Platelet-activating factor (PAF) receptor-mediated calcium mobilization and phosphoinositide turnover in neurohybrid NG108-15 cells: studies with BN50739, a new PAF antagonist.

Platelet-activating factor (PAF) is an unusually potent lipid autacoid with a variety of biological activities. The growing body of evidence suggests that PAF might play an important role in modulation of central nervous system function, particularly during ischemia- and trauma-induced neuronal damage. However, the mechanisms involved in PAF actions on neuronal or other brain cells is virtually unknown. Therefore, this study was designed to characterize PAF receptor-mediated cellular signal transduction in neurohybrid NG108-15 cells with the aid of a new potent PAF antagonist, BN 50739. PAF induced an immediate and concentration-dependent increase in [Ca++]i with an EC50 of 6.8 nM. PAF-induced [Ca++]i mobilization was inhibited by several structurally unrelated PAF antagonists such as BN 50739, WEB 2086, SRI 63-441 and BN 52021, in a dose-dependent manner with IC50 values of 4.8, 6.9, 809 and 98500 nM, respectively. The calcium channel blockers nifedipine (5 microM) and diltiazem (10 microM) had no effect on the PAF-induced increase in [Ca++]i, but omission of CA++ from the incubation buffer caused an 82% reduction of PAF-induced [Ca++]i elevation; the remainder contributed from intracellular sources was completely inhibited by 10 microM TMB-8, an intracellular Ca++ blocker. NG108-15 cells exhibited homologous desensitization to sequential addition of PAF, but no heterologous desensitization between PAF and other agonists such as bradykinin, endothelin, angiotensin II and ATP was observed. PAF stimulated phosphoinositide metabolism in a dose-dependent manner with an EC50 of 5.1 nM for IP3 formation, which was also inhibited by the PAF antagonist BN 50739 in a dose-dependent manner (IC50 = 3.6 nM).(ABSTRACT TRUNCATED AT 250 WORDS)     

Protective effect of BN 50739, a new platelet-activating factor antagonist, in endotoxin-treated rabbits

Platelet-activating factor (PAF) has been demonstrated in the circulation and organs of animals exposed to gram negative endotoxins, whereas PAF antagonists have been shown to exhibit some efficacy in modifying the course of endotoxemia. In this study we evaluated BN 50739, a novel specific PAF antagonist, for its capacity to block PAF or lipopolysaccharide endotoxin (LPS)-mediated effects in rabbits. Pretreatment with BN 50739 (3 and 10 mg/kg i.p.) inhibited PAF (500 pmol/kg i.v.)-induced thrombocytopenia, leukopenia and plasma thromboxane B2 elevation in a dose-dependent manner. The inhibitory effect lasted 3.5 to 4.5 hr. BN 50739 (10 mg/kg) prevented the early phase of LPS (50 micrograms/kg i.v.)-induced thrombocytopenia and thromboxane B2 elevation, and reduced the 24-hr mortality rate from 75 to 22% (P less than .05). Post-treatment with BN 50739 increased the 10-hr survival rate from 33 to 87% (P less than .05); however, it had no effect on the 24-hr mortality. BN 50739 did not affect LPS-induced leukopenia or the elevation in plasma tumor necrosis factor. Our data support possible therapeutic efficacy of PAF antagonists in septic shock despite their inability to prevent the generation of tumor necrosis factor.     

Tumor necrosis factor (TNF)-soluble high-affinity receptor complex as a TNF antagonist

A novel high-affinity inhibitor of tumor necrosis factor (TNF) is described, which is created by the fusion of the extracellular domains of TNF-binding protein 1 (TBP-1) to both the alpha and beta chains of an inactive version of the heterodimeric protein hormone, human chorionic gonadotropin. The resulting molecule, termed TNF-soluble high-affinity receptor complex (SHARC), self-assembles into a heterodimeric protein containing two functional TBP-1 moieties. The TNF-SHARC is a potent inhibitor of TNF-alpha bioactivity in vitro and has a prolonged pharmacokinetic profile compared with monomeric TBP-1 in vivo. Consistent with the long half-life, the duration of action in an lipopolysaccharide-mediated proinflammatory mouse model is prolonged similarly. In a collagen-induced arthritis mouse model, this molecule demonstrates improved efficacy over monomeric TBP-1. Based on these results, we demonstrated that inactivated heterodimeric protein hormones are flexible and efficient scaffolds for the creation of soluble high-affinity receptor complexes.     

A tumor necrosis factor (TNF) receptor-IgG heavy chain chimeric protein as a bivalent antagonist of TNF activity

Using a multistep polymerase chain reaction method, we have produced a construct in which a cDNA sequence encoding the extracellular domain of the human 55-kD tumor necrosis factor (TNF) receptor is attached to a sequence encoding the Fc portion and hinge region of a mouse IgG1 heavy chain through an oligomer encoding a thrombin-sensitive peptide linker. This construct was placed downstream from a cytomegalovirus promoter sequence, and expressed in Chinese hamster ovary cells. A secreted protein, capable of binding TNF and inactivating it, was produced by the transfected cells. Molecular characterization revealed that this soluble version of the TNF receptor was dimeric. Moreover, the protein could be quantitatively cleaved by treatment with thrombin. However, the monovalent extracellular domain prepared in this way has a greatly reduced TNF inhibitory activity compared with that of the bivalent inhibitor. Perhaps because of its high affinity for TNF, the chimeric protein is far more effective as a TNF inhibitor than are neutralizing monoclonal antibodies. This molecule may prove very useful as a reagent for the antagonism and assay of TNF and lymphotoxin from diverse species in health and disease, and as a means of deciphering the exact mechanism through which TNF interacts with the 55-kD receptor.     

Functional switching of macrophage responses to tumor necrosis factor-alpha (TNF alpha) by interferons. Implications for the pleiotropic activities of TNF alpha.

Recent work conducted in our laboratory has been directed towards understanding the role of TNF alpha in stimulating the synthesis of two macrophage gene products, namely IGF-1, a growth factor implicated in wound repair and fibrosis, and complement component factor B (Bf), an alternative pathway complement component. The expression of these proteins is induced by hyaluronic acid and poly (I:C), respectively, although TNF alpha plays a requisite role in the expression of both proteins. The objective of this study was to determine the mechanism governing the dichotomy in the expression of IGF-1 and Bf by TNF alpha. First, we questioned if the diversity in IGF-1 and Bf synthesis was regulated at the level of TNF receptor usage. Second, based on earlier findings that IFNs contribute to the initiation of Bf expression, we determined if IFNs modulate the response of macrophages to TNF alpha. Our data show that differences in TNF receptor usage cannot fully explain the dichotomy in the expression of IGF-1 and Bf. However, prior exposure to IFN-beta or IFN-gamma was found to be a dominant factor controlling the expression of these proteins, suppressing IGF-1, and enhancing Bf. These findings indicate that IFNs mediate a functional "switch" in the response of macrophages to TNF alpha and suggest that the pattern of cytokine expression by diverse macrophage stimuli is an important determinant of the eventual responses of macrophages to TNF alpha.     

Dual role of the p75 tumor necrosis factor (TNF) receptor in TNF cytotoxicity

Whereas there is ample evidence for involvement of the p55 tumor necrosis factor (TNF) receptor (p55-R) in the cytocidal effect of TNF, the role of the p75 TNF receptor (p75-R) in this effect is a matter of debate. In this study, we probed the function of p75-R in cells sensitive to the cytotoxicity of TNF using a wide panel of antibodies (Abs) against the receptor's extracellular domain. Two distinct Ab effects were observed. The Abs triggered signaling for cytotoxicity. This effect: (a) was correlated with the extent of p75-R expression by the cells; (b) was dependent on receptor cross-linking by the Abs; (c) occurred in HeLa cells, but not in A9 cells transfected with human p75- R or in HeLa cells expressing cytoplasmically truncated p75-R mutants, indicating that it involves cell-specific activities of the intracellular domain of the receptor; (d) was synergistic with the cytocidal effect of Abs against p55-R. Moreover, it seemed to reverse induced desensitization to the cytocidal effect of anti p55-R Abs, suggesting that it involves mechanisms different from those of the signaling by the p55 TNF-R. In addition, the Abs affected the response to TNF in a way that does not involve the signaling activity of p75-R. These effects: (a) could be observed also in cells in which only p55-R signaled for the cytocidal effect; (b) were not dependent on receptor cross-linking by the Abs; (c) varied according to the site at which the Abs bound to the receptor; and (d) were correlated inversely with the effects of the Abs on TNF binding to p75-R. That is, Abs binding to the membrane-distal part of the receptor's extracellular domain displaced TNF from the p75 receptor and enhanced cytocidal effect, whereas Abs that bind to the membrane-proximal part of the extracellular domain--a region at which a conformational change seems to take place upon TNF binding--decreased the dissociation of TNF from p75-R and inhibited its cytocidal effect. The above findings suggest that p75-R contributes to the cytocidal effect of TNF both by its own signaling and by regulating the access of TNF to p55-R.     

Identification of Grb2 as a novel binding partner of tumor necrosis factor (TNF) receptor I.

Tumor necrosis factor alpha (TNF-alpha) is a proinflammatory cytokine. Its pleiotropic biological properties are signaled through two distinct cell surface receptors: the TNF receptor type I (TNFR-I) and the TNF receptor type II. Neither of the two receptors possesses tyrosine kinase activity. A large majority of TNF-alpha-dependent activities can be mediated by TNFR-I. Recently, c-Raf-1 kinase was identified as an intracellular target of a signal transduction cascade initiated by binding of TNF-alpha to TNFR-I. However, the mechanism engaged in TNF-alpha-dependent activation of c-Raf-1 kinase is still enigmatic. Here we report that the cytosolic adapter protein Grb2 is a novel binding partner of TNFR-I. Grb2 binds with its COOH-terminal SH3 domain to a PLAP motif within TNFR-I and with its NH2-terminal SH3 domain to SOS (son of sevenless). A PLAP deletion mutant of TNFR-I fails to bind Grb2. The TNFR-I/Grb2 interaction is essential for the TNF-alpha-dependent activation of c-Raf-1 kinase; activation of c-Raf-1 kinase by TNF-alpha can be blocked by coexpression of Grb2 mutants harboring inactivating point mutations in the NH2- or COOH-terminal SH3 domain, cell-permeable peptides that disrupt the Grb2/TNFR-I interaction or transdominant negative Ras. Functionality of the TNFR-I/Grb2/SOS/Ras interaction is a prerequisite but not sufficient for TNF-alpha-dependent activation of c-Raf-1 kinase. Inhibition of the TNFR-I/FAN (factor associated with neutral sphingomyelinase) interaction, which is essential for TNF-alpha-dependent activation of the neutral sphingomyelinase, either by cell-permeable peptides or by deletion of the FAN binding domain, prevents activation of c-Raf-1 kinase. In conclusion, binding of the Grb2 adapter protein via its COOH-terminal SH3 domain to the nontyrosine kinase receptor TNFR-I results in activation of a signaling cascade known so far to be initiated, in the case of the tyrosine kinase receptors, by binding of the SH2 domain of Grb2 to phosphotyrosine.     

急性白血病骨髓基质细胞肿瘤坏死因子α分泌活性及其意义

目的：探讨急性白血病（ＡＬ）骨髓基质细胞肿瘤坏死因子α（ＴＮＦα）分泌活性。方法：应用体外骨髓基质细胞培养法及ＴＮＦα生物活性检测法，对ＡＬ患者２０例及正常对照者１０名的骨髓基质细胞培养上清中的ＴＮＦα活性水平进行检测，并同步检测了患者血清、骨髓白血病细胞培养上清中的ＴＮＦα活性。结果：ＡＬ骨髓基质细胞形成能力较差，但其ＴＮＦα活性明显高于对照组（Ｐ＜０．０５）；少部分急性髓系白血病（ＡＭＬ）骨髓白血病细胞能自发分泌ＴＮＦα；患者血清中的ＴＮＦα活性显著高于正常对照（Ｐ＜０．００１）。结论：ＡＬ患者骨髓基质细胞存在量和质的异常，ＴＮＦα的异常分泌可能是导致ＡＬ增殖的一个重要因素     

Implication of Toll-like receptor and tumor necrosis factor alpha signaling in septic shock

Septic shock is initiated by a systemic inflammatory response to microbial infection that frequently leads to impaired perfusion and multiple organ failure. Because of its high risk of death, septic shock is a major problem particularly for patients in the intensive care unit. In general, bacterial lipopolysaccharide (LPS) is a strong activator of various immune responses and stimulates monocytes/macrophages to release a variety of inflammatory cytokines. However, overproduction of inflammatory factors in response to bacterial infections is known to cause septic shock, similar to that induced by LPS. Studies of LPS-signaling pathways and downstream inflammatory cytokines may have critical implications in the treatment of sepsis. In recent years, there has been significant progress in understanding the signaling pathways activated by LPS and its receptor Toll-like receptor 4 (TLR4), as well as by tumor necrosis factor alpha (TNFalpha), a potent inflammatory cytokine induced by LPS stimulation. This review briefly summarizes our current knowledge of these signaling pathways and critical signal transducers. Characterization of key signal transducers may allow us to identify tractable, novel targets for the therapeutic interventions of sepsis.     

A human tumor necrosis factor (TNF) alpha mutant that binds exclusively to the p55 TNF receptor produces toxicity in the baboon

A number of recent studies have demonstrated that cellular responses to tumor necrosis factor (TNF) mediated by the p55 and the p75 TNF receptors are distinct. To evaluate the relative in vivo toxicities of wild-type TNF alpha (wtTNF alpha) and a novel p55 TNF selective receptor agonist, healthy, anesthetized baboons (Papio sp.) were infused with a near-lethal dose of either wtTNF alpha or a TNF alpha double mutant (dmTNF alpha) that binds specifically to the p55, but not to the p75, TNF receptor. Both wtTNF alpha and dmTNF alpha produced comparable acute hypotension, tachycardia, increased plasma lactate, and organ dysfunction in Papio. However, administration of wtTNF alpha produced a marked granulocytosis and loss of granulocyte TNF receptors, whereas little if any changes in neutrophil number or cell surface TNF receptor density were seen after dmTNF alpha mutant administration. Infusion of dmTNF alpha resulted in a plasma endogenous TNF alpha response that peaked after 90-120 min. We conclude that selective p55 TNF receptor activation is associated with early hemodynamic changes and the autocrine release of endogenous TNF alpha. Significant systemic toxicity results from p55 TNF receptor activation, but the role of the p75 TNF receptor in systemic TNF toxicity requires further study.     

Platelet response to the aggregatory effect of platelet activating factor (PAF) ex vivo in patients with acute myocardial infarction

Platelets from patients with acute myocardial infarction exhibit an increased sensitivity to the aggregatory effect of PAF, in vitro, the first 48 h after the onset of the symptoms. This sensitivity, expressed as PAF EC50 values, seems to be transient after the 2 day period. Also, a remarkable decreased sensitivity to the inhibitory effect of PGI2 against the aggregation induced by PAF appears to the platelets of those patients the first hours after the onset of the symptoms, and persists for at least 14 days. Treatment of patients by drugs with a known inhibitory effect on platelet aggregation in vivo and in vitro (aspirin, nifedipine, indomethacin), does not influence the increase in platelet sensitivity to PAF, but inhibits the secondary aggregation induced by the released aggregating factors from the PAF activated platelets. The increase in platelet sensitivity to PAF is not unique to the AMI since it is also observed in patients with acute bacterial pneumonia. However, we cannot support the theory that it is a general phenomenon of acute tissue injury since it is general phenomenon of acute tissue injury since it is not observed in patients with acute muscular injury.     

Increased tumor necrosis factor alpha (TNF alpha), interleukin 1, and interleukin 6 (IL-6) levels in the plasma of stored platelet concentrates: relationship between TNF alpha and IL-6 levels and febrile transfusion reactions

Increased interleukin 6 (IL-6) levels were found in 8 of 12 platelet concentrates (PCs) after 3 days of storage and in 10 of 12 PCs after 5 and 7 days of storage. Most of the PCs with an increased IL-6 level also showed increased tumor necrosis factor alpha (TNF alpha) and interleukin 1 beta (IL-1 beta) levels. Levels of IL-6 increased by 3 log10 over the base level during storage. Increased levels were found when the PC white cell count exceeded 3 × 10(9) per L. A linear correlation was found among the levels of TNF alpha, IL-1 beta, IL-1 alpha, and IL-6 in the PCs (r > 0.885). Comparison of the TNF alpha, IL- 1 beta, and IL-6 levels in samples taken at various storage times indicates that the increased levels are the result of an active synthesis and release of interleukins during storage. In a second part of the study, 45 transfusions of white cell-reduced PCs were studied. Six transfusions were complicated by a febrile reaction. These reactions were related to high levels of IL-6 and TNF alpha in the PCs (p < 0.0001). These cytokines are known as endogenous pyrogens. These findings indicate that transfusion reactions might be due to the intravenous administration of plasma with high cytokine levels and might not always result from an antigen-antibody reaction.     

Identification of a 60-kD tumor necrosis factor (TNF) receptor as the major signal transducing component in TNF responses

We describe here a monoclonal antibody (H398) that immunoprecipitates a human 60-kD tumor necrosis factor (TNF) membrane receptor (p60) and competes with TNF binding to p60 but not to p85 TNF receptors. Despite partial inhibition of TNF binding capacity of cells coexpressing both TNF receptor molecules, H398 uniformly and completely inhibits very distinct TNF responses on a variety of cell lines. These data suggest a limited structural heterogeneity in those components actually contributing to TNF responsiveness and identify p60 as a common receptor molecule essential for TNF signal transduction. As H398 is a highly effective TNF antagonist in vitro, it might be useful as a therapeutic agent in the treatment of TNF-mediated acute toxicity.     

Antidepressant drug-induced alterations in neuron-localized tumor necrosis factor-alpha mRNA and alpha(2)-adrenergic receptor sensitivity

The pleiotropic cytokine tumor necrosis factor-alpha (TNF) and alpha(2)-adrenergic receptor activation regulate norepinephrine (NE) release from neurons in the central nervous system. The present study substantiates the role of TNF as a neuromodulator and demonstrates a reciprocally permissive relationship between the biological effects of TNF and alpha(2)-adrenergic receptor activation as a mechanism of action of antidepressant drugs. Immunohistochemical analysis and in situ hybridization reveal that administration of the antidepressant drug desipramine decreases the accumulation of constitutively expressed TNF mRNA in neurons of the rat brain. Superfusion and electrical field stimulation were applied to a series of rat hippocampal brain slices to study the regulation of [(3)H]NE release. Superfusion of hippocampal slices obtained from rats chronically administered the antidepressant drug zimelidine demonstrates that TNF-mediated inhibition of [(3)H]NE release is transformed, such that [(3)H]NE release is potentiated in the presence of TNF, an effect that occurs in association with alpha(2)-adrenergic receptor activation. However, chronic zimelidine administration does not alter stimulation-evoked [(3)H]NE release, whereas chronic desipramine administration increases stimulation-evoked [(3)H]NE release and concomitantly decreases alpha(2)-adrenergic autoreceptor sensitivity. Collectively, these data support the hypothesis that chronic antidepressant drug administration alters alpha(2)-adrenergic receptor-dependent regulation of NE release. Additionally, these data demonstrate that administration of dissimilar antidepressant drugs similarly transform alpha(2)-adrenergic autoreceptors that are functionally associated with the neuromodulatory effects of TNF, suggesting a possible mechanism of action of antidepressant drugs.     

Inhibition of the platelet activating factor (PAF)-induced in vivo responses in rats by trans-2,5-(3,4,5-trimethoxyphenyl) tetrahydrofuran (L-652,731), a PAF receptor antagonist

Trans-2,5-bis-(3,4,5-trimethoxyphenyl)tetrahydrofuran (L652, 731), a potent and specific receptor antagonist of platelet activating factor (PAF) (Hwang et al., J. Biol. Chem. 260: 15639-15645, 1985), potently inhibits several PAF-induced in vivo responses in rats. Intravenously and p.o. administered L-652,731 gave a dose-response inhibition of PAF-induced lysosomal hydrolase secretion and extravasation with ED50 values of 1 and 3 mg/kg, respectively. Inhibitions of 87% were achieved with 50 mg/kg p.o. After a single 5-mg/kg p.o. dose, L-652,731 achieved 50 to 60% inhibition of PAF-induced lysosomal hydrolase secretion and extravasation by 0.5 to 1.5 hr with near maximum inhibition lasting through 6 hr. A 20-mg/kg p.o. dose of L-652,731 inhibited PAF-induced leukopenia and neutropenia by 96 and 73%, respectively. The most substantial inhibitions of the extravasation and lysosomal hydrolase secretion induced by PAF or soluble immune complexes were achieved by p.o. L-652,731 (20 mg/kg) with moderate inhibition by dexamethasone and little or no inhibition by antagonists/inhibitors of histamine H1 or H2 or serotonin receptors or cyclooxygenase. Intravenous infusion of a 0.4 mg of L-652,731 bolus inhibited the hypotensive responses from subsequent PAF infusions by a maximum of 72% and with a half-life duration of action of 2.5 hr. Intravenous infusion of L-652,731 results in an immediate 87% reversal of the extreme hypotension induced by a previous endotoxin injection. Thus, with its good p.o. activity, long duration of action and specificity in inhibiting PAF-induced responses in vivo, L-652,731 is a very useful tool in determining the role of PAF in mediating different pathophysiological processes.     

Serum neopterin, tumor necrosis factor-alpha and soluble tumor necrosis factor receptor II (p75) levels and disease activity in Egyptian female patients with systemic lupus erythematosus

OBJECTIVE: To determine the clinical value of assaying serum levels of neopterin, tumor necrosis factor-alpha (TNF-alpha) and soluble tumor necrosis factor receptor II (p75) (sTNFRII) in patients with systemic lupus erythematosus (SLE), manifested clinically with lupus nephritis (LN), neuropsychiatric lupus erythematosus (NPLE), and/or vasculitis compared with established parameters (complements C3 and C4). PATIENTS AND METHODS: Serum concentrations of neopterin, TNF-alpha and sTNFRII were studied in 40 female patients with SLE at various degrees of disease activity and in 10 healthy controls, matched for age and sex, using an ELISA kit. Disease activity was assessed by the SLE disease activity index (SLEDAI) score. Thirty-five, 30 and 28 of our patients presented with LN, NPLE and/or vasculitis, respectively, as the main clinical manifestation. RESULTS: Serum levels of neopterin, TNF-alpha and sTNFRII were significantly increased, while the TNF-alpha/sTNFRII ratio, C3 and C4 levels of SLE patients were significantly lower than those of healthy controls. Neopterin and sTNFRII were the only parameters that showed significantly higher levels in SLE patients with mild activity compared to normal subjects and were the only parameters that showed a significant elevation in membranous nephritis and in mild NPLE compared to patients without nephritis and NPLE. Patients with vasculitis had significant elevation of serum neopterin, TNF-alpha and sTNFRII levels compared to patients without vasculitis. We found significant correlations between all measured variables and the SLEDAI score. Also, serum neopterin levels showed significant positive correlation with serum TNF-alpha, sTNFRII and TNF-alpha/sTNFRII levels. Serum neopterin and sTNFRII could be used to identify SLE patients from normals with a sensitivity and specificity of 100%. Multivariate linear regression analysis showed that serum sTNFRII was the only significant independent variable among parameters for prediction of SLE disease activity. CONCLUSION: We suggest that serum sTNFRII and neopterin are more sensitive markers of disease activity than TNF-alpha, C3 or C4. However, sTNFRII may be a clinically useful independent marker for prediction of SLE disease activity and to differentiate normal subjects from those having mild SLE.     

Release of soluble receptors for tumor necrosis factor (TNF) in relation to circulating TNF during experimental endotoxinemia.

Serial plasma samples from human volunteers obtained after intravenous administration of Escherichia coli endotoxin were analyzed for the presence of circulating soluble tumor necrosis factor receptors (sTNFR). A four- to fivefold increase of type A (p75) and type B (p55) sTNFR was observed 3 h after endotoxin challenge. Pretreatment of the volunteers with ibuprofen before the injection of endotoxin resulted in a slight increase (3.87 +/- 0.2 vs. 3.27 +/- 0.3 ng/ml) and temporal shift of sTNFR-A release concurrent to a marked augmentation of TNF levels (603 +/- 118 vs. 338 +/- 56 pg/ml) as compared to the group without ibuprofen pretreatment. There was a significant correlation between peak sTNFR-A levels and peak TNF levels in the individual probands (r = 0.52, P = 0.04). On the contrary, release kinetics and plasma concentrations of sTNFR-B were identical in both groups (7.38 +/- 0.69 vs. 7.44 +/- 0.33 ng/ml) and no correlation with individual TNF levels was observed. The amount of sTNFR liberated upon endotoxin challenge was not sufficient to block TNF-mediated cytotoxic effects. Our data indicate that the release in vivo of type A and type B sTNFR upon a short exposure to endotoxin is regulated differently.     

Beneficial effects of TCV-309, a novel potent and selective platelet activating factor antagonist in endotoxin and anaphylactic shock in rodents.

Pharmacological profiles of a novel specific platelet activating factor (PAF) antagonist, TCV-309 (3-bromo-5-[N-phenyl-N-[2-[2- (1,2,3,4-tetrahydro-2-isoquinolycarbonyloxy)ethyl] carbamoyl]ethyl] carbamoyl]-1-propylpyridinium nitrate] and its beneficial effects in shock were examined. TCV-309 specifically inhibited PAF-induced aggregation of rabbit and human platelets, and [3H]PAF binding to rabbit platelet microsomes with IC50 values of 33, 58 and 27 nM, respectively. It was as potent as WEB 2086 and more potent than CV-6209 and CV-3988. TCV-309 did not cause hemolysis in human or rat blood due to a detergent-like action. In rats, TCV-309 selectively inhibited the PAF-induced hypotension, hemoconcentration and death with ED50 values of 2.7, 6.4 and 1.7 micrograms/kg (i.v.), respectively. TCV-309 most potently protected mice from death induced by PAF and due to anaphylactic shock with ED50 values of 2.1 and 2.6 micrograms/kg (i.v.), respectively, when compared with CV-3988, CV-6209, WEB 2086 (i.v.) and L-652731 (p.o.). TCV-309 also reversed PAF-induced hypotension and endotoxin-induced hypotension in rats with ED50 values of 3.3 and 1.2 micrograms/kg (i.v.), respectively. There was a significant linear relationship between the ability (ED50 value) of these PAF antagonists to prevent death induced by PAF and death due to anaphylactic shock in mice, and between their reversing ability (ED50 value) for the hypotension induced by PAF and endotoxin in rats. TCV-309 (100 micrograms/kg i.v.) protected rats from death induced by endotoxin. Thus, PAF may be a lethal mediator in anaphylactic shock and a hypotensive mediator in endotoxin shock in rodents.     

Identification of a novel cyclosporin-sensitive element in the human tumor necrosis factor alpha gene promoter

Tumor necrosis factor alpha (TNF-alpha), a cytokine with pleiotropic biological effects, is produced by a variety of cell types in response to induction by diverse stimuli. In this paper, TNF-alpha mRNA is shown to be highly induced in a murine T cell clone by stimulation with T cell receptor (TCR) ligands or by calcium ionophores alone. Induction is rapid, does not require de novo protein synthesis, and is completely blocked by the immunosuppressant cyclosporin A (CsA). We have identified a human TNF-alpha promoter element, kappa 3, which plays a key role in the calcium-mediated inducibility and CsA sensitivity of the gene. In electrophoretic mobility shift assays, an oligonucleotide containing kappa 3 forms two DNA protein complexes with proteins that are present in extracts from unstimulated T cells. These complexes appear in nuclear extracts only after T cell stimulation. Induction of the inducible nuclear complexes is rapid, independent of protein synthesis, and blocked by CsA, and thus, exactly parallels the induction of TNF-alpha mRNA by TCR ligands or by calcium ionophore. Our studies indicate that the kappa 3 binding factor resembles the preexisting component of nuclear factor of activated T cells. Thus, the TNF-alpha gene is an immediate early gene in activated T cells and provides a new model system in which to study CsA-sensitive gene induction in activated T cells.     

Platelet activating factor-induced shock and intestinal necrosis in the rat: role of endogenous platelet-activating factor and effect of saline infusion.

BACKGROUND AND METHODS: The mechanism of ischemic bowel necrosis induced by platelet-activating factor is unclear. Since intestinal hypoperfusion is observed after platelet-activating factor injection, we hypothesized that mesenteric vasoconstriction is the mechanism of bowel injury. The present study investigated the effects of saline infusion on platelet activating factor-induced bowel necrosis and its mechanism. Male Sprague-Dawley rats were divided into four groups: group A consisted of sham-operated rats; group B received platelet-activating factor (1.5 micrograms/kg iv); group C received platelet-activating factor and saline (0.097 mL/min iv); group D received platelet-activating factor and WEB 2086 (platelet-activating factor antagonist). RESULTS: Saline infusion largely reversed platelet activating factor-induced hypotension, hemoconcentration, and reduction of the superior mesenteric arterial blood flow. Saline infusion also ameliorated platelet activating factor-induced bowel injury, although a mild-to-moderate degree of necrosis still developed focally. In addition, saline prevented the platelet activating factor-induced increase in intestinal platelet-activating factor production. Saline also prevented the increase in intestine leukocyte number, as estimated by myeloperoxidase activity. CONCLUSIONS: Saline infusion is an effective treatment for platelet activating factor-induced shock and intestinal necrosis. However, focal bowel injury is still observed, suggesting that other factors besides hemodynamic changes contribute to the development of tissue injury. We also showed that, in vivo, platelet-activating factor stimulates its own synthesis via a positive feedback loop, which could be blocked by intravascular volume expansion with saline.