Albrecht Wendel

Tumor necrosis factor is a terminal mediator in galactosamine/endotoxin-induced hepatitis in mice

Intravenous injection of murine recombinant tumor necrosis factor alpha(TNF-alpha) to male NMRI albino mice in doses greater than 4 micrograms/kg (specific activity 4 x 10(7) U/mg) resulted in a fulminant hepatitis when animals had been sensitized 1 hr before by intraperitoneal administration of 700 mg/kg galactosamine. Liver injury was assessed by measurement of serum transaminases as well as sorbitol dehydrogenase activity 8 hr after administration of TNF-alpha. Pretreatment with either galactosamine or 40 micrograms/kg TNF-alpha alone did not cause hepatitis. Pretreatment of galactosamine/TNF-alpha-injured mice with 800 mg/kg uridine or with 6 mg/kg calmidazolium fully protected the animals, while administration of either verapamil or nifedipine (100 mg/kg, respectively) had no significant effect. The following inhibitors of generation or action of leukotriene D4, which were previously shown to block galactosamine/endotoxin-induced hepatitis in mice, failed to protect against galactosamine/TNF-alpha-induced intoxication: 200 micrograms/kg dexamethasone, 174 mg/kg BW 755 C or 13 x 10 mg/kg FPL 55712. In addition, unlike in the galactosamine/endotoxin model no prevention was achieved by pretreatment of galactosamine/TNF-alpha-injured animals with the following substances blocking the development of an ischemia/reperfusion syndrome: 2 x 100 mg/kg allopurinol, 3.3 x 10(4) U/kg superoxide dismutase, 10(6) U/kg catalase or 10 micrograms/kg iloprost. We conclude from our results that tumor necrosis factor alpha is likely to act as a final mediator of endotoxin action in a sequence of events which includes formation of leukotriene D4 and reactive oxygen species.

In vitro differentiation of human monocytes to macrophages: change of PDE profile and its relationship to suppression of tumour necrosis factor‐α release by PDE inhibitors

During in vitro culture in 10% human AB serum, human peripheral blood monocytes acquire a macrophage‐like phenotype. The underlying differentiation was characterized by increased activities of the macrophage marker enzymes unspecific esterase (NaF‐insensitive form) and acid phosphatase, as well as by a down‐regulation in surface CD14 expression. In parallel, a dramatic change in the phosphodiesterase (PDE) profile became evident within a few days that strongly resembled that previously described for human alveolar macrophages. Whereas PDE1 and PDE3 activities were augmented, PDE4 activity, which represented the major cyclic AMP‐hydrolysing activity of peripheral blood monocytes, rapidly declined. Monocytes and monocyte‐derived macrophages responded to lipopolysaccharide (LPS) with the release of tumour necrosis factor‐α (TNF). In line with the change in CD14 expression, the EC50 value of LPS for induction of TNF release increased from approximately 0.1 ng ml−1 in peripheral blood monocytes to about 2 ng ml−1 in macrophages. Both populations of cells were equally susceptible towards inhibition of TNF release by cyclic AMP elevating agents such as dibutyryl cyclic AMP, prostaglandin E2 (PGE2) or forskolin, which all led to a complete abrogation of TNF production in a concentration‐dependent manner and which were more efficient than the glucocorticoid dexamethasone. In monocytes, PDE4 selective inhibitors (rolipram, RP73401) suppressed TNF formation by 80%, whereas motapizone, a PDE3 selective compound, exerted a comparatively weak effect (10–15% inhibition). Combined use of PDE3 plus PDE4 inhibitors resulted in an additive effect and fully abrogated LPS‐induced TNF release as did the mixed PDE3/4 inhibitor tolafentrine. In monocyte‐derived macrophages, neither PDE3‐ nor PDE4‐selective drugs markedly affected TNF generation when used alone (<15% inhibition), whereas in combination, they led to a maximal inhibition of TNF formation by about 40–50%. However, in the presence of PGE2 (10 nM), motapizone and rolipram or RP73401 were equally effective and blocked TNF release by 40%. Tolafentrine or motapizone in the presence of either PDE4 inhibitor, completely abrogated TNF formation in the presence of PGE2. Thus, an additional cyclic AMP trigger is necessary for PDE inhibitors to become effective in macrophages. Finally, the putative regulatory role for PDE1 in the regulation of TNF production in macrophages was investigated. Zaprinast, at a concentration showing 80% inhibition of PDE1 activity (100 μmol l−1), did not influence TNF release. At higher concentrations (1 mmol l−1), zaprinast became effective, but this inhibition of TNF release can be attributed to a significant inhibitory action of this drug on PDE3 and PDE4 isoenzymes. In summary, the in vitro differentiation of human peripheral blood monocytes to macrophages is characterized by a profound change in the PDE isoenzyme pattern. The change in the PDE4 to PDE3 ratio is functionally reflected by an altered susceptibility towards selective PDE inhibitors under appropriate stimulating conditions.

ICE-PROTEASE INHIBITORS BLOCK MURINE LIVER INJURY AND APOPTOSIS CAUSED BY CD95 OR BY TNF-ALPHA

The two apoptosis receptors of mammalian cells, i.e. the 55 kDa TNF receptor (TNF-R1) and CD95 (Fas/APO1) are activated independently of each other, however, their signaling involves a variety of ICE-related proteases [I]. We used a cell-permeable inhibitor of ICE-like protease activity to examine in vivo whether post-receptor signaling of TNF and CD95 are fully independent processes. Mice pretreated with the inhibitor, Z-VAD-fluoromethylketone (FMK) were dose-dependently protected from liver injury caused by CD95 activation as determined by plasma alanine aminotransferase and also from hepatocyte apoptosis assessed by DNA fragmentation (ID50 = 0.1 mg/kg). A dose of 10 mg/kg protected mice also from liver injury induced by TNF-alpha. Similar results were found when apoptosis was initiated via TNF-alpha or via CD95 in primary murine hepatocytes (IC50 = 1.5 nM) or in various human cell lines. In addition to prevention, an arrest of cell death by Z-VAD-FMK was demonstrated in vivo and in vitro after stimulation of apoptosis receptors. These findings show in vitro and in vivo in mammals that CD95 and the TNF-alpha receptor share a distal proteolytic apoptosis signal.

Leukotriene-mediated liver injury

The pathogenic mechanism of fulminant hepatitis induced by 700 mg/kg D-galactosamine plus 33 micrograms/kg endotoxin was investigated in male NMRI mice. The extent of liver injury was assessed by measurement of serum transaminases and sorbitol dehydrogenase activities 9 hr after intoxication, as well as by histopathological evaluation. When the hepatic glutathione content of galactosamine endotoxin-treated animals had been decreased by more than 90% following administration of 250 mg/kg phorone or 400 mg/kg diethyl maleate given three times, no signs of liver injury were observed. Since different agents interfering with the leukotriene synthesis pathway also prevented galactosamine/endotoxin-induced hepatitis, we suspected that a glutathione-derived peptidoleukotriene may be the pathogenic metabolite. In vivo inhibition of the catabolism of leukotriene C4 by administration of 50 mg/kg of the glutamyl transpeptidase inhibitor AT 125 (Acivicin) also protected the animals against liver injury. In order to elucidate which metabolite of leukotriene C4 was responsible for the observed hepatotoxicity we intravenously injected leukotrienes into animals that had received only galactosamine. Injection of 50 micrograms/kg leukotriene E4 1 hr after galactosamine had no effect. The same dose of leukotriene D4 led to a fulminant hepatitis which was prevented when the leukotriene D4 antagonist FPL 55712 had been given before. In contrast, lipoxygenase inhibitors or AT 125 did not protect against galactosamine + LTD4. Galactosamine/endotoxin-induced and galactosamine/leukotriene D4-induced hepatitis resulted in similarly localized histopathological changes, i.e. diffuse necrosis in the organ. We conclude from our results that galactosamine/endotoxin-induced hepatitis is mediated by a leukotriene D4-dependent mechanism.

Improved Innate Immunity of Endotoxin-Tolerant Mice Increases Resistance to Salmonella enterica Serovar Typhimurium Infection despite Attenuated Cytokine Response

ABSTRACT During infection with gram-negative bacteria, exposure of immune cells to lipopolysaccharide (LPS) from the bacterial cell membrane induces a rapid cytokine response which is essential for the activation of host defenses against the invading pathogens. Administration of LPS to mice induces a state of hyporesponsiveness, or tolerance, characterized by reduced cytokine production upon subsequent LPS challenge. In the model of experimental Salmonella entericaserovar Typhimurium infection of mice, we assessed the question of whether complete LPS tolerance induced by repetitive doses of LPS interfered with cytokine production and host defense against gram-negative bacteria. Although production of various cytokines in response to serovar Typhimurium was attenuated by LPS pretreatment, LPS-tolerant mice showed improved antibacterial activity, evidenced by a prolongation of survival and a continuously lower bacterial load. We attribute this protective effect to three independent mechanisms. (i) Peritoneal accumulation of leukocytes in the course of LPS pretreatment accounted for enhanced defense against serovar Typhimurium during the first 6 h of infection but not for decreased bacterial load in late-stage infection. (ii) LPS-tolerant mice had an increased capacity to recruit neutrophilic granulocytes during infection. (iii) LPS-tolerant mice showed threefold-increased Kupffer cell numbers, enhanced phagocytic activity of the liver, and strongly improved clearance of blood-borne serovar Typhimurium. These results demonstrate that despite attenuated cytokine response, acquired LPS tolerance is associated with enhanced resistance to infections by gram-negative bacteria and that this effect is mainly mediated by improved effector functions of the innate immune system.

Peripheral infusion of rat bone marrow derived endothelial progenitor cells leads to homing in acute lung injury

BackgroundBone marrow-derived progenitors for both epithelial and endothelial cells have been observed in the lung. Besides mature endothelial cells (EC) that compose the adult vasculature, endothelial progenitor cells (EPC) are supposed to be released from the bone marrow into the peripheral blood after stimulation by distinct inflammatory injuries. Homing of ex vivo generated bone marrow-derived EPC into the injured lung has not been investigated so far. We therefore tested the hypothesis whether homing of EPC in damaged lung tissue occurs after intravenous administration.MethodsEx vivo generated, characterized and cultivated rat bone marrow-derived EPC were investigated for proliferation and vasculogenic properties in vitro. EPC were tested for their homing in a left-sided rat lung transplant model mimicking a severe acute lung injury. EPC were transplanted into the host animal by peripheral administration into the femoral vein (106 cells). Rats were sacrificed 1, 4 or 9 days after lung transplantation and homing of EPC was evaluated by fluorescence microscopy. EPC were tested further for their involvement in vasculogenesis processes occurring in subcutaneously applied Matrigel in transplanted animals.ResultsWe demonstrate the integration of intravenously injected EPC into the tissue of the transplanted left lung suffering from acute lung injury. EPC were localized in vessel walls as well as in destructed lung tissue. Virtually no cells were found in the right lung or in other organs. However, few EPC were found in subcutaneous Matrigel in transplanted rats.ConclusionTransplanted EPC may play an important role in reestablishing the endothelial integrity in vessels after severe injury or at inflamatory sites and might further contribute to vascular repair or wound healing processes in severely damaged tissue. Therapeutic applications of EPC transplantation may ensue.

Cyclic nucleotide phosphodiesterase isoenzyme activities in human alveolar macrophages

Background Alveolar macrophages and their precursors, the monocytes are involved in airway inflammation in asthma. An increase in intraceliular cAMP by PDE inhibitors is known to suppress macrophage and monocyte functions. A comparison of the PDE‐isoenzyine profiles of human alveolar macrophages from normal and atopic donors and of human peripheral blood monocytes might form a basis to differentially affect functions of these cells by PDE inhibitors.

Strong inhibition of mammalian lipoxygenases by the antiinflammatory seleno-organic compound ebselen in the absence of glutathione

Both human recombinant 5-lipoxygenase (EC 1.13.11.34) and 15-lipoxygenase (EC 1.13.11.33, mammalian enzyme) purified from rabbit reticulocytes were inhibited in the absence of glutathione (GSH) by submicromolar concentrations of the seleno-organic compound ebselen. These concentrations were comparable to those of the enzymes. Soybean lipoxygenase-1 (EC 1.13.11.33, plant enzyme) was not inhibited, whereas prostaglandin endoperoxide synthase-1 (EC 1.14.99.1) was inhibited only at much higher concentrations of ebselen (IC50 = 37.7 +/- 4.3 microM). The action of ebselen on reticulocyte 15-lipoxygenase (IC50 = 0.17 +/- 0.01 microM) was studied in detail. Inhibition occurred instantaneously and appeared to be reversible and was largely abolished by a 20-fold molar excess of GSH over ebselen. In the presence of 1 mM GSH 50% inhibition was observed only at ebselen concentrations as high as 234 +/- 27 microM. 13S-hydroperoxy-9Z, 11E-octadecadienoic acid, the lipoxygenase product formed from linoleic acid, augmented the inhibitory effect at low concentrations and caused a partial reversal at high concentrations. A variety of derivatives or structural analogues of ebselen were also tested and proved to be either inactive or weaker inhibitors of 15-lipoxygenase. We have concluded that the potent inhibition of 15-lipoxygenase by ebselen is due neither to GSH peroxidase-like activity nor to lowering of the hydroperoxide tone. The pharmacological implications of these unique characteristics of the action of ebselen on lipoxygenases are then discussed.

Comparison of the skin sensitizing potential of unsaturated compounds as assessed by the murine local lymph node assay (LLNA) and the guinea pig maximization test (GPMT)

The skin sensitization potential of eight unsaturated and one saturated lipid (bio)chemicals was tested in both the LLNA and the GPMT to address the hypothesis that chemicals with unsaturated carbon-carbon double bonds may result in a higher number of unspecific (false positive) results in the LLNA compared to the GPMT. Seven substances (oleic acid, linoleic acid, linolenic acid, undecylenic acid, maleic acid, squalene and octinol) gave clear positive results in the LLNA (stimulation index (SI)> or = 3) and thus would require labelling as skin sensitizer. Fumaric acid and succinic acid gave clearly negative results. In the GPMT, besides some sporadic skin reactions, reproducible skin reactions indicating an allergic response were found in a few animals for four test substances. Based on the GPMT results, only undecylenic acid would have to be classified and labelled as a skin sensitizer according to the European Dangerous Substance Directive (67/548/EEC) (results for linoleic acid were inconclusive), while the other seven test substances would not require labelling. Possible mechanisms for unspecific skin cell stimulation and lymph node responses are discussed. In conclusion, the suitability of the LLNA for unsaturated compounds bearing structural similarity to the tested substances should be carefully considered and the GPMT should remain available as an accepted test method for skin sensitization hazard identification.

Phosphodiesterase profile of human B lymphocytes from normal and atopic donors and the effects of PDE inhibition on B cell proliferation

1 CD19+ B lymphocytes were purified from the peripheral blood of normal and atopic subjects to analyse and compare the phosphodiesterase (PDE) activity profile, PDE mRNA expression and the importance of PDE activity for the regulation of B cell function. 2 The majority of cyclic AMP hydrolyzing activity of human B cells was cytosolic PDE4, followed by cytosolic PDE7‐like activity; marginal PDE3 activity was found only in the particulate B cell fraction. PDE1, PDE2 and PDE5 activities were not detected. 3 By cDNA‐PCR analysis mRNA of the PDE4 subtypes A, B (splice variant PDE4B2) and D were detected. In addition, a weak signal for PDE3A was found. 4 No differences in PDE activities or mRNA expression of PDE subtypes were found in B cells from either normal or atopic subjects. 5 Stimulation of B lymphocytes with the polyclonal stimulus lipopolysaccharide (LPS) induced a proliferative response in a time‐ and concentration‐dependent manner, which was increased in the presence of interleukin‐4 (IL‐4). PDE4 inhibitors (rolipram, piclamilast) led to an increase in the cellular cyclic AMP concentration and to an augmentation of proliferation, whereas a PDE3 inhibitor (motapizone) was ineffective, which is in accordance with the PDE profile found. The proliferation enhancing effect of the PDE4 inhibitors was partly mimicked by the cyclic AMP analogues dibutyryl (db) cyclic AMP and 5,6‐dichloro‐1‐β‐D‐ribofuranosylbenzimidazole‐3′,5′‐cyclic monophosphorothioate, Sp‐isomer (dcl‐cBIMPS), respectively. However, at concentrations exceeding 100 μM db‐cyclic AMP suppressed B lymphocyte proliferation, probably as a result of cytotoxicity. Prostaglandin E2 (PGE2, 1 μM) and forskolin (10 μM) did not affect B cell proliferation, even when given in combination with rolipram. 6 Inhibition of protein kinase A (PKA) by differentially acting selective inhibitors (KT 5720, Rp‐8‐Br‐cyclic AMPS) decreased the proliferative response of control cells and reversed the proliferation enhancing effects of rolipram. 7 Importantly, PDE4 activity in LPS/IL‐4‐activated B lymphocytes decreased by about 50% compared to unstimulated control values. 8 We conclude that an increase in cyclic AMP, mediated by down‐regulation of PDE4 activity, is involved in the stimulation of B cell proliferation in response to LPS/IL‐4. B cell proliferation in response to a mitogenic stimulus can be further enhanced by pharmacological elevation of cyclic AMP.