Hypochlorous acid – a potential secondary messenger in the process of neutrophils’ respiratory burst development

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Abstract

Hypochlorous acid and hypochlorite ions are formed in the halogenating cycle of myeloperoxidase, localized mainly in neutrophils, and play a primary role in antimicrobial protection. The paper presents the results of a study of the effect of exogenous HOCl/OCl– in micromolar concentrations on the mechanisms of the “respiratory burst” formation by neutrophils stimulated to phagocytosis. It is shown that this oxidizer is capable of stimulating the functional activity of neutrophils, which is expressed in an increase in the yield of reactive oxygen and chlorine species (ROСS) and secretory degranulation of cells. Enhancement of the “respiratory burst” is associated with activation of NADPH oxidase, PI-3K, MAP kinase ERK1/2 and a decrease in the contribution of intracellular myeloperoxidase to ROСS production by neutrophils. It was found that HOCl/OCl– in the studied concentrations is capable of inhibiting myeloperoxidase activity. It is suggested that hypochlorous acid should be considered as a new potential secondary messenger regulating neutrophil functions.

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About the authors

G. N. Semenkova

Belarusian State Medical University

Email: n.amaegberi@gmail.com
Belarus, Minsk, 220083

I. I. Zholnerevich

Belarusian State University

Email: n.amaegberi@gmail.com
Belarus, Minsk, 220030

M. A. Murina

Lopukhin Federal Scientific and Clinical Center for Physical-Chemical Medicine of the Federal Medical and Biological Agency

Email: n.amaegberi@gmail.com
Russian Federation, Moscow, 119435

N. V. Amaegberi

Belarusian State University

Author for correspondence.
Email: n.amaegberi@gmail.com
Belarus, Minsk, 220030

D. I. Roshchupkin

Pirogov Russian National Research Medical University

Email: n.amaegberi@gmail.com
Russian Federation, Moscow, 117513

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2. Fig. 1. Kinetic curves of intensity of luminol-dependent chemiluminescence (CL) of rabbit neutrophils stimulated by PMA in control (1) and under the action of sodium hypochlorite (2-5), respectively, at final concentrations of 1, 10, 20 and 40 microns. PMA (5 micrograms/ml) was injected into a suspension of neutrophils (106 cells in 1 ml) immediately after luminol (20 micrograms). The time of pre-incubation of cells with sodium hypochlorite is 5 minutes. In, (%) is the normalized light intensity: in control (1), the maximum luminous intensity is assumed to be 100%. The averaged dependences of CL intensity according to 10 independent experiments are presented; the data spread is represented by the root-mean-square error of the average value.

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3. Fig. 2. The dependence of the total intensity of luminol-dependent chemiluminescence (CL) of human neutrophils stimulated by latex and adhesion (a), as well as fMLP and LPS (b), on the concentration of NaOCl. The integral intensity of CL cells in the presence of (ΣIHL) and in the absence of (ΣIHLO) NaOCl was measured within 10 minutes after the addition of luminol.The concentration of fMLP is 0.1 microns, LPS is 25 micrograms/ml. The pre-incubation time of neutrophils with NaOCl is 10 min. The results are presented as averages and their standard deviations.

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4. Fig. 3. Dependence of the maximum chemiluminescence intensity (Imax) on the concentration of sodium hypochlorite (NaOCl) in the luminol oxidation reaction. The results are presented as averages and their standard deviations.

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5. Fig. 4. The effect of enzyme inhibitors on the degree of inhibition of luminol-dependent CL of human neutrophils in the presence (light columns) and in the absence (dark columns) of NaOCl (15 microns). The cells were stimulated by adhesion to the glass surface. The time of pre–incubation of neutrophils with NaOCl is 30 minutes, the registration time is 10 minutes; temperature is 37 °C, pH 7.4. Inhibitors are shown: NADPH oxidase (DPI, 1 µm); myeloperoxidase (AVAN, 50 µm); phosphatidylinositol-3-kinase (LY-294002, 3.5 µm) and MAP kinase ERK1/2 (PD-98059, 25 microns). Vertically, the degree of CL inhibition is indicated ((ΣI0 – ΣIi) / ΣI0) · 100%), where ΣI0 and ΣIi are the total intensity of cell luminescence in the absence and presence of the inhibitor, respectively. The results are presented as averages and their standard deviations.

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6. Fig. 5. The effect of sodium hypochlorite on lysozyme secretion from human blood neutrophils. Incubation time of cells with NaOCl is 15 min. Lysozyme secretion was assessed by the rate of lysis of the cell walls of Micrococcus lysodeikticus bacteria. The results are presented as averages and their standard deviations.

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7. Fig. 6. MPO activity under the influence of sodium hypochlorite. The pre–incubation time of NaOCl/AVAN with MPO is 10 min. The concentration of AVAN is 1 micron. The results are presented as averages and their standard deviations.

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8. Fig. 7. The effect of reduced glutathione (GSH) on hypochlorite-enhanced luminol chemiluminescence in rabbit blood neutrophil suspension. Curve 1 – control (luminol and PMA were added to the neutrophils); 2 – GSH (0.2 mM) was introduced into the neutrophil suspension before PMA; 3 – NaOCl (5 microns) was introduced into the neutrophil suspension 3 minutes before PMA; 4 – NaOCl (5 microns) was introduced into the neutrophil suspension 3 minutes before GSH (0.2 mM) and FMA. In, – normalized to control (100% at maximum) the intensity of XL. The averaged dependences of CL intensity according to the data of five independent experiments are presented.; The spread of the data is represented by the root-mean-square error of the average value.

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