Control of the oxidative burst of human neutrophils by staphylococcal leukotoxins

Abstract

The ability of staphylococcal two-component leukotoxins to induce an oxidative burst and/or to prime human polymorphonuclear cells (PMNs) was studied by using spectrofluorometry or flow cytometry. At sublytic concentrations, the HlgA-HlgB, HlgA-LukF-PV, LukS-PV-LukF-PV, and HlgC-LukF-PV combinations of leukotoxins, but not the LukS-PV-HlgB and HlgC-HlgB combinations, were able to induce H(2)O(2) production similar to the H(2)O(2) production induced by 1 micro M N-formyl-Met-Leu-Phe (fMLP). In addition, when added at sublytic concentrations, all of the leukotoxin combinations primed PMNs for H(2)O(2) production induced by fMLP. Leukotoxin activation was dependent on the presence of Ca(2+) and was inhibited by wortmannin, an inhibitor of phosphatidylinositol 3-kinase, but not by N-methyl-L-arginine, an inhibitor of NO generation, which eliminates the possibility that NO plays a role in the action of leukotoxins. At higher concentrations, all leukotoxins inhibited H(2)O(2) production by PMNs activated by fMLP, phorbol 12-myristate 13-acetate (PMA), or the leukotoxins themselves. This inhibition was not related to the pore formation induced by leukotoxins. Intracellular release of H(2)O(2) induced by fMLP and PMA was not primed by leukotoxins but was inhibited. It seems that leukotoxin inhibition of H(2)O(2) release is independent of pore formation but secondary to an intracellular event, as yet unknown, triggered by leukotoxins.

FIG. 1.

Fluorometric determination of the influence of six leukotoxin combinations (HlgA-HlgB, HlgA-LukF-PV, LukS-PV-HlgB, LukS-PV-LukF-PV, HlgC-HlgB, and HlgC-LukF-PV) on the H₂O₂ produced by human PMNs in a microplate assay in the presence of DHR. The concentration of HlgA was 3 nM, the concentration of LukS-PV was 1 nM, and the concentration of HlgC was 1 nM, and these toxins were associated with different concentrations of HlgB and LukF-PV. The data are means ± standard errors (n = 4) for four identical experiments.

FIG. 2.

Fluorometric determination of the effect of fMLP on the H₂O₂ produced by human PMNs in the presence of six leukotoxin combinations (see the legend to Fig. 1) in a microplate assay in the presence of DHR. fMLP (1 μM) was added to PMNs 10 min after the leukotoxins were added. The data are means ± standard errors (n = 4) for four identical experiments.

FIG. 3.

Fluorometric determination of the effect of PMA on the H₂O₂ produced by human PMNs in the presence of six leukotoxin combinations (see the legend to Fig. 1) in a microplate assay in the presence of DHR. PMA (10 nM) was added to PMNs 10 min after the leukotoxins were added. The data are means ± standard errors (n = 4) for four identical experiments.

FIG. 4.

Spectrofluorometric determination of the influence of extracellular Ca²⁺, fMLP, and PMA in the presence of LukS-PV-LukF-PV on the time course of the intracellular Ca²⁺ concentration (A) and H₂O₂ production (B) of human PMNs. LukS-PV (1 nM)-LukF-PV (0.6 nM), 1 μM fMLP, and 1 μM PMA were added to PMNs loaded with Fluo3 or incubated with DHR. The arrows indicate the times when LukS-PV-LukF-PV (line a), LukS-PV-LukF-PV plus fMLP (lines b and f), LukS-PV-LukF-PV plus PMA (lines c and g), fMLP (line d), and PMA (line e) were added in the presence of 1 mM free Ca²⁺ (lines a, b, c, d, and e) or in the absence of Ca²⁺ (lines f and g). a.u., arbitrary units.

FIG. 5.

Spectrofluorometric determination of the influence of different concentrations of LukF-PV in the presence of LukS-PV on the time course of the intracellular Ca²⁺ concentration (A) and H₂O₂ production (B) of human PMNs. LukS-PV (1 nM) and different concentrations of LukF-PV (line a, 0.4 nM; line b, 0.75 nM; line c, 1 nM; line d, 1.5 nM; line e, 2 nM) were added to PMNs loaded with Fura-2 or incubated with DHR. a.u., arbitrary units.

FIG. 6.

Fluorometric determination by microplate assay of the effects of different concentrations of wortmannin on the H₂O₂ produced by human PMNs activated by fMLP and subjected to different concentrations of LukF-PV in the presence of 2 nM LukS-PV. PMNs were incubated with leukotoxins in the presence of DCFH-DA 10 min prior to the addition of 1 μM fMLP. The data are means ± standard errors (n = 4) for four identical experiments.

FIG. 7.

Spectrofluorometric determination of the influence of LukS-PV-LukF-PV and HlgA-HlgB on pore formation and H₂O₂ production of human PMNs. PMNs were preincubated with DHR or ethidium bromide. (A) Time course of pore formation and H₂O₂ production induced by 1 nM LukS-PV-1 nM LukF-PV and 1 nM HlgA-1.75 nM HlgB added at the times indicated by the arrows. The maximum ethidium fluorescence intensity obtained in the presence of Triton X-100 was 99,000 absorbance units (a.u.). (B) Plot of the average slopes of the curves of ethidium fluorescence intensity versus DHR fluorescence intensity calculated during the time delimited by the dotted lines in panel A after addition of different concentrations of LukS-PVl-LukF-PV and HlgA-HlgB. The concentrations of LukS-PV-LukF-PV used were 1 nM LukS-PV-1 nM LukF-PV, 1 nM LukS-PV-1.5 nM LukF-PV, and 1 nM LukS-PV-1.75 nM LukF-PV, and the concentrations of HlgA-HlgB used were 1 nM HlgA-0.625 nM HlgB, 1 nM HlgA-1.25 nM HlgB, 1 nM HlgA-1.75 nM HlgB, and 1 nM HlgA-2.5 nM HlgB.

FIG. 8.

Flow cytometric determination of the intracellular H₂O₂ produced by human PMNs induced by different concentrations of LukS-PV-LukF-PV in the absence of fMLP and PMA (A) or in the presence of fMLP (B) or PMA (C). fMLP (1 μM) and 10 nM PMA were added to PMNs 10 min after 1 nM LukS-PV and different concentrations of LukF-PV in the presence of DHR were added.