Edna J. Bates

Polyunsaturated fatty acids increase neutrophil adherence and integrin receptor expression

Fish oils are abundant in polyunsaturated fatty acids of the n‐3 series (in particular eicosapen‐ taenoic, 20:5 and docosahexaenoic acid, 22:6). Such fatty acids are generally considered to be beneficial in the prevention of cardiac disease and to have anti‐inflammatory properties. Neutrophil adherence is an essential early event in an acute inflammatory response, and we have demonstrated that both 20:5 and 22:6 stimulate adherence in vitro. Arachidonic acid (20:4, n‐6) was also stimulatory. Significant stimulation of adherence was seen from 5 to 80 μM (nontoxic concentrations) 22:6, 20:5, or 20:4. At the lower fatty acid concentrations tested (≤40 μM) 20:5 was less active than 22:6 or 20:4 at stimulating adherence. Above 40 μM there was no difference in the ability of the three fatty acids to stimulate adherence. At the lower fatty acid concentrations tested (≤10 μM) 22:6 was less active than 20:4, whereas above 10 μM they were equally active. Immunofluorescent flow cytometric analysis of neutrophil integrin (adherence) receptors showed that the complement G3bi receptor (GDI lb) was up‐regulated by these fatty acids. There was no change in GDI la or GDllc. Saturated fatty acids of the same chain length were without effect on adherence or receptor expression. The findings suggest that these polyunsaturated fatty acids may, under certain conditions, be proinflammatory with respect to their acute effects on the interaction of neutrophils with microbes, endothelium, and other tissues.

Inhibition of Stimulus-Induced Endothelial Cell Intercellular Adhesion Molecule-1, E-Selectin, and Vascular Cellular Adhesion Molecule-1 Expression by Arachidonic Acid and Its Hydroxy and Hydroperoxy Derivatives

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 (20:4n-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-alpha (TNF-alpha) 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-alpha-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-alpha-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-alpha 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-alpha receptor binding level. The 15-HPETE was found to inhibit the TNF-alpha-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.

Effect of fatty acid structure on neutrophil adhesion, degranulation and damage to endothelial cells

Neutrophils have been implicated in ischaemic heart disease, unstable angina pectoris and acute myocardial infarction. Alterations in dietary levels of specific 18- and 20-carbon polyunsaturated fatty acids have significant clinical benefits in cardiovascular disease. However, to date there has been no concerted effort to identify the structural basis for polyunsaturated fatty acid-induced alterations in key neutrophil functions. We have investigated the influence of fatty acid structure and involvement of lipoxygenase/cyclooxygenase pathways on fatty acid-induced neutrophil functions. When neutrophils were incubated with 18-carbon fatty acids containing one to four double bonds (10-33 mumol/l), a significant increase in adherence and release of specific granule constituents occurred compared with control cells. In general, as the number of double bonds in the 18-carbon fatty acid increased, so did its ability to stimulate these functions. There was less stimulation of adherence and specific granule release by 18:3(n-3) than its isomer 18:3(n-6). Smaller effects were seen on azurophilic granule release. A further increase in adherence and degranulation was observed with increasing carbon chain length (20:3(n-6) and 20:4(n-6)). Differences were found in the ability of isomers of 20:3 to stimulate neutrophil function. Of the fatty acids tested only 20:4(n-6) was able to induce significant neutrophil-mediated endothelial detachment. Introduction of either internal hydroperoxy or hydroxyl groups into 20:4(n-6) abolished its adherence stimulating activity and considerably reduced its ability to stimulate release of both specific and azurophilic granules. Preincubation of neutrophils with either lipoxygenase (caffeic acid) or cyclooxygenase (indomethacin) inhibitors had no effect on 20:4(n-6) stimulated function. These studies show that the number and position of double bonds, carbon chain length and oxidation state can be critical to the neutrophil stimulatory properties of these fatty acids.

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 μΜ 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 μΜ) 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.

Augmentation of the human monocyte/macrophage chemiluminescence response during short‐term exposure to interferon‐gamma and tumour necrosis factor‐alpha

The effects of short‐term (30 min) pre‐incubation of human monocytes and macrophages (3‐day cultured monocytes) with leucocyte‐derived human interferon‐gamma (IFN‐γ) and recombinant human tumour necrosis factor‐alpha (rTNF‐α) were examined. Pre‐incubation of either monocytes or macrophages with rTNF‐α or IFN‐γ (100 U/5 × 105 cells) augmented their respiratory burst to formyl‐L‐methionyl‐L‐leucyl‐L‐phenylalanine (fMLP), measured by the luminol‐ and lucigenin‐dependent chemiluminescence assay. In addition, both cell types showed a burst of respiratory activity in the presence of rTNF‐α or IFN‐γ only. The effects of IFN‐γ were removed by adsorption with an anti‐ IFN‐γ monoclonal antibody and those of rTNF‐α were abolished by heating at 100°C, or by the addition of anti‐TNF‐α monoclonal antibody. The results demonstrate that both IFN‐γ and rTNF‐α are stimulators of monocytes and macrophages, and rapidly alter the capacity of the cells to respond to fMLP, which binds to cell surface receptors.

Inhibitory effects of arachidonic acid (20:4,n-6) and its monohydroperoxy- and hydroxy-metabolites on procoagulant activity in endothelial cells

The procoagulant response of endothelium to pathophysiological agents such as tumour necrosis factor alpha (TNF alpha) and phorbol myristate acetate (PMA) alters the expression of proteins such as tissue factor. The modulation of such procoagulant activity (PCA) by the polyunsaturated fatty acid arachidonic acid (20:4,n-6) and its 15-hydroperoxy (15-HPETE) and 15-hydroxy (15-HETE) metabolites was examined since this may have important implications in cardiovascular disease and atherosclerosis. Treatment of human umbilical vein endothelial cells (HUVEC) for 30 min with 20:4, 15-HPETE or 15-HETE before induction of PCA with TNF alpha (100 U) or PMA (10(-7) M) caused a significant inhibition of PCA. This inhibition was seen at 2-5 microM fatty acids. Dose response curves with TNF alpha indicated that the inhibition was greatest at higher concentrations of TNF alpha (> or = 250U TNF alpha/ml). The mode of administration of the fatty acid was not critical as fatty acids presented as DPC-fatty acid micelles or solubilised in ethanol gave similar inhibitions of PCA. 20:4, 15-HPETE or 15-HETE did not alter the binding of I125-labelled TNF alpha to its surface receptors on HUVEC, suggesting that the effect of these fatty acids was not mediated by events at the cell surface receptor level. In support of this, these fatty acids were found to inhibit PCA induced by PMA which bypasses cell surface receptors to activate protein kinase C directly.(ABSTRACT TRUNCATED AT 250 WORDS)

Neutrophil-mediated cartilage injury in vitro is enhanced by tumour necrosis factor alpha.

SummaryNeutrophil functions relevant to tissue damage are altered by cytokines such as tumour necrosis factor alpha (cachectin, TNFα), known to be present in inflammatory foci. In this study we examined the effect of TNFα on neutrophil-mediated cartilage damage in vitro. Human neutrophils were able to injure both human and bovine articular cartilage slices by degrading proteoglycan and inhibiting its synthesis. Recombinant human TNFα enhanced neutrophil-mediated degradation of proteoglycan, even when neutrophils were preincubated with TNFα and washed before incubating with cartilage. TNFα alone degraded proteoglycan and inhibited its synthesis. Neutrophil-mediated inhibition of proteoglycan biosynthesis was increased after incubating cartilage together with neutrophils and TNFα, but was unaltered when neutrophils were preincubated with TNFα. We conclude that TNFα enhances neutrophil injury to articular cartilage.

Tumour Necrosis Factor‐β Modulates Human Neutrophil‐Mediated Cartilage Damage

Human neutrophils, when cultured with human articular cartilage coated with heal aggregated immunoglobulin G, degraded proteoglycan and inhibited its synthesis. Neutrophil‐mediated degradation of cartilage was potentiated by recombinant human tumour necrosis factor‐β (TNFβ), although TNFβ alone did not alter proteoglycan degradation. This effect was seen when TNFβ, neutrophils, and cartilage were incubated together, and also when neutrophils were preincubated with TNFβ and washed before being added to cartilage. Similar results were obtained with living and killed cartilage, in contrast, ptetreatment of neutrophils with TNFβ abrogated the neutrophil‐mediated inhibition of proteoglycan biosynthesis. There was no effect of TNFβ alone on synthesis of proteoglycan.

Inhibition of neutrophil respiratory burst and degranulation responses to platelet-activating factor by antagonists WEB 2086, CV 6209 and CV 3988

The effect of three platelet-activating factor (PAF) antagonists, WEB 2086, CV 6209 and CV 3988, on neutrophil respiratory burst activity and degranulation in response to PAF was investigated. Both WEB 2086 and CV 6209 significantly inhibited the respiratory burst and degranulation in response to 400 nM PAF in a dose-dependent manner (10(-8)-10(-5) M). Higher concentrations of CV 3988 were required to inhibit these functions (10(-5) M and above). The three antagonists inhibited both the release of beta-glucuronidase (from azurophilic granules) and vitamin B12 binding protein (from specific granules) in response to PAF. Only a small nonsignificant inhibition of neutrophil function occurred in the absence of PAF. There was no loss of viability after incubation with the three antagonists at the concentrations tested. These antagonists will be useful tools to study the involvement of PAF in neutrophil-mediated tissue damage and inflammation.

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.