Docosahexaenoic Acid and Eicosapentaenoic Acid Antagonize the Proinflammatory Interactions of Pneumolysin with Human Neutrophils

Abstract

Pneumolysin (4.18 ng/ml)-mediated influx of Ca²⁺ and augmentation of the chemoattractant-activated generation of reactive oxidants was antagonized by pretreatment of human neutrophils with the omega-3 polyunsaturated fatty acids docosahexaenoic acid and eicosapentaenoic acid (1.25 to 5 μg/ml). These agents may have potential in attenuating the proinflammatory properties of this pneumococcal toxin.

Notwithstanding complement-activating properties, pore-forming interactions with neutrophils and monocytes, resulting in influx of Ca²⁺, have been implicated in the proinflammatory activities of the pneumococcal toxin, pneumolysin (1, 2, 3, 4, 11). Rather than contributing to the eradication of the infection, however, the resultant, predominantly neutrophil-mediated inflammatory response appears to favor persistence and extrapulmonary dissemination of the pneumococcus (7, 9).

Although no pharmacological antagonists of pneumolysin have, to our knowledge, been described, we reasoned that agents, such as the omega-3 polyunsaturated fatty acids docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA), which increase neutrophil membrane fluidity and stability may interfere with the proinflammatory, pore-forming activities of the toxin. The present study was designed to investigate the potential of DHA and EPA to neutralize two related proinflammatory activities of pneumolysin, these being the toxin-mediated influx of Ca²⁺ into human neutrophils and Ca²⁺-dependent sensitization of these cells for increased production of toxic reactive oxidants (4).

Endotoxin-free (<2 pg/ml) recombinant pneumolysin (specific activity, 4 × 10⁶ hemolytic units/mg) was expressed in Escherichia coli and purified from cell extracts as previously described (12).

Neutrophils were isolated from heparinized (5 U of preservative-free heparin/ml) venous blood of healthy, adult human volunteers (4) and resuspended to 10⁷ neutrophils/ml in phosphate-buffered saline (0.15 M, pH 7.4).

cis-4,7,10,13,16,19-DHA and cis-5,8,11,14,17-EPA were purchased from Sigma Chemical Co. (St. Louis, Mo.) and made to stock concentrations of 25 mg/ml in ethanol. Dilutions were made in ethanol, and these agents were used at final concen-trations of 1.25, 2.5, and 5 μg/ml in a final ethanol concentration of 0.1%. All control systems included in the experiments described below contained 0.1% ethanol, which did not interfere with the biological activity of pneumolysin.

Fura-2/AM (Sigma) was used as the fluorescent Ca²⁺-sensitive indicator to measure alterations in neutrophil cytosolic Ca²⁺ as previously described (4). The neutrophils were preloaded with fura-2/AM (final concentration, 2 μM) for 30 min at 37°C in phosphate-buffered saline, washed, and resuspended in Hanks balanced salt solution (pH 7.4, 1.25 mM CaCl₂; Highveld Biological Pty. Ltd., Johannesburg, South Africa). The cells (1 × 10⁶/ml) were then preincubated for 10 min at 37°C with or without DHA or EPA (1.25 to 5 μg/ml) and transferred thereafter to cuvettes, maintained at 37°C in a Hitachi 650 10S fluorescence spectrophotometer with excitation and emission wavelengths set at 340 and 500 nm, respectively. Pneumolysin (fixed; final concentration, 4.18 ng/ml) was added to the cells, and fluorescence intensity was monitored over a 5-min period. At this concentration, pneumolysin promotes influx of Ca²⁺ into the neutrophils with no detectable cytotoxicity over the time course of the experiments (4), and it is also well within the range (0.83 to 180 ng/ml) of those reported in the cerebrospinal fluid of patients with pneumococcal meningitis (16). In an additional series of experiments, fura-2-loaded neutrophils were exposed to DHA (2.5 and 5 μg/ml) for 10 min at 37°C, followed by washing and measurement of Ca²⁺ influx following addition of pneumolysin.

Luminol (5-amino-2,3-dihydro-1,4-phthalazinedione) (final concentration, 0.1 mM; Sigma)-enhanced chemiluminescence (LECL) was used to investigate the effects of DHA and EPA (1.25 to 5 μg/ml) on the generation of reactive oxidants by chemoattractant-activated neutrophils in the absence and presence of pneumolysin (4.18 ng/ml). Neutrophils (2 × 10⁵/ml) were preincubated with DHA or EPA for 5 min at 37°C, followed by addition of pneumolysin or an equal volume of Hanks balanced salt solution for control systems and a further 5 min of incubation at 37°C. Basal LECL values were then recorded using a Lumac Biocounter (Lumac Systems, Titusville, Fla.) followed by activation of the cells with the synthetic chemotactic tripeptide, N-formyl-l-methionyl-l-leucyl-l-phenylalanine (FMLP) (final concentration, 1 μM; Sigma) and measurement of LECL. The final volume in each vial was 1 ml. The results are expressed as the mean peak LECL values ± standard errors of the means in relative light units attained 40 to 50 s after the addition of FMLP and have been corrected for background (i.e., subtraction of the value for the corresponding unstimulated system). Statistical significance was calculated by the Mann-Whitney U test (two-tailed).

As shown in Fig. 1, treatment of neutrophils with pneumolysin was accompanied, after a short lag, by a striking increase in fura-2 fluorescence, which is a consequence of influx of Ca²⁺ (4). Pretreatment of the cells with DHA, in particular, and EPA resulted in dose-related antagonism of pneumolysin-mediated influx of Ca²⁺, which was also observed when DHA-treated neutrophils were washed prior to addition of pneumolysin (not shown).

FIG. 1.

Traces of the fura-2 fluorescence responses of neutrophils exposed to pneumolysin (4.18 ng/ml, added as denoted by the arrows) in the absence and presence of DHA or EPA. The results shown are typical traces from two experiments, with a total of seven in the series. The paired responses for matched neutrophils with DHA or EPA are shown vertically.

The effects of DHA and EPA on the generation of reactive oxidants (LECL) by FMLP-activated neutrophils in the absence and presence of pneumolysin are shown in Fig. 2. As reported previously, the LECL responses of FMLP-activated neutrophils were significantly (P < 0.05) increased by pneumolysin (4), while DHA and EPA, at concentrations of 2.5 and 5 μg/ml, respectively, not only decreased the LECL responses of FMLP-activated neutrophils but also abolished the pneumolysin-mediated increments in these responses.

FIG. 2.

Effects of DHA and EPA on the LECL responses of FMLP-activated neutrophils in the absence and presence of pneumolysin (Pln; 4.18 ng/ml). The results of two experiments with six replicates for each system are shown as the mean values ± the standard errors of the means in relative light units (rlu). *, P < 0.05.

In the present study, EPA and especially DHA antagonized pneumolysin-mediated influx of Ca²⁺ into neutrophils. Both agents, as reported previously (13, 15), also inhibited the FMLP-activated generation of reactive oxidants by these cells and were particularly effective in neutralizing the Ca²⁺-dependent, prooxidative interactions of pneumolysin with neutrophils, which are secondary to the pore-forming actions of the toxin (4). Although the exact molecular and biophysical mechanisms by which DHA and EPA enable the neutrophil plasma membrane to resist pneumolysin remain to be established, effects on membrane fluidity and stability are likely to be involved (6, 8, 15). This contention is supported by the observation that DHA-mediated resistance of neutrophils to the toxin was essentially unaffected by washing of the cells. Moreover, DHA and EPA have been reported to exclude proteins from lipid rafts in the plasma membrane (5), which may explain both the antagonism of pore formation by pneumolysin and interference with NADPH oxidase (14). Interestingly, interaction of the pore-forming beta toxin of Clostridium perfringens with the plasma membrane of eukaryotic cells has been reported to involve binding to lipid rafts (10).

In conclusion, DHA and EPA antagonize the Ca²⁺-dependent proinflammatory interactions of pneumolysin with neutrophils. Further evaluation of these agents, either alone or as adjuncts to antimicrobial chemotherapy, in models of experimental pneumococcal infection is warranted.