Reassessment of the microbicidal activity of reactive oxygen species and hypochlorous acid with reference to the phagocytic vacuole of the neutrophil granulocyte

Abstract

During phagocytosis, neutrophils undergo a burst of respiration in which oxygen is reduced to superoxide (O(-)(2)), which dismutates to form H(2)O(2). Myeloperoxidase (MPO) is discharged from the cytoplasmic granules into the phagosome following particle ingestion. It is thought to utilize H(2)O(2) to oxidize halides, which then react with and kill ingested microbes. Recent studies have provided new information as to the concentration of O(-)(2) and proteins, and the pH, within the vacuole. This study was conducted to examine the antimicrobial effect of O(-)(2), H(2)O(2) and hypochlorous acid under these conditions and it was found that the previously described bactericidal effect of these agents was reversed in the presence of granule proteins or MPO. To establish which cellular proteins were iodinated by MPO, cellular proteins and bacterial proteins, iodinated in neutrophils phagocytosing bacteria in the presence of (125)I, were separated by 2D gel electrophoresis. Iodinated spots were detected by autoradiography and the oxidized proteins were identified by MS. The targets of these iodination reactions were largely those of the host cell rather than those of the engulfed microbe.

Fig. 1

Effect of pH on bacterial viability, destruction by stimulated neutrophils and bactericidal effects of ${\mathrm{O}}_2^{-}$. (a) IgG opsonized S. aureus (■) or E. coli (□) (1 × 10⁸ c.f.u. ml⁻¹) was mixed at a ratio of one target organism to five neutrophils in 1 ml PBS (pH 7·3) for the indicated periods of time and bacterial viability was determined. The mean (±se) of three experiments is shown. No significant difference was observed between killing of S. aureus and E. coli. (b) Bactericidal effect of ${\mathrm{O}}_2^{-}$ was determined by suspending S. aureus (1 × 10⁷ c.f.u.) in 0· 01 M phosphate buffer (pH 7·5) (●) or buffer containing different concentrations of KO₂ (○). Each point is the mean of triplicate experiments (±se). (c, d) To determine the effect of pH on bacterial viability, S. aureus or E. coli (1 × 10⁷ c.f.u. ml⁻¹) was incubated at 37 °C in 0·01 M phosphate buffer, pH 5·5 (□), 6·5 (○) or 7·5 (●). Reduction in survival of S. aureus at pH 5·5 compared to 6·5 was found to be significant, P < 0·033.

Fig. 2

Bactericidal activity of H₂O₂ in the presence and absence of granule protein. The reaction mixture contained S. aureus (a, c, e) or E. coli (b, d, f) (2 × 10⁷ c.f.u. ml⁻¹) in 0·01 M phosphate buffer at pH 5·5, 6·5 or 7·5 with 1 (●), 10 (○) or 100 (□) mM H₂O₂ added at 37 °C. Aliquots were removed at the times indicated. The experiment was repeated with 100 mM H₂O₂ and granule protein (g, h) (25 mg ml⁻¹) at pH 7·5 (●), 6·5 (○) or 5·5 (□). Each value is derived from triplicate plating. The mean values (±se) from four experiments are shown. Changes in viability greater than 50% were always significant (P ≤ 0·05).

Fig. 3

Bactericidal activity of HOCl in the presence and absence of granule protein. The reaction mixture 0·01 M phosphate buffer, pH 7·5 (●), 6·5 (○) or 5·5 (□), contained S. aureus (a, c) or E. coli (b, d) (2 × 10⁷ c.f.u. ml⁻¹) and 1 or 5 μM HOCl. Inhibition of killing of S. aureus (e, g) (2 × 10⁷ c.f.u. ml⁻¹) or E. coli (f, h) by HOCl was observed in 0·01 M phosphate buffer, pH 7·5 or 5·5, with added granule protein. Bacteria (2 × 10⁷ c.f.u. ml⁻¹) were exposed to 100 μM(●), 250 μM(○), 500 μM(▲) or 1 mM (△) HOCl in the presence of granule protein (25 mg ml⁻¹). Each line is representative of the mean (±SE) of three experiments.

Fig. 4

Bacterial killing by the MPO/H₂O₂/Cl⁻ system. S. aureus (1 × 10⁷ c.f.u. ml⁻¹) in 0·01 M phosphate buffer at pH 7·5 (●), 6·5 (○) or 5·5 (□) was mixed with MPO (5 mg ml⁻¹). H₂O₂ at a concentration of 1 (a), 10 (b) or 100 (c) mM was added. (d) MPO itself had no effect on bacterial viability (□), whilst the bactericidal effect of 100 mM H₂O₂ (●) was prevented in the presence of MPO (5 mg ml⁻¹) (○) at pH 7·5. Each line is representative of the mean (±se) of three experiments.

Fig. 5

2D gel electrophoresis and autoradiographs of neutrophils before and after phagocytosis of S. aureus. Neutrophils (1 × 10⁷) in 1 ml PBS (pH 7·3) containing 100 μCi (3700 kBq) ¹²⁵I were mixed in a rapidly stirring oxygenated chamber at 37 °C without (a, c) or with (b, d) IgG opsonized S. aureus (1 × 10⁸ c.f.u.). After 4 min the suspension was taken into 10% TCA. Coomassie blue 2D stained gels (a, b) and corresponding autoradiographs (c, d) (216 h exposure) are shown. Iodinated proteins (labelled 1–27) were excised from the SDS gel and identified.