Fatty acid modulation of cytokine release from human monocytic cells.

The effect of individual fatty acids on the release of interleukin-1-like (IL-1-like) cytokine activity was investigated on human monocytic cells, primarily the cell line U937 and also peripheral blood monocytes. IL-1-like bioactivity was estimated by assessing the effect of supernatants from monocytic cells on the proliferation of thymocytes as measured by [3H]thymidine incorporation into the acid insoluble fraction of the thymocytes. A pronounced concentration-dependent increase (in the range 1-100 microM) in the release of IL-1-like activity was observed with arachidonic acid, dihomo-gamma-linolenic acid (DGLA) and eicosapentaenoic acid (EPA) in absence and presence of bacterial endotoxin (LPS). A much less pronounced concentration-dependent increase in the release of IL-1-like activity was observed with oleic acid and linoleic acid had only a small effect at 100 microM. The potencies of each fatty for this effect using the EC50 concentrations were arachidonic acid > EPA > or = DGLA > linoleic acid > oleic acid with palmitic acid having no effect. The IL-1-like activity was confirmed by the attenuation of the monocytic-cell-supernatant-induced increase in thymocyte proliferation by anti-IL-1 beta antiserum. An increase in the release of anti-IL-1 beta-antiserum-precipitable radioactivity from U937 cells prelabelled with [35S]methionine then incubated with fatty acids in the presence of LPS further confirmed that IL-1 release was increased. Arachidonic acid and EPA also increased the release of IL-1-like activity from peripheral blood monocytes demonstrating that normal monocytes can respond in a similar manner and that this effect of the fatty acids is not restricted to the U937 cell line.

Inhibition of cytokine-stimulated thymic lymphocyte proliferation by fatty acids: the role of eicosanoids.

The effect of individual fatty acids on the proliferation of thymic lymphocytes in response to interleukin-1 (IL-1) was investigated. Proliferation was estimated by measuring [3H]thymidine incorporation into the acid insoluble fraction of the thymocytes. A concentration-dependent inhibition (in the range 1-100 microM) in the IL-1-stimulated proliferation was observed with the C20 fatty acids dihomo-gamma-linolenic acid (DGLA), arachidonic acid and eicosapentaenoic acid (EPA). A less pronounced concentration-dependent inhibitory response was observed with the C18 fatty acids linoleic acid, alpha-linolenic acid and gamma-linolenic acid. Palmitic acid and oleic did not have any effect on either basal or IL-1-stimulated proliferation at concentrations up to 100 microM. The potencies of each fatty acid for this effect at a concentration of 100 microM were: arachidonic acid > EPA > or = DGLA > linoleic acid. DGLA, arachidonic acid and EPA also attenuated IL-2-stimulated proliferation. The inhibitory action of these fatty acids was not mediated by conversion to prostaglandins or other eicosanoids as the cyclooxygenase inhibitor, ketoprofen and NDGA did not alter their action. Incubation of thymocytes with radiolabelled DGLA and EPA followed by reverse-phase HPLC analysis revealed that DGLA is predominantly converted to a more polar metabolite which is not PGE1 whereas EPA does not appear to be converted to any other detectable metabolite. The data indicate that the inhibitory actions of fatty acids on cell proliferation do not occur as a result of conversion to other metabolites but may be direct effects. The inhibition of cytokine-stimulated lymphocyte proliferation by unsaturated fatty acids would imply that they may attenuate cell-mediated immune reactions.

Interleukin-1 inhibits PGE2 binding to macrophage-like P388D1 cells by a cyclic AMP-independent process.

The interaction between interleukin IL-1 alpha and PGE2 on P388D1 cells has been investigated. Preincubation of murine macrophage-like cells, P388D1, with IL-1 alpha (0-73 pM) reduced the binding of PGE2 to these cells in a concentration-dependent manner. Scatchard analysis showed that IL-1 alpha decreased the PGE2 binding by lowering both the high and low affinity receptor binding capacities (from 0.31 +/- 0.02 to 0.12 +/- 0.01 fmol/10(6) cells for the high affinity receptor binding sites and from 2.41 +/- 0.12 to 1.51 +/- 0.21 fmol/10(6) cells for the low affinity receptor binding sites). However, the dissociation constants of the receptors of the IL-1 alpha-treated cells remained unchanged. Inhibition of PGE2 binding by IL-1 alpha did not involve changes in either protein phosphorylation or intracellular cyclic AMP levels. Our data clearly show that IL-1 alpha inhibits the binding of PGE2 to monocytes/macrophages and may thereby counter the immunosuppressive actions of PGE2.

Characterization of prostaglandin E2 receptors expressed on human monocytic leukaemic cell line, U937.

Prostaglandin E2 (PGE2) receptors of a human monocytic leukaemic cell line, U937 cells, have been identified. [3H]PGE2 binding to these cells was found to be saturable and highly specific. Scatchard analysis of binding data revealed a non-linear plot indicating the presence of two independent classes of binding sites with different affinities and capacities. The high-affinity class had Kd1 = 3.1 nM and binding capacities n1 = 0.6 fmol/10(6) cells, whereas the low-affinity class had Kd2 = 137 nM and capacities n2 = 16 fmol/10(6) cells. Incubation of U937 cells with 3 microM PGE2 stimulated a 15-fold increase in cAMP formation compared to basal levels. Prior exposure of these cells with 10 microM PGE2 for 60 min induced both homologous and heterologous desensitization of adenylate cyclase activity. PGE2 (3 microM) or histamine (100 microM) showed reduced stimulation of cAMP formation in these desensitized cells compared to controls. The desensitized cells also showed 80% reduction of specific PGE2 binding compared to control cells. Our data suggest that U937 cells have PGE2 receptors which are linked to the adenylate cyclase system.

Regulation of prostaglandin E2 binding to a murine macrophage cell line, P388D1, by insulin.

Preincubation of murine macrophage-like P388D1 cells with physiological amounts of insulin resulted in an increase in prostaglandin E2 binding to these cells, by approximately 2-fold, when compared to untreated cells. Scatchard analysis of the binding of PGE2 to insulin-treated cells indicated that the enhanced binding was due to an increase in receptor number (from 0.30 +/- 0.02 to 0.63 +/- 0.03 fmol/10(6) cells for the high affinity receptor binding sites, and from 2.4 +/- 0.31 to 5.0 +/- 0.41 fmol/10(6) cells for the low affinity receptor binding sites) rather than to an increase in the affinity of the binding sites. The insulin-stimulation of PGE2 binding appeared to be associated with a lowering of the cAMP level in these cells; treatment of cells with insulin lowered the cAMP level by increasing the cAMP phosphodiesterase activity of both the membrane and cytosolic fractions. However, enhanced PGE2 binding to the cells resulted in an increase in cAMP level in the cells. This increase in cAMP level may help to enhance the immunosuppressive action of this prostanoid, as PGE2 is known to suppress many steps in the immune response, including interleukin-1 expression, by raising cAMP levels via activation of receptor-linked adenylate cyclase. Our data suggest that insulin at physiological concentrations may enhance the immunosuppressive action of PGE2.

Lipopolysaccharide, muramyl dipeptide and polyinosinic: polycytidylic acid induce the accumulation of inositol phosphates in blood monocytes and lymphocytes.

Rabbit macrophages (Mø) and lymphocytes (Ly) incubated with three structurally dissimilar immunomodulators, lipopolysaccharide (bacterial endotoxin, LPS), polyinosinic: polycytidylic acid (poly-I:C) and muramyl dipeptide (MDP), were found to accumulate inositol phosphates (IPs) in a concentration- and time-dependent manner. The threshold concentration of LPS necessary for an increase in IPs in both cell types was less than 1 ng/ml and a maximum effect was observed between 1 and 10 micrograms/ml. The threshold concentrations for poly-I:C and MDP were between 0.1 and 1 microgram/ml for both cell types. Significant increases in the concentration of inositol phosphates occurred between 30 and 60 min after challenge of either cell type with any of the three agents studied. In addition, all three immunomodulators produced a greater accumulation of IPs in macrophages than in mixed lymphocytes and after 2 h appeared to approach a maximum in macrophages, whereas the IPs level in lymphocytes appeared to be still rising after 2 h. In Mø and Ly the IPs level was increased within 10 min of incubation in the presence of either PGE2 or medium previously obtained from cells incubated with LPS. In addition, anisomycin (a protein synthesis inhibitor) and ketoprofen (a cyclo-oxygenase inhibitor) inhibited the LPS-stimulated increase of IPs accumulation in both cell types. These two observations suggest that the LPS-stimulated increase in IPs in macrophages and lymphocytes is mediated by a protein intermediate and possibly a prostanoid.

Effect of CLA supplementation on immune function in young healthy volunteers.

OBJECTIVES: This study investigated the effect of dietary CLA supplementation (3g/day; 50:50 mix of the two major isomers) on the immune system and plasma lipids and glucose of healthy human (male and female) volunteers. DESIGN: Double-blind, randomized, reference-controlled study. SUBJECT AND INTERVENTION: A total of 28 healthy male and female participants aged 25-50 y received either high oleic sunflower oil (reference) or 50% CLA 9-11 and 50% CLA 10-12 CLA isomers (50:50 CLA-triglyceride form). The treatments were given as supplements in soft-gel capsules providing a total 3 g (6 x 500 mg capsules) per day in treatment groups for 12 weeks. A 12-week washout period followed the intervention period. RESULTS: Levels of plasma IgA and IgM were increased (P < 0.05 and 0.01 respectively), while plasma IgE levels were decreased (P < 0.05). CLA supplementation also decreased the levels of the proinflammatory cytokines, TNF-alpha and IL-1beta (P < 0.05), but increased the levels of the anti-inflammatory cytokine, IL-10 (P < 0.05). Another aspect of immune function, delayed type hypersensitivity (DTH) response, was decreased during and after CLA supplementation (P < 0.05). However, plasma glucose, lipids, lymphocyte phenotypic results were not affected significantly by CLA. CONCLUSION: This is the first study to show that CLA, a fatty acid naturally found in dairy and meat products, can beneficially affect immune function in healthy human volunteers. SPONSORSHIP: This study was supported by Loders-Croklaan, The Netherlands and SEERAD (Scottish Executive Environmental Rural and Agriculture Department).

A study of the pyrogenic actions of interleukin-1 alpha and interleukin-1 beta: interactions with a steroidal and a non-steroidal anti-inflammatory agent.

1. The pyrogenic effects of intravenously administered human recombinant interleukin-1 alpha (IL-1 alpha) and IL-1 beta were studied in the rabbit. 2. Both cytokines produced dose-related increases in body temperature. At all doses studied (100-5000 u kg-1) both cytokines elicited a monophasic increase in body temperature, beginning 15 min and reaching a maximum 45 min after administration. 3. A comparison of thermal response index (TRI2, the magnitude of febrile responses over 2 h obtained by integrating the change in temperature in degrees C against time in hours) values indicated that IL-1 beta (500 u kg-1, TRI2 = 0.69 +/- 0.04, n = 4) was approximately 5 fold more potent than IL-1 alpha (2500 u kg-1, TRI2 = 0.73 +/- 0.07, n = 4, all values are means +/- s.e.means) in elevating body temperature. delta Tmax values for the above doses of IL-1 beta and IL-1 alpha were 0.60 +/- 0.06 and 0.61 +/- 0.03 respectively. When IL-1 alpha and IL-1 beta were heated for 30 min at 60 degrees C prior to administration no biological activity was observed. 4. A cyclo-oxygenase inhibitor, ketoprofen (3 mg kg-1) administered 15 min before either cytokine completely abolished the fever induced by both IL-1 alpha (2500 u kg-1) and IL-1 beta (500 u kg-1). 5. Intravenous administration of the steroidal anti-inflammatory agent dexamethasone (3 mg kg-1) 1 h before either cytokine attenuated the fever induced by IL-1 alpha (2500 u kg-1) and IL-1 beta (500 u kg-1). 6. The effects of ketoprofen and dexamethasone on IL-I pyrogenicity indicate that prostanoids are almost certainly involved in the responses. The different potencies of IL-l alpha and IL-1 beta may be related to their relative ability to stimulate prostanoid biosynthesis.

Antipyretic actions of human recombinant lipocortin-1.

The effect of human recombinant lipocortin-1 (hrLC-1) on the pyrogenic actions of the synthetic polyribonucleotide polyinosinic:polycytidylic acid (poly I:C) has been studied in conscious rabbits. Poly I:C (2.5 micrograms kg-1) given i.v. produced a biphasic fever with a first peak after 90-105 min and a second peak between 225-240 min. hrLC-1 (50 micrograms kg-1) given i.v. simultaneously with the poly I:C produced a significant reduction in the febrile response but without complete suppression. The thermal response index over 5 h (TRI5) was 4.69 +/- 0.51 for poly I:C given with saline and the TRI5 for poly I:C given with hrLC-1 was 2.66 +/- 0.45 (values are for n = 5 +/- s.e. mean, P less than 0.05). hrLC-1 administered alone had no effect on body temperature and its antipyretic activity was lost on heating. In a separate series of experiments 1 h pretreatment with dexamethasone (1 mg kg-1) given i.v. reduced the pyrogenic response (TRI5) to poly I:C (2.5 micrograms kg-1) from 4.87 +/- 0.54 without dexamethasone to 2.00 +/- 0.25 (n = 5, P less than 0.05) and dexamethasone given alone had no effect on body temperature. These data demonstrate that LC-1 possesses antipyretic actions and raises the possibility that the antipyretic actions of dexamethasone are mediated through the induction of LC-1.

Is cholecystokinin therapeutic in chronic schizophrenia?

Cholecystokinin (CCK) has been implicated as a neurotransmitter, and recent research has identified CCK as having potential antipsychotic effects in patients with chronic schizophrenia. Nine chronic schizophrenic patients with prominent psychotic symptoms and a history of resistance to conventional neuroleptic treatment were administered ceruletide, a synthetic decapeptide of cerulein, intramuscularly. No clinically significant short-term or long-term therapeutic effects were demonstrated, despite the results of statistical analysis which indicated significant improvement. The implications of this open clinical trial for a new treatment modality of chronic schizophrenia are discussed.

The pyrogenic actions of the interferon-inducer, polyinosinic:polycytidylic acid are antagonised by ketoprofen.

The effect of various doses of polyinosinic:polycytidilic acid (Poly I:C) in the range (0.25-250 micrograms/kg) administered i.v. on the body temperature of rabbits was investigated. The effect of ketoprofen on the febrile responses was also studied. The threshold dose of Poly I:C administered which produced a fever was between 0.25 and 0.5 micrograms/kg and a double-sigmoidal dose-response relationship was observed. A sigmoidal relationship was obtained with lower doses from 0.25 micrograms/kg to an apparent maximum with 2.5 micrograms/kg and another with higher doses from 5 micrograms/kg to another apparent maximum with 250 micrograms/kg. Ketoprofen (3 mg/kg) given s.c. 15 min prior to Poly I:C inhibited the production of fever. Ketoprofen administered after the onset of fever resulted in an immediate deffervescence. These results suggest that febrile responses to Poly I:C involve prostaglandin biosynthesis.

Pyrogenic immunomodulators increase the level of prostaglandin E2 in the blood simultaneously with the onset of fever.

Blood prostaglandin E2 (PGE2) levels, estimated by radioimmunoassay, and body temperatures of conscious rabbits were measured simultaneously during fever in response to polyinosinic: polycytidylic acid, lipopolysaccharide and interleukin 1/endogenous pyrogen. The effects of the antipyretic agent ketoprofen on both parameters was also studied. Significant rises (in the order of 6- to 8-fold) in the PGE2 level were observed after injection of either of the three pyrogens and occurred simultaneously with the rise in temperature. Ketoprofen given after the onset of fever in response to the pyrogens produced an immediate defervescence and a simultaneous decrease in plasma PGE2. Ketoprofen given before the pyrogens prevented any rise in either body temperature or plasma PGE2 level. When animals were subjected to an environmental temperature of 34 degrees C a hyperthermia was observed without any change in the blood PGE2 level. These results suggest that an increase in the blood PGE2 level may contribute to the pathogenesis of fever.

Dexamethasone pre-treatment is antipyretic toward polyinosinic: polycytidylic acid, lipopolysaccharide and interleukin 1/endogenous pyrogen.

The effect of intravenously injected dexamethasone on the febrile response of rabbits to Polyinosinic: Polycytidylic acid (Poly I:C), lipopolysaccharide (LPS) and interleukin 1/endogenous pyrogen (IL1/E.P.) was studied. Dexamethasone (1 mg/kg) attenuated the febrile response to Poly I:C (5 micrograms/kg) but only if administered between 0.5 to 2 h before Poly I:C. If it was given after Poly I:C this resulted in a potentiation of the fever. Antagonism of the febrile response to Poly I:C by dexamethasone pre-treatment was dose-dependent and a maximal effect was observed with 3 mg/kg, a higher dose (6 mg/kg) resulted in a lesser effect on the Poly I:C fever. DEX injected alone (0.5-6 mg/kg) did not have any effect on body temperature. Fevers in response to LPS (50 ng/kg) and IL1/E.P. were also attenuated by dexamethasone. It is concluded that Poly I:C, LPS and IL1/E.P. induce fever by a common mechanism which is either directly or indirectly inhibited by dexamethasone.

Therapeutic implications of disorders of cell death signalling: membranes, micro-environment, and eicosanoid and docosanoid metabolism.

Disruptions of cell death signalling occur in pathological processes, such as cancer and degenerative disease. Increased knowledge of cell death signalling has opened new areas of therapeutic research, and identifying key mediators of cell death has become increasingly important. Early triggering events in cell death may provide potential therapeutic targets, whereas agents affecting later signals may be more palliative in nature. A group of primary mediators are derivatives of the highly unsaturated fatty acids (HUFAs), particularly oxygenated metabolites such as prostaglandins. HUFAs, esterified in cell membranes, act as critical signalling molecules in many pathological processes. Currently, agents affecting HUFA metabolism are widely prescribed in diseases involving disordered cell death signalling. However, partly due to rapid metabolism, their role in cell death signalling pathways is poorly characterized. Recently, HUFA-derived mediators, the resolvins/protectins and endocannabinoids, have added opportunities to target selective signals and pathways. This review will focus on the control of cell death by HUFA, eicosanoid (C20 fatty acid metabolites) and docosanoid (C22 metabolites), HUFA-derived lipid mediators, signalling elements in the micro-environment and their potential therapeutic applications. Further therapeutic approaches will involve cell and molecular biology, the multiple hit theory of disease progression and analysis of system plasticity. Advances in the cell biology of eicosanoid and docosanoid metabolism, together with structure/function analysis of HUFA-derived mediators, will be useful in developing therapeutic agents in pathologies characterized by alterations in cell death signalling.

Fatty acids and endothelial cell function: regulation of adhesion molecule and redox enzyme expression.

Different types of dietary fatty acids have distinct, and sometimes opposing, effects on endothelial cell function. Long-chain n-3 polyunsaturated fatty acids from fish oil (eicosapentaenoic-acid-20:5n-3 and docosahexaenoic-acid-22:6n-3) attenuate cytokine-induced adhesion molecule expression (mRNA and protein) while the n-6 polyunsaturated fatty acid arachidonic acid (eicosatetraenoic-acid-20:4n-6) either has no effect, or elicits as up- or down-regulation. Conjugated linoleic acids derived from 18:2n-6, linoleic acid also reduced the expression of adhesion molecules. These polyunsaturated fatty acids also induced redox enzyme expression (mRNA and protein) in endothelial cells and modulation of redox-sensitive transcription factors (e.g. nuclear factor kappa B, activated protein-1) which regulate the gene expression of adhesion molecules, redox enzymes and various stress proteins. The induction of redox enzyme expression by n-3 polyunsaturated fatty acids and conjugated linoleic acids could explain their inhibitory effects on gene transcription and adhesion molecule protein expression. Individual adhesion molecule genes and transcription factors appear to differ in their responsiveness to various oxidant stimuli and non-redox, more direct regulatory mechanisms (e.g. peroxisome proliferator activation receptor activation) might also be involved in their regulation.

Alpha-melanocyte-stimulating hormone suppresses fever and increases in plasma levels of prostaglandin E2 in the rabbit.

1. The effect of alpha-melanocyte-stimulating hormone (alpha-MSH) on changes in body temperature and plasma levels of prostaglandin E2 (PGE2) were measured in the rabbit following intravenous injection of bacterial lipopolysaccharide (LPS), rabbit endogenous pyrogen (EP), human recombinant tumour necrosis factor-alpha (TNF-alpha), human recombinant interleukin-1 beta (IL-1 beta) and intracerebroventricular injection of PGE2. 2. LPS (25 ng kg-1), EP (25 microliters kg-1), TNF-alpha (11 micrograms kg-1) and IL-1 beta (5 ng kg-1) produced increases in body temperature simultaneously with increases in plasma PGE2 levels. alpha-MSH (5 or 10 micrograms kg-1) attenuated both the increase in body temperature and increases in plasma levels of PGE2. 3. Intracerebroventricular injection of PGE2 (500 ng) produced a monophasic increase in body temperature. alpha-MSH (5 micrograms kg-1) administered 20 min after PGE2 had no effect on the hyperthermic response. 4. alpha-MSH (10 micrograms kg-1) had no effect on either body temperature or plasma levels of PGE2 in response to I.V. injection of sterile saline. 5. These data demonstrate that alpha-MSH inhibits both the pyrogenic actions of LPS, EP, TNF-alpha and IL-1 beta and their ability to increase PGE2 release without affecting the direct actions of PGE2, suggesting the possibility that alpha-MSH may prevent the synthesis of PGE2 either by preventing the actions or release of mediators such as TNF-alpha and IL-1 in response to LPS.

A study of cyclic AMP-dependent protein phosphorylation in Schistocerca gregaria CNS: a comparison to that in mammalian CNS.

1. The effect of cyclic AMP (10 microM) on the incorporation of 32P into protein was studied in cell-free preparations of Schistocerca gregaria cerebral ganglia. 2. Cyclic AMP-dependent phosphorylation of total protein was maximal after 60 sec, had a pH optimum of 7 to 8, was not affected by temperature (22-37 degrees C) and had a Km of 77 microM ATP. 3. Cyclic AMP increased the phosphorylation of total and specific protein in soluble fractions greater than synaptosomal greater than microsomal greater than crude membrane fractions. 4. In a direct comparison of locust brain to rat cerebral cortex, cyclic AMP stimulated the increased phosphorylation of only three protein bands, whereas in identical fractions of locust brain the phosphorylation of at least 12 protein bands was observed.

Prostaglandin and fatty acid modulation of Escherichia coli O157 phagocytosis by human monocytic cells.

Phagocytosis by human monocytes is an important primary survival mechanism particularly during bacterial infection. However, the processes that control the events and mediators involved in the activation of monocytes and their impact on the phagocytosis of bacteria are poorly understood. The effect of bacterial endotoxin, interleukin-1 beta (IL-1 beta), fatty acids and prostaglandin E2 (PGE2) on the phagocytosis of fluoroscein isothiocyanate (FITC)-labelled Escherichia coli (O157) by human blood monocytes and U937 cells was studied by flow cytometry. Endotoxin increased the phagocytosis of labelled bacteria by both monocytes and U937 cells. IL-1 beta and the polyunsaturated fatty acids; dihomo-gamma-linolenic and arachidonic acids also increased the phagocytic activity of both monocytes and U937 cells. In contrast, PGE2 suppressed phagocytosis in a concentration-dependent manner. The cyclo-oxygenase inhibitor, ketoprofen, further enhanced the increased phagocytic activity in the presence of endotoxin and interleukin-1 (IL-1) indicating suppression by endogenous prostaglandins. This was confirmed by the data which showed that lipopolysaccharide (LPS) and IL-1 increased PGE2 release and ketoprofen inhibited release. Endotoxin and fatty acids increased IL-1 beta release also, whereas PGE2 inhibited release. The data suggest that phagocytic activity may be linked to changes in IL-1 levels. The data presented in this study also suggest that monocyte phagocytosis in the course of bacterial infection would be altered during pathophysiological events which result in elevation of extracellular fatty acids.