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 g/ml). These agents may have potential in attenuating the proinflammatory properties of this pneumococcal toxin.Notwithstanding complement-activating properties, poreforming interactions with neutrophils and monocytes, resulting in influx of Ca 2ϩ , 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 toxinmediated influx of Ca 2ϩ into human neutrophils and Ca 2ϩ -dependent sensitization of these cells for increased production of toxic reactive oxidants (4).Endotoxin-free (Ͻ2 pg/ml) recombinant pneumolysin (specific activity, 4 ϫ 10 6 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 10 7 neutrophils/ml in phosphate-buffered saline (0.15 M, pH 7.4).cis 7,10,13,16,8,11,14,17-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 concentrations of 1.25, 2.5, and 5 g/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 Ca 2ϩ -sensitive indicator to measure alterations in neutrophil cytosolic Ca 2ϩ as previously described (4). The neutrophils were preloaded with fura-2/AM (final concentration, 2 M) for 30 min at 37°C in phosphate-buffered saline, washed, and resuspended in Hanks balanced salt solution (pH 7.4, 1.25 mM CaCl 2 ; Highveld Biological Pty. Ltd., Johannesburg, South Africa). The cells (1 ϫ 10 6 /ml) were then preincubated for 10 min at 37°C with or without DHA or EPA (1.25 to 5 g/ml) and transferred thereafter to cuvettes, maintained at 37°C in a Hitachi 650 10S fluorescence spectrophotometer with e...

Pneumolysin-mediated activation of NFκB in human neutrophils is antagonized by docosahexaenoic acid

“…The effects of DHA (5 µg/ml) and EGTA (10 m m ) on the influx of Ca 2+ into neutrophils following exposure to pneumolysin (8·37 ng/ml) are shown in Tables 1 and 2, respectively. Treatment of neutrophils with pneumolysin was accompanied by influx of Ca 2+ , which is in agreement with previous studies using spectrofluorimetric procedures [1,12]. Pneumolysin‐mediated influx of Ca 2+ was completely eliminated by inclusion of EGTA in the cell‐suspending medium and substantially decreased by pretreatment of the cells with DHA.…”

“…Such a mechanism appears to be operative in the case of pneumolysin‐activated neutrophils. This contention is based on our previous findings that pneumolysin, but not a mutant version of the toxin inactivated with respect to pore‐forming activity, causes influx of Ca 2+ into neutrophils [1,12], while activation of NFκB by the toxin, as reported here, as well as synthesis of IL‐8, as reported previously [2], are attenuated by the Ca 2+ ‐chelating agent, EGTA. The absence of effects of DPI appears to exclude involvement of NADPH oxidase and intracellular oxidative stress in the pneumolysin‐mediated activation of NFκB in neutrophils.…”

“…One such group of agents is the omega‐3 polyunsaturated fatty acids, which have been reported to possess beneficial immunomodulatory and anti‐inflammatory properties in acute and chronic inflammatory disorders of both infective and noninfective origin [8–10], and to attenuate the pro‐inflammatory interactions of Escherichia coli haemolysin with rabbit macrophages [11]. In agreement with these observations, we have recently reported that these agents antagonize the pro‐oxidative interactions of pneumolysin with human neutrophils by interfering with Ca 2+ influx [12]. Interestingly, omega‐3 polyunsaturated fatty acids have also been reported to exclude proteins from the lipid rafts of eukaryotic cell membranes [13].…”

Mechanisms by which fatty acids regulate leucocyte function

“…Although the effect of FAs on ROS production has been studied extensively, several points remain to be fully established, as some authors have found an increase [130,131], whereas others have found a decrease [132,133], in ROS production by neutrophils in response to FA treatment. Recently, we have investigated the effect of OA, LA and γ -linolenic acid on intra-and extra-cellular content of ROS by unstimulated and PMA-stimulated neutrophils by using five techniques: luminol-amplified chemiluminescence, lucigenin-amplified chemiluminescence, cytochrome c reduction, hydroethidine and phenol red reduction [134].…”

Papel dos ácidos graxos na função e morte de neutrófilos de humanos: utilização do exercício intenso como modelo.

“…Embora os efeitos dos AGs na produção de EROS por leucócitos tenham sido amplamente estudados, vários pontos devem ser esclarecidos, pois há muitos dados controversos quanto à produção destas moléculas. Alguns trabalhos demonstram estimulação (Bellinati-Pires et al, 1993;Moriuchi et al, 1998) enquanto outros demonstram inibição (Ishida-Okawara et al, 1996;Cockeran et al, 2004) da produção de EROS por neutrófilos.…”