The polyunsaturated fatty acids docosahexaenoic acid (C22.,63), eicosapentaenoic acid, arachidonic acid, and linoleic acid caused marked in vitro growth inhibition of Plasmodium falciparum, assessed by a radiometric assay. In contrast, negligible parasite killing was seen with oleic acid or docosanoic acid. Parasite killing was significantly increased when oxidized forms of polyunsaturated fatty acids were used. Antioxidants greatly reduced the fatty acid-induced killing. Mice infected with P. berghei and treated for 4 d with C22:6,,.3 showed marked reduction in parasitemia. The anemia associated with the infection was also alleviated by treatment with C22A6,,3. The data provide new information that could be explored in order to develop new strategies in malaria treatment. (J. Clin. Invest. 1992. 89:961-967.)

Although unesterified polyunsaturated fatty acids (PUFA) have been shown to elicit marked changes in neutrophil function, the associated signal transduction processes require clarification. In this study we examined the effect of PUFA on the sphingomyelin (SM)‐signalling cycle in human neutrophils. Treatment of neutrophils with eicosatetraenoic acid [ arachidonic acid, 20:4(n‐6)] caused a decrease in the mass of cellular SM and an increase in the level of ceramide. 20:4(n‐6)‐stimulated neutral sphingomyelinase (SMase) activity of the leucocytes in a time‐ and concentration‐dependent manner. Other unsaturated fatty acids, docosahexaenoic [22:6(n‐3)], eicosapentaenoic [20:5(n‐3)], octadecenoic [oleic, 18:1(n‐9)] and octadecadienoic [linoleic, 18:2(n‐6)] acids also had the capacity to activate neutral SMase; however, certain 20:4(n‐6) derivatives {20:4(n‐6) methyl ester [20:4(n‐6)ME], 15‐hydroperoxyeicosatetraenoic (15‐HPETE) and 15‐hydroxyeicosatetraenoic (15‐HETE) acids}, very‐long‐chain PUFA {tetracosatetraenoic [24:4(n‐6)] and octacosatetraenoic [28:4(n‐6)] acids} and saturated fatty acids [octadecanoic (stearic, 18:0) and eicosanoic (arachidic, 20:0) acids] had no significant effect. Activation of neutral SMase by 20:4(n‐6) appeared to involve metabolism via 20:4(n‐6)CoA (arachidonoyl CoA) and was not dependent on prostaglandin and leukotriene synthesis. All of the fatty acids and derivatives tested failed to activate acidic SMase of neutrophils. Ceramide was found to inhibit 20:4(n‐6)‐induced superoxide generation by the cells. It is envisaged that the PUFA‐induced ceramide production in neutrophils plays a role in the regulation of biological responses.

The n-3 polyunsaturated fatty acids (PUFA) appear to have antiinflammatory properties that can be partly explained by their biological activity on leukocytes. Since leukocyte emigration is an essential component of the inflammatory response, we have examined the effects of the n-3 PUFA (eicosapentaenoic and docosahexaenoic acids) on neutrophil random and chemotactic movement. Preexposure of neutrophils for 15-30 min to 1-10 gg/ml PUFA reduced the random and chemotactic migration to both FMLP-and fungi-activated complement. The inhibitory effect diminished with increasing saturation and carbon chain length, and methylation abolished this activity. Arachidonic and docosahexaenoic acids were the most active fatty acids. The PUFA concentration required to inhibit migration was dependent on cell number, suggesting that the fatty acid effects on leukocyte migration in vivo may be governed by the stage of the inflammatory response. It was concluded that the PUFA rather then their metabolites were responsible for the inhibition since: (a) antioxidants did not prevent the PUFA-induced migration inhibition and the hydroxylated intermediates were less active, and (b) inhibitors of the cyclooxygenase and lipoxygenase pathways were without effect. Inhibitors of protein kinases and calmodulin-dependent enzyme system did not prevent the PUFA-induced migration inhibition, which was also independent of phospholipase D-catalyzed hydrolysis of phospholipids. It is also shown that PUFA decrease the FMLP-induced Ca2+ mobilization. (J. Clin. Invest. 1994.

Although polyunsaturated fatty acids (PUFA) have been shown to stimulate neutrophil responses such as the oxygen-dependent respiratory burst (superoxide production), the mechanisms involved still remain undefined. Here we investigate the effect of PUFA on the phospholipase A2 (PLA2)-signal transduction process in human neutrophils. Exogenous eicosatetraenoic acid [arachidonic acid; C20:4(n-6)] or docosahexaenoic acid [C22:6(n-3)] promoted the release of [3H]C20:4(n-6) from prelabelled neutrophils in a time- and dose-dependent manner, which is indicative of PLA2 activation. The release of [3H]C20:4(n-6) from the cells by C20:4(n-6) and C22:6(n-3) was suppressed by PLA2 inhibitors. Other PUFA ¿eicosapentaenoic [C20:5(n-3)], octadecatrienoic [gamma-linolenic; C18:3(n-6)] and octadecadienoic [linoleic; C18:2(n-6)] acids¿ also had the ability to release [3H]C20:4(n-6); however, certain C20:4(n-6) derivatives [15-hydroperoxyeicosatetraenoic acid, 15-hydroxyeicosatetraenoic acid and C20:4(n-6) methyl ester] and saturated fatty acids [octadecanoic (stearic; C18:0) and eicosanoic (arachidic; C20:0) acids] had no significant effect. Treatment of the neutrophils with exogenous C22:6(n-3) caused the mass of endogenous unesterified C20:4(n-6) to increase. Incubation of the leucocytes with C20:4(n-6) or C22:6(n-3) evoked activation of the 85 kDa cytosolic PLA2 (cPLA2) and the 14 kDa secretory PLA2 (sPLA2), but not the cytosolic Ca2+-independent PLA2. In contrast, C20:0 did not activate any of the PLA2 isoforms. Activation of cPLA2 by PUFA was found to precede that of sPLA2. C22:6(n-3), C20:4(n-6) and other PUFA induced punctate localization of cPLA2 in the cells, which was not observed with saturated fatty acids. Pretreatment of the leucocytes with PLA2 inhibitors markedly decreased superoxide production induced by C20:4(n-6). These results show that PUFA activate PLA2 in neutrophils, which might have a mandatory role in biological responses.

Localized adhesion of peripheral blood leukocytes to the endothelial lining is essential for their exit from the blood under both physiological and pathological conditions. The establishment, development, and resolution of the inflammatory response is regulated by an array of mediators, many of which remain to be categorized. These include arachidonic acid (20:4 n -6) and its hydroperoxy (HPETE) and hydroxy (HETE) derivatives, which are released during inflammation. The data show that human umbilical vein endothelial cells, pretreated with these fatty acids, have a reduced ability to be stimulated by tumor necrosis factor-α (TNF-α) for enhanced neutrophil and monocyte adhesion; the order of inhibitory activity being 15-HPETE>15-HETE>20:4 ( n -6). This fatty acid–induced inhibitory activity was reflected in the ability of the mediators to decrease the TNF-α–induced expression of the following endothelial adhesion molecules: intercellular adhesion molecule-1 (ICAM-1), E-selectin, and vascular cell adhesion molecule-1 (VCAM-1), measured by both enzyme-linked immunosorbent assay and flow cytometric analysis. TNF-α–induced increased expression of ICAM-1, E-selectin, and VCAM-1 mRNA was significantly depressed by 15-HPETE. Constitutively expressed ICAM-1 and ICAM-1 mRNAs were unchanged by the fatty acids. The saturated fatty acid 20:0 and the methyl ester of 20:4( n -6) had no inhibitory activity. The binding of TNF-α to its receptors was not altered by these fatty acids. The fatty acids also inhibited the expression of ICAM-1 and E-selectin induced by phorbol 12-myristate 13-acetate, showing that inhibition occurred at a post–TNF-α receptor binding level. The 15-HPETE was found to inhibit the TNF-α–induced increase in adhesion molecule expression in the early stage of the incubation, but expression returned to normal after 18 hours. An effect of 15-HPETE on the early cell signaling system was demonstrated by the ability of this fatty acid to inhibit agonist-induced protein kinase C translocation.

The effect of albumin on the release of [3H]lysophosphatidylcholine from cultured rat hepatocytes prelabelled with [Me-3H]choline was studied. In the absence of serum and albumin from the medium, the cells released essentially no [3H]lysophosphatidylcholine. Albumin stimulated this process dramatically, and it reached a plateau at 2 mg/ml. After an initial lag of 30 min, the release of [3H]lysophosphatidylcholine was linear for at least 4 h. At low concentrations, albumin slightly stimulated [3H]phosphatidylcholine release. The albumin had no measurable effect on the metabolism of cellular [3H]phosphatidylcholine, [3H]lysophosphatidylcholine or [3H]glycerophosphocholine. In addition, albumin did not alter the release of 3H-labelled water-soluble compounds, including [3H]glycerophosphocholine, into the medium. The possibility that the [3H]lysophosphatidylcholine was arising from catabolism of [3H]phosphatidylcholine in the medium by secreted enzymes was excluded. The effect on [3H]lysophosphatidylcholine secretion was also observed when the cells were incubated with alpha-cyclodextrin, a cyclic polysaccharide that has the ability to bind lysophosphatidylcholine. The albumin-released lysophosphatidylcholine was enriched in unsaturated fatty acids. Alteration of the fatty acid composition of cellular phosphatidylcholine gave rise to parallel changes in phosphatidylcholine and lysophosphatidylcholine in the medium. It is concluded that phosphatidylcholine is constantly being degraded in the rat hepatocyte to lysophosphatidylcholine which is released into the medium only when a suitable acceptor is present.

The regulation of allergic and autoimmune inflammatory reactions by polyunsaturated fatty acids and their metabolic products (eicosanoids) continues to be of major interest. Our data demonstrate that arachidonic acid 5,8,11,14-eicosatetraenoic acid (20:4n-6) and its hydroxylated derivatives

The oxygen-dependent respiratory burst is a key neutrophil function required for the killing of bacteria. However, despite intensive investigation, the molecular events which initiate the respiratory burst remain unclear. Recent reports have suggested the agonist-induced hydrolysis of cellular phosphatidylcholine (PtdCho) by phospholipase D may be an essential requirement for initiating or mediating the respiratory burst. We have investigated the effects of the chemotactic peptide N-formylmethionylleucylphenylalanine (fMLF), the phorbol ester 12-0-tetradecanoyl-phorbol 13-acetate (TPA) and the polyunsaturated fatty acids arachidonic [20 : 4 (n-6)] and docosahexaenoic [22 : 6 (n-3)] acids in light of this hypothesis. Ethanol-inhibited superoxide production in response to 20:4, 22:6 and fMLF, in a dose-dependent fashion, suggesting an involvement of phospholipase D. The phosphatidic-acid phosphohydrolase inhibitor DL-propranolol completely inhibited superoxide production induced by both 20:4 and 22:6, and partially inhibited the response to TPA. In contrast, superoxide production in response to fMLF was increased by propranolol. fMLF and TPA, but not the fatty acids, stimulated phospholipase D as indicated by the accumulation of phosphatidic acid and, in the presence of ethanol, phosphatidylethanol derived from PtdCho. Extracellular Ca2+ was found to be an essential requirement for fMLF-induced superoxide production. However, responses to the fatty acids were dramatically enhanced under Ca2',-free conditions. Responses to TPA were independent of the extracellular Ca2+ concentration. Both fatty acids and fMLF, but not TPA, mobilised Ca2+ from intracellular stores, a response insensitive to the effects of both ethanol and propranolol. These results show that, unlike fMLF and TPA, the fatty acids do not cause hydrolysis of PtdCho by phospholipase D. However, the data indirectly suggests that the fatty acids may initiate the phospholipase-D-catalysed hydrolysis of phospholipids other than PtdCho.The oxygen-dependent respiratory burst is a major biochemical response in neutrophils that is associated with their interaction with various types of soluble and particulate stimuli. This response is mediated by a membrane-bound flavoproteinlb-type cytochrome complex, the NADPH oxidase, which catalyses the reduction of molecular oxygen to superoxide (reviewed by Rossi, 1986; Bdbior, 1988;Segel, 1989). However, the sequence of events following ligand interaction with the cell surface which ultimately lead to oxyradical production are not well understood. Receptor binding has been shown to initiate the hydrolysis of inositol phosphates, resulting in the production of the intracellular messengers, inositol 1,4,5-trisphosphate and diacylglycerol (reviewed by Cockcroft, 1989). Inositol 1,4,5-trisphosphate has been reported to mobilise Ca2+ from intracellular stores, whileCorrespondence to A.