Altered responses of human macrophages to lipopolysaccharide by hydroperoxy eicosatetraenoic acid, hydroxy eicosatetraenoic acid, and arachidonic acid. Inhibition of tumor necrosis factor production.

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 15(s)-hydroxy-5,8,11,13-eicosatetraenoic acid (15-HETE) and 15(s)-hydroperoxy-5,8,11,13-eicosatetraenoic acid (15-HPETE) regulate agonist-induced tumor necrosis factor alpha (TNF) production, a cytokine that plays a role in inflammatory diseases. Although 20:4n-6 and 15-HETE caused a reduction in production of TNF in mononuclear leukocytes stimulated with phytohaemagglutinin, pokeweed mitogen, concanavalin A, and Staphylococcus aureus, 15-HPETE was far more active. 15-HPETE was also found to dramatically depress the ability of bacterial lipopolysaccharide to induce TNF production in monocytes and the monocytic cell line Mono Mac 6. These fatty acids depressed the expression of TNF mRNA in Mono Mac 6 cells stimulated with LPS; 15-HPETE was fivefold more active than 20:4n-6 and 15-HETE. While 15-HPETE treatment neither affected LPS binding to Mono Mac 6 cells nor caused a decrease in CD14 expression, the fatty acid significantly reduced the LPS-induced translocation of PKC (translocation of alpha, betaI, betaII, and epsilon isozymes), suggesting that 15-HPETE acts by abrogating the early signal transduction events. The findings identify another molecule that could form the basis for development of antiinflammatory pharmaceuticals.

Cord blood neutrophil responses to polyunsaturated fatty acids: effects on degranulation and oxidative respiratory burst.

Lipid mediators such as arachidonic acid (AA) generated during inflammation play an important role in stimulating phagocytic cell responses. Since cord blood neutrophils show reduced responses to agonists such as the bacterial tripeptide f-Met-Leu-Phe (fMLP), it would be of interest to know whether cord blood neutrophils show normal or reduced responses to AA and other fatty acids. The data showed that the polyunsaturated n-6 fatty acid (PUFA) AA stimulated cord blood neutrophils to produce a respiratory response (measured by chemiluminescence) and degranulation. Other PUFAs, eicosapentanoic acid and docosahexaenoic acid, elicited similar responses in cord blood neutrophils. Specific granule release and chemiluminescence response in cord blood neutrophils were evident at 0.1-0.5 microgram/ml of PUFA, concentrations normally found in vivo during inflammation or following diets enriched with n-3 fatty acids. Neutrophil responses to PUFA were significantly better than those to either fMLP or phorbol myristate acetate. Cord blood neutrophils primed with PUFA showed enhanced responses to fMLP. These results suggest that cord blood neutrophils respond to a similar degree to adult neutrophils to the AA which is generated during the inflammatory response and to the n-3 fatty acids eicosapentaenoic acid/docosahexaenoic acid, both of which may be used in diet manipulation of neurological function and immunological reactions.

Docosahexanoic acid (22:6, n-3) but not eicosapentaenoic acid (20:5, n-3) can induce neutrophil-mediated injury of cultured endothelial cells: involvement of neutrophil elastase.

Previously published work has indicated that polyunsaturated fatty acids (PUFA) may enhance neutrophil-mediated damage to host tissues. We have found that endothelial detachment was significantly increased by neutrophils pretreated with docosahexaenoic (22:6, n-3) and arachidonic (20:4, n-6) acids at 10-40 microM but not by eicosapentaenoic acid (20:5, n-3). Endothelial cell lysis as measured by 51Cr release was unaffected. The extent of detachment was dependent on both fatty acid and neutrophil pretreatment concentrations. A specific leukocyte elastase inhibitor abrogated the increased detachment but catalase had no effect. Measurement of prostaglandin I2 synthesis as an alternative nonlytic assay of endothelial function indicated that 20:4 but not 20:5 was able to stimulate neutrophil-induced endothelial PGI2 synthesis. Although all three PUFA (3-33 microM) were found to stimulate release from neutrophil-specific granules, only 22:6 and 20:4 could stimulate release of the azurophilic granules containing elastase to any significant extent. Saturated fatty acids (20:0 and 22:0) and the methyl ester of 22:6 did not cause either neutrophil-mediated endothelial detachment or degranulation. We conclude that neutrophils pretreated with 22:6 or 20:4 but not 20:5 can decrease endothelial integrity through detachment involving neutrophil elastase. These findings may have important implications for the dietary use of fish oils rich in n-3 fatty acids.

Induction of tumor necrosis factor (TNF) and interleukin-1 (IL-1) by Pseudomonas aeruginosa and exotoxin A-induced suppression of lymphoproliferation and TNF, lymphotoxin, gamma interferon, and IL-1 production in human leukocytes.

Pseudomonas aeruginosa is a dominant pathogen in infection in cystic fibrosis. This bacterium is thought to play a major role in the chronic bronchial infection-induced pathophysiology. Our data showed that whole formalin-fixed heat-killed P. aeruginosa was mitogenic for human lymphocytes and induced production of substantial amounts of tumor necrosis factor alpha (TNF) in peripheral blood mononuclear leukocytes in cultures. Significant amounts of TNF were produced at 10(3) bacteria per 2 x 10(5) mononuclear leukocytes. Treatment of P. aeruginosa with polymixin B did not affect its ability to stimulate TNF production, suggesting that bacterial lipopolysaccharide is not involved. P. aeruginosa, however, did not stimulate production of the T-cell lymphokine lymphotoxin (TNF beta). Exotoxin A, considered to be an important virulence factor produced by P. aeruginosa, did not stimulate either lymphoproliferation or production of TNF. In fact, this toxin, at nontoxic concentrations, was found to depress lymphoproliferation induced by phytohemagglutinin and Staphylococcus aureus and decreased production of TNF, lymphotoxin, and gamma interferon in either lymphocytes or macrophages. This toxin similarly inhibited the production of interleukin-1 beta (IL-1 beta) and IL-1 alpha, but for the inhibition of the latter, 25-fold-less toxin was required than for inhibition of the former. Inhibition of production of TNF was as sensitive as the IL-1 alpha to exotoxin A. The effects of exotoxin A on lymphoproliferation and cytokine production could be neutralized by the addition of anti-exotoxin A antibodies. These results suggest that two mechanisms by which P. aeruginosa could contribute to the chronic bronchial infection-induced pathophysiology are the nonspecific stimulation of TNF and IL-1 and the release of exotoxin A, a toxin which depresses immune responses.

Neutrophil stimulating activity released by Staphylococcus-stimulated mononuclear leukocyte conditioned medium. Further characterization and partial purification.

Culture medium conditioned by human mononuclear leukocytes (MNL) stimulated with formalin fixed heat-killed Staphylococcus aureus induces a small respiratory burst in human neutrophils, and dramatically increases the response of neutrophils to stimuli such as N-formyl-L-methionyl-L-leucyl-L-phenylalanine. The data presented show that the activity is not unique to Staphylococcus aureus. Similar neutrophil modulating activities were produced by medium conditioned by MNL cultured in the presence of Streptococcus pneumonia, and Group B streptococcus. The activity was relatively resistant to heating; significant reduction of activity was observed only when 80 degrees C was reached. Neutrophil stimulating activity production by stimulated MNL was dependent on protein and RNA synthesis and the activity appeared to be released by the non-adherent fraction of the MNL, suggesting that it is not of macrophage origin. The activity was not sensitive to soya bean trypsin inhibitor, but was sensitive to trypsin and was not removed when stimulated conditioned medium was depleted of immunoglobulin and albumin by affinity chromatography. Purification by gel filtration on Sephadex G-100 and high-performance liquid chromatography with Bio-Sil TSK250 columns showed that the major activity had an apparent molecular weight of 35,000-43,000 under conditions in which ionic interactions and association with albumin were reduced; by using polyethylene glycol or high salt (0.46 M Na+) in the elution buffer.

Tumour necrosis factor beta (lymphotoxin) inhibits locomotion and stimulates the respiratory burst and degranulation of neutrophils.

The data presented here demonstrate that recombinant human tumour necrosis factor beta (rHuTNF beta; lymphotoxin) is a neutrophil modulator. The lymphokine inhibited the locomotion of neutrophils and augmented the neutrophil oxygen-dependent respiratory burst in response to N-formyl-L-methionyl-L-leucyl-L-phenylalanine (FMLP) and phorbol myristate acetate (PMA), as measured by their capacity to produce chemiluminescence, H2O2 and superoxide. The effects on the respiratory burst occurred at a tenth of the concentration of TNF beta required to inhibit locomotion. After incubation with TNF beta, the neutrophils could be washed without any reduction in their capacity to show augmented responses. The TNF beta enhanced granule enzyme (lysozyme and beta-glucuronidase) release of neutrophils stimulated with cytochalasin B-FMLP.

Effects of tumour necrosis factor alpha and interleukin-1 alpha and beta on human neutrophil migration, respiratory burst and degranulation.

Recombinant human tumour necrosis factor alpha (rHuTNF alpha) was shown to inhibit human neutrophil migration in the presence or absence of a chemotactic gradient generated with the tripeptide, N-formyl-L-methionyl-L-leucyl-L-phenylalanine (fMLP), at doses of 20-100 U/10(6) cells. In contrast, neither recombinant human interleukin-1 alpha (rHuIL-1 alpha), rHuIL-1 beta, human leucocyte-derived IL-1 alpha (1HuIL-1 alpha) nor 1HuIL-1 beta contained neutrophil migration inhibition properties. However, both the interleukins (1HuIL-1 alpha, 1HuIL-1 beta and rHuIL-1 alpha) and rHuTNF alpha stimulated a neutrophil respiratory burst and significantly elevated the neutrophil respiratory response to fMLP (measured as chemiluminescence and H2O2 production). The stimulatory effects were observed at doses of between 5 and 100 U/5 x 10(5) cells. A characteristic feature of the effects of the cytokines was the range of variation observed in neutrophil responses from different individuals. However, a concentration-related effect was observed with each experiment, delineating suboptimal, optimal and supra-optimal cytokine concentrations. Neutrophils treated with rHuTNF alpha and rHuIL-1 alpha and washed free of exogenous cytokine retained the capacity to show an enhanced response to fMLP. Pretreatment of cells with cytochalasin B enhanced their response to fMLP, and this response was further increased if the cells had also been pretreated with the cytokines. The response to phorbol myristate acetate was also enhanced by rHuTNF alpha and rHuIL-1 alpha. The effects of these cytokines on neutrophils could be abolished by boiling the preparation but not by treating it with polymixin B, suggesting that bacterial lipopolysaccharide was not responsible for the activity of these preparations. The rHuIL-1 alpha increased the release of lysozyme, beta-glucuronidase and myeloperoxidase initiated by cytochalasin B/fMLP, while rHuTNF alpha only increased lysozyme release.

Staphylococcus aureus-stimulated human mononuclear leucocyte-conditioned medium augments the basal and stimuli-induced neutrophil respiratory burst and degranulation.

Culture medium conditioned by mononuclear leucocytes (MNL) stimulated by formalin-fixed heat-killed Staphylococcus aureus (sCM) modulated a number of neutrophil functions. The sCM inhibited the locomotion of human neutrophils in both the presence and absence of a chemotactic gradient generated with N-formyl-L-methionyl-L-leucyl-L-phenylalanine (FMLP). It also stimulated the oxygen-dependent respiratory burst as assessed by its ability to stimulate basal H2O2, superoxide and chemiluminescence production by neutrophils. Neutrophils treated with sCM also showed increased release of lysozyme but not beta-glucuronidase. In addition, the sCM-treated neutrophils showed a potentiated response to stimuli that bind surface receptors, i.e. FMLP and opsonized zymosan. The effects of sCM and either of the stimuli were synergistic. Examination of lysosomal enzyme release showed that sCM enhanced the release of lysozyme and beta-glucuronidase induced by either FMLP/cytochalasin B or zymosan. The response to phorbol myristate acetate (PMA), which bypasses the surface receptor, was also stimulated but compared poorly with the FMLP response. The sCM effects on the FMLP-induced chemiluminescence response occurred even when FMLP addition was delayed for 4 hr. Cells treated with sCM and washed retained the ability to show an enhanced FMLP response. The neutrophil-modulating activity was not produced by MNL cultured in the absence of bacteria.

Inhibition of human monocyte respiratory burst, degranulation, phospholipid methylation and bactericidal activity by pneumolysin.

The interaction between the pneumococcal toxin pneumolysin and human monocytes was examined. At non-cytotoxic concentrations (0.5-2.5 HU/10(6) cells) pneumolysin depressed the oxygen-dependent respiratory burst in monocytes, induced by opsonized zymosan or phorbol myristate acetate (PMA). This included depressed hexose-monophosphate shunt activity and hydrogen peroxide production. The toxin also depressed the ability of monocytes to degranulate (measured by release of lysozyme) in response to the above stimuli. Phospholipid transmethylation was also markedly decreased by pretreating monocytes with pneumolysin. These effects on monocyte functions were accompanied by a decreased ability of pneumolysin-treated monocytes to kill Streptococcus pneumoniae, the organism that produces the toxin. Cholesterol, which inhibits the haemolytic activity of the toxin, was shown to abrogate the effects of pneumolysin on monocytes.