Recombinant human tumour necrosis factor alpha (rHuTNFα) 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/10⁶ cells. In contrast, neither recombinant human interleukin-1 alpha (rHuIL-1α), rHuIL-1β, human leucocyte-derived IL-lα (lHuIL-lα) nor lHuIL-1β contained neutrophil migration inhibition properties. However, both the interleukins (lHuIL-1α, lHuIL-1β and rHuIL-1α) and rHuTNFα 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 × 10⁵ 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 rHuTNFα and rHuIL-1α 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 rHuTNFα and rHuIL-1α. 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-1α increased the release of lysozyme, β-glucuronidase and myeloperoxidase initiated by cytochalasin B/fMLP, while rHuTNFα only increased lysozyme release.

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

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.

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.

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 μg/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.