In vivo evidence for protease-catalysed mechanism providing bioactive tumor necrosis factor alpha.

Mice pretreated by intravenous injection of 42 mg/kg of the serine protease inhibitor alpha 1-antitrypsin prior to a hepatotoxic dose of D-galactosamine/lipopolysaccharide (GalN/LPS) were fully protected against hepatitis. Pretreatment with alpha 1-antitrypsin with doses up to 300 mg/kg at different times failed to protect galactosamine sensitized animals against tumor necrosis factor alpha (TNF alpha)-induced hepatitis. No bioactive TNF alpha was detectable in serum of mice protected against GalN/LPS-induced hepatitis by pretreatment with alpha 1-antitrypsin. In contrast, abundant amounts of TNF were found in sera of GalN/LPS-treated control animals. It is concluded that a serine protease sensitive to alpha 1-antitrypsin provides bioactive TNF alpha by proteolytic cleavage of a TNF alpha precursor.

Stimulation of human and murine adherent cells by bacterial lipoprotein and synthetic lipopeptide analogues.

Lipoprotein from the outer membrane of Escherichia coli and its synthetically prepared N-terminal lipopeptide segments Pam3Cys-Ser-Ser-Asn-Ala and Pam3Cys-Ser, as well as lipoprotein from other Enterobacteriaceae, constitute potent polyclonal B lymphocyte activators. Here, we demonstrate that these compounds were also able to stimulate human and murine leukocytes: in murine macrophages, we could show the induction of interleukin 1 release by the mitogens, as measured in the thymocyte proliferation assay. Moreover, murine peritoneal exudate cells were stimulated to secrete prostaglandins E2 (PGE2) and F2 alpha (PGF2 alpha). The effect of Pam3Cys-Ser on the murine macrophage cell line P388D1 was also tested: the compound induced an increase in proliferation, as measured by a thymidine incorporation assay. In addition, the cell line could be induced to release IL 1 into the supernatant. Correspondingly, induction of IL 1 release could also be demonstrated in human mononuclear cells. Our results demonstrate that the two novel synthetic lipopeptides are potent stimulators for human monocytes and murine macrophages. These findings may be important for the elucidation of the role of these bacterial surface components in the course of bacterial infections.

Bacterial endotoxin: molecular relationships between structure and activity.

The significance of endotoxins in bacterial infection and their role as bacterial surface antigens (O antigens) have stimulated investigations into their chemical nature and the mechanisms of their biologic action during the last few decades. This article summarizes some of the recent results and emphasizes structure-activity relationships.

Ascorbic acid induces lipid peroxidation on neuroectodermal SK-N-LO cells with high endogenous ferritin content and loaded with MAb-ferritin immunoconjugates.

Neuroblasma-and other malignant cells often contain elevated amounts of iron-rich ferritin and H2O2 and may therefore be a potential target for pro-oxidative effects of ascorbic acid (AA), generating cytotoxic products e.g. by lipid peroxidation (LPO). The influence of H2O2 and iron, either in its free form or bound to ferritin, on AA induced LPO was first investigated using erythrocyte ghosts as a model system. Results of these experiments showed that AA induced LPO not only in the presence of free available iron but also in the presence of ferritin. Similarly, AA induced significant LPO in neuroectodermal SK-N-LO cells with elevated intracellular ferritin levels. These LPO promoting effects of ferritin in the presence of AA on SK-N-LO cells could also be observed using ferritin-immunoconjugates: for this purpose, ferritin was bound to human monoclonal antibodies (MAb-ferritin) recognizing ganglioside GD2 which is present in large quantities on cell surfaces of SK-N-LO and many neuroblastoma cells. We conclude that the pro-oxidative effects of AA could be exploited in the treatment of ferritin rich neuroblastoma in combination with chemotherapy or with MAb-ferritin immunoconjugates.

Involvement of lipoxygenases in the activation of mouse macrophages by endotoxin.

Preincubation with lipopolysaccharides stimulated the phagocytic capacity of mouse peritoneal macrophages. Treatment of cell cultures with inhibitors of lipoxygenases which affect this pathway specifically (diethylcarbamazine) or compounds which block lipoxygenases and cyclooxygenase (BW 755 C, eicosatetraynoic acid) inhibited the increase, while inhibitors of cyclooxygenase (indomethacin, aspirin) did not affect the LPS induced enhancement of the phagocytic capacity. It is, therefore, concluded that lipoxygenase products are required for the activation of macrophages by endotoxins.

Pentoxifylline increases survival in murine endotoxin shock and decreases formation of tumor necrosis factor.

Pentoxifylline (POF) was found to possess high protecting potential in murine endotoxin shock. POF was tested in endotoxic shock in normal, LPS-tolerant, LPS-hyperreactive, and D-galactosamine-sensitized mice. A dose of 50 mg/kg increased survival in normal and tolerant mice from 50% in LPS-controls to 90%, and in LPS-hypersensitized and D-galactosamine-sensitized mice from 20% in LPS-controls to 90%. POF did not interfere with the development of endotoxin tolerance. The protective effects of POF were dose-dependent and were observed, even when POF was given up to 4 h post endotoxin. POF was found to inhibit the appearance of TNF in serum of LPS-treated, D-galactosamine-sensitized mice and in the supernatants of LPS-stimulated, thioglycollate-induced mouse peritoneal macrophages.

The role of prostacyclin in the protective effects of pentoxifylline and other xanthine derivatives in endotoxin action in mice.

The xanthine derivative pentoxifylline (POF, Trental) and its metabolically more stable structural analogues, HWA 138 and HWA 448, were compared for their capacity to prevent leukopenia provoked in mice by injection of bacterial lipopolysaccharide (LPS). It was found that HWA 138 and HWA 448 counteracted LPS-induced leukopenia more effectively than POF. Indomethacin diminished the action of HWA 138 and HWA 448, and the stable prostacyclin analogue CG-4203 (Taprosten) mimicked the effect of the xanthine derivatives. Since pentoxifylline and its structural analogues induced synthesis of PGI2 in endothelial cell cultures, it is suggested that its effect on LPS leukopenia is mediated by endogenous prostacyclin production. All of the xanthine derivatives tested and CG-4203 blocked the leukopenia induced by recombinant tumor necrosis factor, which is a major endogenous mediator for endotoxin toxicity.

The effect of endotoxin on the lipoxygenase-mediated conversion of exogenous and endogenous arachidonic acid in mouse peritoneal macrophages.

The influence of lipopolysaccharide (LPS, endotoxin) or its lipid A component (bacterial and synthetic) on the synthesis of zymosan induced leukotriene C4, prostaglandin E2 and prostacyclin and on the conversion of exogenous arachidonic acid was studied in mouse peritoneal macrophages. It was found that following preincubation with LPS the amount of leukotriene C4 released during phagocytosis of zymosan was substantially decreased. The levels of prostaglandin E2 and prostacyclin, however, were the same in LPS-treated cells and controls. Likewise, pretreatment with LPS impaired the capacity to convert exogenously added arachidonic acid to mono- and di-HETE's. Lipid A (bacterial and synthetic) exhibited the same activity as LPS. LPS had no effect on macrophages of the endotoxin low responder mouse strain (C3H/HeJ). Several explanations could be possible for the observed LPS effect. The finding that low doses of alpha-tocopheryl acetate prevented the LPS-induced decrease of LTC4 synthesis indicates a protective role of this agent. We would, therefore, favour the idea that lipoxygenases undergo oxidative selfinactivation during LPS action.

The effect of pentoxifylline in septic shock--new pharmacologic aspects of an established drug.

Pentoxifylline (Trental) is a well-known vasoactive drug with proven clinical efficacy in various circulatory disorders. It improves the microcirculation due to its rheologic effects on red blood cells, platelets, and plasmatic components, resulting in a decrease of whole blood viscosity. Surprisingly, it has been found that pentoxifylline will also be of great benefit in different models of animal sepsis, including both gram positive and gram negative bacteria. In these experiments, survival rates are significantly increased in the pentoxifylline group when compared with the controls, which is paralleled by a decrease in germ counts. By different experimental approaches it could be shown that this drug interferes with pathologic granulocyte-endothelium interactions which are closely related to septic symptoms, both downregulating intravasal granulocyte hyperreactivity as well as stimulating antiaggregatory activity of the vessel endothelium. Through this way, beneficial effects of pentoxifylline may be expected in various diseases related to infection, sepsis, and shock which, however, have still to be proven in detailed clinical studies.

Differential protective activities of site specific lipoxygenase inhibitors in endotoxic shock and production of tumor necrosis factor.

Lipoxygenase inhibitors have been shown to exert beneficial effects in experimental models of endotoxin shock. In the present study it was found that lipoxygenase inhibitors prevented LPS, but not tumor necrosis factor alpha (TNF alpha)-evoked leukopenia in mice. These inhibitors protected against endotoxin lethality but not against TNF alpha induced lethality. When the protective potency of the specific 5-lipoxygenase inhibitors (MK 886, CGS 81585) was tested in endotoxin-induced leukopenia and shock, they were found to be ineffective. Site specificity of the inhibitors was assessed by comparison of their effects on the formation of LTC4 and the conversion of linoleic acid to 13-hydroxyoctadecadienoic acid (13-HODD) by macrophages. The 5-lipoxygenase inhibitors interfered with LTC4 formation in macrophages, however, they did not affect endotoxin-induced TNF alpha formation, neither in cell cultures nor in mice. The inhibitory strength of other, less specific lipoxygenase blockers to suppress TNF alpha formation correlated quantitatively with their ability to interfere with 13-HODD synthesis. From these findings it is concluded that lipoxygenase inhibitors interfere with endotoxic effects because they block TNF alpha formation. Since 5-lipoxygenase inhibitors neither prevented the formation of TNF alpha nor endotoxin leukopenia and lethality, it is suggested that a lipoxygenase product distinct from the leukotrienes is involved in TNF alpha synthesis. Based on the fact that a tight correlation exists between inhibition of TNF alpha synthesis and 13-HODD formation, activation of 15-lipoxygenase might be important for TNF alpha formation.

Increased 13-hydroxyoctadecadienoic acid content in lipopolysaccharide stimulated macrophages.

Endotoxin-stimulated mouse peritoneal macrophages were found to contain 13-hydroxyoctadecadienoic acid, which was released upon alkaline hydrolysis of the cells. Compared to untreated cells, incubation with LPS increased the content of 13-hydroxyoctadecadienoic acid in macrophage hydrolysates to about 8-fold. Analysis of the material on chiralphase HPLC revealed that it consisted prevalently of 13(S)-hydroxyoctadecadienoic acid. This indicates its enzymatic origine.

Metabolism of exogenous arachidonic acid by mouse peritoneal macrophages.

On incubation of resident mouse peritoneal macrophages with arachidonic acid several hydroxyacyl derivatives detectable in cellular supernatants are formed. As main products monohydroxyarachidonic acids (monoHETE's) were identified. In addition, smaller amounts of dihydroxyarachidonic acids (diHETE's) were formed. A detailed analysis of cell culture supernatants by reversed phase HPLC, normal phase HPLC in combination with UV-spectroscopy and combined gas-chromatography/masspectrometry revealed the presence of 5-, 8-, 12- and 15- monoHETE's, two distinct 5,12-diHETE's, several 8,15-diHETE's and 14,15-diHETE. Among the 5,12-diHETE's, only small amounts of a compound with the characteristics of LTB4 were detected. Under the conditions employed, the cyclooxygenase products PGE2 and PGI2 (as 6-keto-PGF1 alpha) were only minor metabolites. In contrast, when macrophage cultures were stimulated with the phagocytic stimulus zymosan, PGI2, PGE2 and LTC4 were found as the major conversion products of arachidonic acid, whereas mono- and diHETE's were not formed in detectable amounts.

The role of lipoxygenases in endotoxin-induced cytokine production.

The LPS induced synthesis of tumor necrosis factor in macrophage cultures, as determined in a fibroblast cytolysis assay was found to be effectively blocked by inhibitors of lipoxygenases. Likewise, the presence of tumor necrosis factor in serum of D-galactosamine sensitized mice after challenge with endotoxin was suppressed by the lipoxygenase inhibitors. From LPS-treated macrophages 13-hydroxylinoleic acid, a lipoxygenase product was isolated, which is significantly increased after LPS treatment of the cells, covalently bound to cellular constituents and was found to counterbalance the suppression of TNF-synthesis by a lipoxygenase inhibitor when added to macrophages exogenously.

Lipoxygenase inhibitors suppress formation of tumor necrosis factor in vitro and in vivo.

Injection of endotoxin in D-galactosamine sensitized mice resulted in increased serum levels of tumor necrosis factor as determined in a fibroblast cytolysis assay. Concomitant injection of different lipoxygenase inhibitors decreased the titer of TNF. When the lipoxygenase inhibitors were tested in macrophage cultures stimulated with LPS, they prevented the production of TNF. Indomethacin, a blocker of cyclooxygenase was neither in vivo nor in vitro effective in the prevention of the endotoxin-induced synthesis of TNF. The involvement of superoxide anion in this effect was excluded by use of superoxide dismutase which did not influence the formation of TNF in macrophages.

Lipoxygenase inhibitors suppress formation of tumor necrosis factor in vitro and in vivo.

The LPS induced synthesis of tumor necrosis factor in macrophage cultures, as determined in a fibroblast cytolysis assay was found to be effectively blocked by inhibitors of lipoxygenases. Likewise, the presence of tumor necrosis factor in serum of D-galactosamine sensitized mice after challenge with endotoxin was suppressed by the lipoxygenase inhibitors. Indomethacin, a blocker of cycclooxygenase was neither in vivo nor in vitro effective in the prevention of the endotoxin-induced synthesis of TNF. From LPS-treated macrophages we were able to isolate 13-hydroxylinoleic acid, a lipoxygenase product, which is significantly increased after LPS treatment of the cells, covalently bound to cellular constituents and may, therefore, be possibly involved in the formation of TNF.

Oxpentifylline in endotoxaemia.

Oxpentifylline (pentoxifylline), which is known to have pharmacological effects in animal models of respiratory distress syndrome, multiorgan failure, and shock, was tested in human beings after injection of endotoxin. Of ten healthy volunteers, nine met the inclusion criterion of a rise in body temperature of at least 1.0 degrees C after 100 ng endotoxin (Salmonella abortus equi) as a bolus injection. Serum levels of tumour necrosis factor alpha (TNF) and interleukin-6 (IL-6) were both significantly higher than baseline levels 2 h and 3 h after endotoxin injection. 3 weeks later the nine volunteers were again injected with 100 ng endotoxin and oxpentifylline (500 mg over 4 h) was also infused. There was no rise in TNF levels, though IL-6 levels rose in parallel with body temperature. These data suggest that oxpentifylline blocks the endotoxin-induced synthesis of TNF in man and, therefore, could possibly have beneficial effects in clinical endotoxaemia.

Mononuclear phagocytes and eicosanoids: aspects of their synthesis and biological activities.

Mononuclear phagocytes convert arachidonic acid and other unsaturated fatty acids from intracellular sources to a variety of oxygenated metabolites such as prostaglandins and leukotrienes which are secreted into the surrounding medium. Other oxidative products such as hydroxylinoleic acids are reacylated into cellular constituents. The underlying metabolic pathways are activated by numerous stimuli of exogenous or endogenous origin. Depending on the state of activation and cell differentiation, the organ of origin and the nature of the stimulus used, macrophages elaborate a distinct spectrum of oxidative arachidonic acid metabolites. The contribution of these metabolites to the proinflammatory properties of macrophages is twofold: As autocrine signals they modulate the synthesis of diverse macrophage products and they influence cellular functions of other cells such as T-lymphocytes.

Effects of pentoxifylline in endotoxinemia in human volunteers.

The following effects of pentoxifylline in endotoxinemia in man could be demonstrated: 1. Il-6 release and Il-6 mediated effects such as clinical responses and leukocytosis are not inhibited by POF treatment. 2. TNF-alpha release is suppressed which might be the most important result, because TNF seems to play the major role in the pathophysiological events of endotoxinemia. Furthermore, pentoxifylline is able to counteract the initial leukocytopenia caused by sticking of leukocytes in the microcirculation. 3. De-novo-synthesis of cytokines by monocytes ex vivo is totally abolished. This may possibly reflect the in vivo situation. In conclusion, beneficial effects of pentoxifylline in respiratory distress syndrome, multi-organ failure and septic shock are suggested. Furthermore, our data indicate that endotoxin-induced formation of Il-6 and TNF-alpha are regulated independently in vivo.