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.

Cytokines and cytokine inducers stimulate prostaglandin E2 entry into the brain.

Cytokine inducers and cytokines increase the circulating level of prostaglandin E2 (PGE2) during the acute-phase immune response. This occurs simultaneously with the onset of fever, indicating that brain levels of PGE2 also increase. This raises the possibility that PGE2 produced in the peripheral circulation, not necessarily at distant sites from the brain, may penetrate the brain and be present in the cerebrospinal fluid (CSF). Blood and CSF levels of PGE2 in rabbits were measured by radioimmunoassay during fever stimulated in response to lipopolysaccharide (LPS), polyinosinic:polycytidylic acid (poly I:C) and interleukin-1 (IL-1) given i.v. The effect of the prostaglandin synthesis inhibitor ketoprofen on these parameters was also studied. In addition, the level of radioactivity in the CSF was measured following the administration of [125I]-labelled PGE2 i.v. during fever induced by LPS, poly I:C, IL-1 or tumor necrosis factor alpha (TNFalpha). Both LPS and poly I:C stimulated an increase in plasma and CSF levels of PGE2 over a 5-h period with a peak at 60 min and 90 min, respectively, which occurred in parallel with the changes in body temperature. Ketoprofen abolished the rise in plasma and CSF PGE2 levels and the rise in body temperature in response to LPS, poly I:C and IL-1. In experiments where animals were given [125I]-labelled PGE2 i.v., radioactivity well above the background level was measured in samples of CSF collected from LPS-, poly I:C-, IL-1- or TNFalpha-pretreated animals. In contrast the radioactivity present in samples of CSF perfusate collected from control (saline-treated) animals was indistinguishable from the background level. These data indicate that cytokine inducers and cytokines increase the mass level of PGE2 in blood and CSF and also increases the entry, from the peripheral circulation, of radiolabelled PGE2 into the third cerebral ventricle.

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.

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.

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.

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.

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.

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.