with microscopic resolution. We covalently attached the ROS-sensitive dye dichlorodihydrofluorescein (DCFH₂) to yeast particles and investigated their fluorescence due to oxidation in vitro and in live phagocytes. In vitro, the dye was oxidized by H₂O₂ plus horseradish peroxidase but also by HOCl. The latter produced a previously unrecognized oxidation product with red-shifted excitation and emission spectra and a characteristic difference in the shape of the excitation spectrum near 480 nm. Millimolar HOCl bleached the DCFH₂ oxidation products. Inside phagosomes, DCFH₂-labeled yeast were oxidized for several minutes in a strictly NADPH oxidase-dependent manner as shown by video microscopy. Inhibition of the NADPH oxidase rapidly stopped the fluorescence increase of the particles. At least two characteristic kinetics of oxidation were distinguished and the variability of DCFH₂ oxidation in phagosomes was much larger than the variability upon oxidation in vitro. We conclude that DCFH₂–yeast is a valuable tool to investigate the kinetics and amplitude of ROS production in individual phagosomes.

Introduction

Quantitative measurement of reactive oxygen species (ROS)¹ has been a challenge ever since the importance of oxidative stress was recognized. Among the numerous organic dyes available for ROS detection, some but not all of them are suitable for fluorescence light microscopy [1], [2]. Indeed, the large majority of these dyes are not perfectly specific and suitable for ROS detection in time and space at the microscopic level. Although new detection methods based on nanoparticles [3] or on fluorescent proteins [4], [5] are under development, the characterization of existing dyes is essential to obtain as much information as possible from them and at the same time recognize their limitations.

By their nature, most ROS have only a short range of diffusion before they react with any biological material. From this point of view, ROS are ideal messengers for subcellular, local biological effects. Information on the subcellular localization of ROS-producing enzymes such as NADPH oxidases and the timing of their activation and inactivation is scarce [6]. Thus, in addition to the spectral properties of the probe and its specificity, its localization needs to be considered. On one hand, most probes do not readily traverse membranes even if some are available in a hydrophobic form (e.g., acetoxymethyl ester) to charge cells. On the other hand, certain ROS, namely hydrogen peroxide (H₂O₂), diffuse across membranes and may reach dyes located in a different cellular compartment. Reactions between radical species are catalyzed by enzymes or ions; the localization of the catalyst with respect to the dye and the ROS could be a critical issue [7]. In conclusion, the localized detection of ROS by fluorescence light microscopy requires an experimental setup that allows spatial control of the fluorescent dyes.

Phagocytes are particularly strong producers of ROS via the NADPH oxidase, NOX2. Strong phagosomal ROS production is essential for efficient host defense against bacterial and fungal infections [8]. Certain bacteria have developed sophisticated methods to avoid or resist phagosomal ROS production [9]. To understand the mechanism and control of phagosomal ROS production, accurate measurement of ROS inside the phagosomes of living cells would be of great use. However, quantitative measurements of the "chemistry of the phagosome" remain a difficult task, the lack of appropriate dyes being one of the problems. The NADPH oxidases produce superoxide, O₂^•−, which dismutates to H₂O₂. Myeloperoxidase (MPO) transforms H₂O₂ into hypochlorous acid (HOCl) and the Fenton reaction generates hydroxyl radical (OH^•) from H₂O₂. These compounds appear in parallel in the phagosome at quite different concentrations and with distinct time courses.

Much of the current knowledge about the phagocyte NADPH oxidase comes from biochemical studies; however, innovative microscopic techniques have become available to investigate the spatial and temporal aspects in living cells [10]. The first microscopic measurement of phagosomal ROS was achieved with Soret band absorption on internalized erythrocytes [11]. The method is based on the decreased intensity of Soret band absorption upon exposure of hemoglobin to ROS. The technique has a good time resolution and allows scoring of oxidized versus nonoxidized phagosomes. It was successfully combined with measurements of NADPH autofluorescence to show that NADPH translocates to phagosomes before its consumption for target oxidation [12]. In combination with exogenous fluorophores it was shown that lactoferrin-containing granules and ROS are delivered in parallel to the phagosome [13]. Nitroblue tetrazolium staining also allowed qualitative ROS detection in individual phagosomes, albeit with low time resolution and without combination with other imaging techniques [14]. In recent years, improved fluorescence microscopy has provided more detailed quantitative data. Advantages and limitations of microscopy methods as well as the importance of targeted probes have been reviewed by Yeung et al. [15].

One of the probes that have been widely used for cellular ROS detection in phagocytes and other cells is the nonfluorescent fluorescein derivative 2′,7′-dichlorodihydrofluorescein, or DCFH₂. After oxidation, DCFH₂ is transformed into the fluorescent 2′,7′-dichlorofluorescein, or DCF. The reactions of oxidation in the presence of a large variety of ROS have been extensively studied in vitro. Most articles agree that O₂^•− reacts poorly with DCFH₂ [16], [17], [18]. H₂O₂ is also weakly reactive; however, in the presence of peroxidases such as horseradish peroxidase (HRP), this compound becomes a major oxidant [16], [17], [19], [20]. HOCl was reported to be a poor oxidant at low concentration [19], whereas OH^• was shown to oxidize DCFH₂ very rapidly [16], [18], [20]. Despite its evident lack of specificity, one particular great advantage of this dye stems from the fact that an amine-reactive derivative, 2′,7′-dichlorodihydrofluorescein diacetate, succinimidyl ester, is commercially available. It can thus be covalently linked to proteins and other biological substances by standard laboratory procedures. DCFH₂ attached to immunoglobulins [21] or particles has been successfully used in a number of publications. For example, labeled polystyrene beads [22] or zymosan particles [23] upon opsonization become internalized and their oxidation reveals phagosomal ROS production.

We attached DCFH₂ to yeast particles and investigated the reactivity of these DCFH₂–yeast toward various ROS in vitro and inside phagosomes. DCFH₂–yeast clearly reported NADPH oxidase-dependent ROS production during the first minutes of phagocytosis. DCFH₂ was oxidized by H₂O₂ as well as HOCl, creating various reaction products that were detectable in phagosomes.

Section snippets

Preparation of solutions

DCFH₂ was prepared from 2′,7′-dichlorodihydrofluorescein diacetate (Sigma) by alkaline hydrolysis with 10 mM NaOH as described by Wrona et al. [18] and dissolved at a final concentration of 100 μM in 50 mM phosphate buffer. DCF (Sigma) solutions for absorption coefficient measurement and spectra acquisition were prepared in 50 mM phosphate buffer, pH 7.4. H₂O₂, sodium hypochlorite (NaOCl), and HRP (P6782) were purchased from Sigma. H₂O₂ was standardized spectrally (ε_240nm = 39.4 M^− 1 cm^− 1) [19]. NaOCl

Chemistry of the dye and identification of a new oxidation product

To use DCFH₂-labeled particles as a quantitative indicator of phagosomal ROS production, it is necessary to understand the influence of the phagosomal chemistry on the dye. Phagocytes liberate large amounts of ROS into the confined phagosomal volume, potentially generating very high concentrations [27]. We concentrated our investigation on the particular conditions of the phagosome.

The known oxidation product of DCFH₂ with ROS is DCF. In agreement with the literature, H₂O₂ in the presence of

Discussion

We have characterized DCFH₂-labeled yeast as a tool for ROS detection in phagosomes. The amplitude and kinetics of the fluorescence increase of these particles within phagosomes was NADPH oxidase dependent and suitable for video microscopy for at least 15 min. DCFH₂–yeast revealed that the ROS production in phagosomes is heterogeneous, with at least two types of kinetics. Several phagosomes in the same cell can be followed individually.

We confirmed that H₂O₂ in the presence of a peroxidase

Conclusion

The attachment of DCFH₂ to particles creates a highly localized ROS detector suitable for detection with standard fluorescence microscopes, spectrofluorimeters, and flow cytometry.

DCFH₂–yeast is a promising tool for kinetic measurements of phagosomal ROS production. The dye is perfectly localized and detects only ROS within the phagosome. However, the interpretation of the DCFH₂ experiments needs to take into account that different ROS may oxidize DCFH₂, catalysts are involved, and several

Acknowledgments

This work was supported by grants from the French Research Ministry, ACI DRAB and Microbiology Program, French League against Cancer, and BQR Program of the University of Paris-Sud. A.T. received a travel grant from the French–Tunisian cooperation program, CMCU. We thank Fabienne Merola, Mickael Bourge, and Jean-Claude Sulpice for help and comments.

References (43)

A. Gomes et al.
Fluorescence probes used for detection of reactive oxygen species
J. Biochem. Biophys. Meth.
(2005)
P. Wardman
Fluorescent and luminescent probes for measurement of oxidative and nitrosative species in cells and tissues: progress, pitfalls, and prospects
Free Radic. Biol. Med.
(2007)
T. Yeung et al.
Quantitative fluorescence microscopy to probe intracellular microenvironments
Curr. Opin. Microbiol.
(2005)
S.L. Hempel et al.
Dihydrofluorescein diacetate is superior for detecting intracellular oxidants: comparison with 2′, 7′-dichlorodihydrofluorescein diacetate, 5(and 6)-carboxy-2′, 7′-dichlorodihydrofluorescein diacetate, and dihydrorhodamine 123
Free Radic. Biol. Med.
(1999)
C. Rota et al.
Evidence for free radical formation during the oxidation of 2′, 7′-dichlorofluorescin to the fluorescent dye 2′, 7′-dichlorofluorescein by horseradish peroxidase: possible implications for oxidative stress measurements
Free Radic. Biol. Med.
(1999)
M. Wrona et al.
Reactivity of 2′, 7′-dichlorodihydrofluorescein and dihydrorhodamine 123 and their oxidized forms toward carbonated nitrogen dioxide, and hydroxyl radicals
Free Radic. Biol. Med.
(2005)
J.P. Crow
Dichlorodihydrofluorescein and dihydrorhodamine 123 are sensitive indicators of peroxynitrite in vitro: implications for intracellular measurement of reactive nitrogen and oxygen species
Nitric Oxide Biol. Chem.
(1997)
O. Myhre et al.
Evaluation of the probes 2′, 7′-dichlorofluorescin diacetate, luminol, and lucigenin as indicators of reactive species formation
Biochem. Pharmacol.
(2003)
T.C. Ryan et al.
Measurement of superoxide release in the phagovacuoles of immune complex-stimulated human neutrophils
J. Immunol. Meth.
(1990)
C.C. Winterbourn et al.
Modeling the reactions of superoxide and myeloperoxidase in the neutrophil phagosome—implications for microbial killing
J. Biol. Chem.
(2006)

P. Bilski et al.

Photosensitized oxidation of 2′, 7′-dichlorofluorescin: singlet oxygen does not contribute to the formation of fluorescent oxidation product 2′, 7′-dichlorofluorescein

Free Radic. Biol. Med.

(2002)

G. Griffiths

On phagosome individuality and membrane signalling networks

Trends Cell Biol.

(2004)

D.L. Clemens

Characterization of the Mycobacterium tuberculosis phagosome

Trends Microbiol.

(1996)

D. Casanova et al.

Single europium-doped nanoparticles measure temporal pattern of reactive oxygen species production inside cells

Nat. Nanotechnol.

(2009)

V.V. Belousov et al.

Genetically encoded fluorescent indicator for intracellular hydrogen peroxide

Nat. Meth.

(2006)

M. Gutscher et al.

Real-time imaging of the intracellular glutathione redox potential

Nat. Meth.

(2008)

T.E. DeCoursey et al.

Regulation and termination of NADPH oxidase activity

Cell. Mol. Life Sci.

(2005)

M. Karlsson et al.

What does the commonly used DCF-test for oxidative stress really show?

Biochem. J.

(2010)

W.M. Nauseef

How human neutrophils kill and degrade microbes: an integrated view

Immunol. Rev.

(2007)

L.A. Allen et al.

To activate or not to activate: distinct strategies used by Helicobacter pylori and Francisella tularensis to modulate the NADPH oxidase and survive in human neutrophils

Immunol. Rev.

(2007)

H.J. van Manen et al.

Single-cell optical imaging of the phagocyte NADPH oxidase

Antioxid. Redox Signaling

(2006)

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The rhodamine-based probe R19-S has been shown to react with hypochlorous acid (HOCl) to yield fluorescent R19, but not with some other oxidants including hydrogen peroxide. Here, we further examined the specificity of R19-S and used it for real-time monitoring of HOCl production in neutrophil phagosomes. We show that it also reacts rapidly with hypobromous acid, bromamines, and hypoiodous acid, indicating that R19-S responds to these reactive halogen species as well as HOCl. Hypothiocyanous acid and taurine chloramine were unreactive, however, and ammonia chloramine and dichloramine reacted only very slowly. MS analyses revealed additional products from the reaction of HOCl with R19-S, including a chlorinated species as a minor product. Of note, phagocytosis of opsonized zymosan or Staphylococcus aureus by neutrophils was accompanied by an increase in R19 fluorescence. This increase depended on NADPH oxidase and myeloperoxidase activities, and detection of chlorinated R19-S confirmed its specificity for HOCl. Using live-cell imaging to track individual phagosomes in single neutrophils, we observed considerable heterogeneity among the phagosomes in the time from ingestion of a zymosan particle to when fluorescence was first detected, ranging from 1 to >30 min. However, once initiated, the subsequent fluorescence increase was uniform, reaching a similar maximum in ∼10 min. Our results confirm the utility of R19-S for detecting HOCl in real-time and provide definitive evidence that isolated neutrophils produce HOCl in phagosomes. The intriguing variability in the onset of HOCl production among phagosomes identified here could influence the way they kill ingested bacteria.
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The neutrophil enzyme, myeloperoxidase, by converting hydrogen peroxide (H₂O₂) and chloride to hypochlorous acid (HOCl), provides important defense against ingested micro-organisms. However, there is debate about how efficiently HOCl is produced within the phagosome and whether its reactions with phagosomal constituents influence the killing mechanism. The phagosome is a small space surrounding the ingested organism, into which superoxide, H₂O₂ and high concentrations of proteins from cytoplasmic granules are released. Previous studies imply that HOCl is produced in the phagosome, but a large proportion should react with proteins before reaching the microbe. To mimic these conditions, we subjected neutrophil granule extract to sequential doses of H₂O₂. Myeloperoxidase in the extract converted all the H₂O₂ to HOCl, which reacted with the granule proteins. 3-Chlorotyrosine, protein carbonyls and large amounts of chloramines were produced. At higher doses of H₂O₂, the extract developed potent bactericidal activity against Staphylococcus aureus. This activity was due to ammonia monochloramine, formed as a secondary product from protein chloramines and dichloramines. Isolated myeloperoxidase and elastase also became bactericidal when modified with HOCl and antibacterial activity was seen with a range of species. Comparison of levels of protein modification in the extract and in phagosomes implies that a relatively low proportion of phagosomal H₂O₂ would be converted to HOCl, but there should be sufficient for substantial protein chloramine formation and some breakdown to ammonia monochloramine. It is possible that HOCl could kill ingested bacteria by an indirect mechanism involving protein oxidation and monochloramine formation.
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They also reconfirm that the direct reaction between roGFP2 and either H2O2 or GSSG is kinetically insignificant at the given concentration and time frame. When phagocytes mount a respiratory burst, a set of highly reactive oxygen species is produced, which includes hypochlorous acid (HOCl) [17,18]. Due to the great difficulty of measuring HOCl in vivo, the actual concentrations reached in phagocytes during the respiratory burst remain unknown.
The genetically encoded probes roGFP2-Orp1 and Grx1-roGFP2 have been designed to be selectively oxidized by hydrogen peroxide (H₂O₂) and glutathione disulfide (GSSG), respectively. Both probes have demonstrated such selectivity in a broad variety of systems and conditions. In this study, we systematically compared the in vitro response of roGFP2, roGFP2-Orp1 and Grx1-roGFP2 to increasing amounts of various oxidant species that may also occur in biological settings. We conclude that the previously established oxidant selectivity is highly robust and likely to be maintained under most physiological conditions. Yet, we also find that hypochlorous acid, known to be produced in the phagocyte respiratory burst, can lead to non-selective oxidation of roGFP2-based probes at concentrations ≥2 µM, in vitro. Further, we confirm that polysulfides trigger direct roGFP2 responses. A side-by-side comparison of all three probes can be used to reveal micromolar amounts of hypochlorous acid or polysulfides.
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Original ContributionKinetic analysis of phagosomal production of reactive oxygen species

Abstract

Introduction

Section snippets

Preparation of solutions

Chemistry of the dye and identification of a new oxidation product

Discussion

Conclusion

Acknowledgments

J. Biochem. Biophys. Meth.

Free Radic. Biol. Med.

Curr. Opin. Microbiol.

Free Radic. Biol. Med.

Free Radic. Biol. Med.

Free Radic. Biol. Med.

Nitric Oxide Biol. Chem.

Biochem. Pharmacol.

J. Immunol. Meth.

J. Biol. Chem.

Free Radic. Biol. Med.

Trends Cell Biol.

Trends Microbiol.

Single europium-doped nanoparticles measure temporal pattern of reactive oxygen species production inside cells

Nat. Nanotechnol.

Genetically encoded fluorescent indicator for intracellular hydrogen peroxide

Nat. Meth.

Real-time imaging of the intracellular glutathione redox potential

Nat. Meth.

Regulation and termination of NADPH oxidase activity

Cell. Mol. Life Sci.

What does the commonly used DCF-test for oxidative stress really show?

Biochem. J.

How human neutrophils kill and degrade microbes: an integrated view

Immunol. Rev.

To activate or not to activate: distinct strategies used by Helicobacter pylori and Francisella tularensis to modulate the NADPH oxidase and survive in human neutrophils

Immunol. Rev.

Single-cell optical imaging of the phagocyte NADPH oxidase

Antioxid. Redox Signaling

Original Contribution
Kinetic analysis of phagosomal production of reactive oxygen species