Effects of dietary supplementation with alpha-linolenic acid on the phospholipid fatty acid composition and quality of spermatozoa in cockerel from 24 to 72 weeks of age

The physiopathology of experimental cerebral malaria (CM), an acute neurological complication of Plasmodium berghei ANKA (PbA) infection, involves interferon-gamma (IFN-gamma) and tumour necrosis factor-alpha (TNF-alpha), two cytokines that are known to modulate major histocompatibility complex (MHC) molecule expression. The aim of this study was to evaluate whether the genetic susceptibility to CM is related to the constitutive or IFN-gamma-induced expression of MHC molecules on brain microvessels. To this end, brain microvascular endothelial cells (B-MVEC) were isolated from CM-susceptible (CM-S, CBA/J) and resistant (CM-R, BALB/c) mice. By flow cytometry, we found that less than 5% of CM-S B-MVEC constitutively expressed MHC class I molecules, in contrast to up to 90% of CM-R B-MVEC. Upon stimulation with IFN-gamma, the percentage of positive cells for MHC class I molecules in CM-S B-MVEC became comparable to CM-R B-MVEC, but a higher fluorescence intensity existed on CM-S B-MVEC compared with CM-R B-MVEC. MHC class II molecules were not constitutively expressed on B-MVEC from either strain. IFN-gamma-induced expression of MHC class II (I-A, I-E) molecules was significantly higher in CM-S than CM-R B-MVEC both in percentage of positive cells and fluorescence intensity. These data demonstrate that absent or low MHC class I and higher inducibility of MHC class II expression on B-MVEC are associated with the genetic susceptibility to CM.     

Crucial role of tumor necrosis factor (TNF) receptor 2 and membrane-bound TNF in experimental cerebral malaria

Tumor necrosis factor (TNF) has been implicated in the pathogenesis of experimental cerebral malaria (CM), but the respective role of its two types of receptors has not been established. A significant increase in the expression of TNF-receptor 2 (TNFR2, p75), but not of TNFR1 (p55), was found on brain microvessels at the time of CM in susceptible animals. Moreover, mice genetically deficient for TNFR2 (Tnfr2null) were significantly protected from experimental CM, in contrast to TNFR1-deficient (Tnfr1null) mice, which were as susceptible as wild-type mice. To identify the factors involved in the protection from CM conferred by the lack of TNFR2, we assessed in both knockout and control mice the serum concentrations of mediators that are critical for the development of CM, as well as the up-regulation of intercellular adhesion molecule-1 (ICAM-1) in the brain microvessels. No significant difference in serum levels of TNF and interferon-gamma was found between infected wild-type and Tnfr1null or Tnfr2null mice. Interestingly, the pronounced ICAM-1 up-regulation and leukocyte sequestration, typically occurring in brain microvessels of CM-susceptible animals, was detected in infected control and Tnfr1null mice-both of which developed CM-whereas no such ICAM-1 up-regulation or leukocyte sequestration was observed in Tnfr2null mice, which were protected from CM. Making use of microvascular endothelium cells (MVEC) isolated from wild-type, Tnfr1null or Tnfr2null mice, we show that soluble TNF requires the presence of both TNF receptors, whereas membrane-bound TNF only needs TNFR2 for TNF-mediated ICAM-1 up-regulation in brain MVEC. Thus, only in MVEC lacking TNFR2, neither membrane-bound nor soluble TNF cause the up-regulation of ICAM-1 in vitro. In conclusion, these results indicate that the interaction between membrane TNF and TNFR2 is crucial in the development of this neurological syndrome.     

Activation of the insulin-like growth factor-I receptor inhibits tumor necrosis factor-induced cell death

The effect of insulin-like growth factor (IGF) on tumor necrosis factor (TNF)-induced cell killing was determined for mouse BALB/c3T3 fibroblasts in vitro. Cells maintained in 0.5% fetal bovine serum (FBS) were killed by TNF within 6 h in a concentration-dependent manner, an effect that was prevented by IGF-I. TNF-induced cytotoxicity of 3T3 cells that overexpress the human IGF-I receptor (p6 cells) was prevented by IGF-I alone in the absence of serum. TNF-induced cell death was associated with the morphologic features of apoptosis and the release of low-molecular-weight DNA, both of which were prevented by IGF-I. Neither epidermal growth factor (EGF) nor platelet-derived growth factor (PDGF) protected p6 cells from TNF-induced apoptosis. The specific protective action of the IGF-I receptor was demonstrated further by the marked sensitivity to TNF of embryo fibroblasts derived from mice with targeted disruption of the IGF-I receptor (R-cells) but not of fibroblasts derived from wild-type littermates or R-cells transfected with the cDNA for the human IGF-I receptor. Cycloheximide or actinomycin D markedly reduced the protection offered by IGF-I. IGF-I protection of BALB/c3T3 cells persisted for up to 5 days in the presence of PDGF and EGF, whereas IGF-I lost its effectiveness after 2 days in the absence of growth factors. IGF-I did not prevent TNF-induced release of arachidonic acid. The results demonstrate a specific role for the IGF-I receptor in the protection against TNF cytotoxicity. This action of the IGF-I receptor is mediated by protective cytosolic proteins that exhibit a high rate of turnover and whose levels are regulated principally by factors within serum other than IGF-I.     

Activation of stress-activated protein kinase/c-Jun N-terminal kinase, but not NF-kappaB, by the tumor necrosis factor (TNF) receptor 1 through a TNF receptor-associated factor 2- and germinal center kinase related-dependent pathway

A key step by which tumor necrosis factor (TNF) signals the activation of nuclear factor-kappaB (NF-kappaB) and the stress-activated protein kinase (SAPK, also called c-Jun N-terminal kinase or JNK) is the recruitment to the TNF receptor of TNF receptor-associated factor 2 (TRAF2). However, the subsequent steps in TRAF2-induced SAPK and NF-kappaB activation remain unresolved. Here we report the identification of a TNF-responsive serine/threonine protein kinase termed GCK related (GCKR) that likely signals via mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK) kinase kinase 1 (MEKK1) to activate the SAPK pathway. TNF, TRAF2, and ultraviolet (UV) light, which in part uses the TNF receptor signaling pathway, all increased GCKR activity. A TRAF2 mutant, which inhibits both TRAF2-induced NF-kappaB and SAPK activation, blocked TNF-induced GCKR activation. Finally, interference with GCKR expression impeded TRAF2- and TNF-induced SAPK activation but not that of NF-kappaB. This suggests a divergence in the TNF signaling pathway that leads to SAPK and NF-kappaB activation, which is located downstream of TRAF2 but upstream of GCKR.     

Interleukin-4 down-regulates both forms of tumor necrosis factor receptor and receptor-mediated apoptosis, NF-kappaB, AP-1, and c-Jun N-terminal kinase. Comparison with interleukin-13

The activity of tumor necrosis factor (TNF), a proinflammatory cytokine, is regulated by a number of other cytokines, including interleukin (IL)-4. How IL-4 regulates various activities of TNF is not fully understood. In the present report, we investigated the effect of IL-4 on the cell surface TNF receptors in human histiocytic lymphoma U-937 cells. Pretreatment of cells with IL-4 down-regulated TNF receptors in a dose- and time-dependent manner; an almost 90% decrease occurred with 10 ng/ml IL-4 treatment for 24 h. Scatchard analysis revealed that the decrease was due to receptor number and not affinity. IL-13, which shares a common receptor subunit and various biological activities with IL-4, had no effect on TNF receptors. IL-4's effect on TNF receptors was not cell type-specific, since decreases also occurred on various epithelial and T cells. Both the p60 and p80 forms of the TNF receptor were down-regulated to the same extent. Western blot showed that IL-4 induced shedding of the TNF receptors. The decrease of TNF receptors by IL-4 was accompanied by down-regulation of TNF-induced activities, including cytotoxicity, caspase-3 activation, NF-kappaB and AP-1 activation, and c-Jun N-terminal kinase induction. Wortmannin reversed the IL-4-induced TNF receptor down-regulation and all other measured cellular responses, indicating a critical role of phosphatidylinositol 3-kinase. Rapamycin also blocked the effect of IL-4-induced regulation, thus suggesting the role of p70 S6 kinase. Overall, our results suggest that TNF receptor down-regulation by IL-4 plays a critical role in the antagonistic effects of IL-4 on TNF-induced cellular responses and that this mechanism differs from that of IL-13.     

Tumor necrosis factor-induced E-selectin expression on vascular endothelial cells

OBJECTIVE: To determine the tumor necrosis factor (TNF) receptor type involved in induction of E-selectin expression on vascular endothelial cells. DESIGN: Prospective, in vitro repeated-measures analysis of cellular responses. SETTING: Research laboratory in an academic medical center. SUBJECTS: Cultured human umbilical vein endothelial cells. INTERVENTIONS: Human umbilical vein endothelial cells were incubated with recombinant human TNF (rhTNF) to induce the expression of E-selectin on their surfaces. To block rhTNF from binding to receptors, the cells were incubated with monoclonal antibodies against TNF receptors (anti-CD120a and anti-CD120b). TNF-induced E-selectin expression of the endothelial cells, with and without blocking antibodies, was then determined using indirect immunofluorescence and flow cytometry. MEASUREMENTS AND MAIN RESULTS: Blocking of either CD120a or CD120b receptors individually resulted in inhibition of TNF-induced E-selectin expression on human umbilical vein endothelial cells. When both antibodies were added, the inhibition of TNF-induced E-selectin expression was synergistic. Inhibition of E-selectin expression was dependent on both TNF concentrations and antibody concentrations. CONCLUSIONS: Both CD120a and CD120b receptors are involved in TNF-induced E-selectin expression on human umbilical vein endothelial cells. Blocking of both or one receptor type can reduce or totally inhibit expression of E-selectin on human umbilical vein endothelial cells, but the response is dependent on both TNF and antibody concentrations.     

Early activation of c-Jun N-terminal kinase and p38 kinase regulate cell survival in response to tumor necrosis factor alpha

Fas ligand and tumor necrosis factor alpha (TNF) bind to members of the TNF receptor superfamily. Stimulation by Fas ligand results in apoptosis, whereas TNF induces multiple effects including proliferation, differentiation, and apoptosis. Activation of the c-Jun N-terminal kinase (JNK) and p38 kinase pathways is common to Fas and TNF signaling; however, their role in apoptosis is controversial. Fas receptor cross-linking induces apoptosis in the absence of actinomycin D and activates JNK in a caspase-dependent manner. In contrast, TNF requires actinomycin D for apoptosis and activates JNK and p38 kinase with biphasic kinetics. The first phase is transient, precedes apoptosis, and is caspase-independent, whereas the second phase is coincident with apoptosis and is caspase-dependent. Inhibition of early TNF-induced JNK and p38 kinases using MKK4/MKK6 mutants or the p38 inhibitor SB203580 increases TNF-induced apoptosis, whereas expression of wild type MKK4/MKK6 enhances survival. In contrast, the Mek inhibitor PD098059 has no effect on survival. These results demonstrate that early activation of p38 kinase (but not Mek) are necessary to protect cells from TNF-mediated cytotoxicity. Thus, early stress kinase activation initiated by TNF plays a key role in regulating apoptosis.     

TNF and IL-6 mediate MIP-1alpha expression in bleomycin-induced lung injury

Previously, macrophage inflammatory protein-1alpha (MIP-1alpha), a member of the C-C chemokine family, has been implicated in bleomycin-induced pulmonary fibrosis, a model of the human disease idiopathic pulmonary fibrosis. Neutralization of MIP-1alpha protein with anti-MIP-1alpha antibodies significantly attenuated both mononuclear phagocyte recruitment and pulmonary fibrosis in bleomycin-challenged CBA/J mice. However, the specific stimuli for MIP-1alpha expression in the bleomycin-induced lesion have not been characterized. In this report, two mediators of the inflammatory response to bleomycin, tumor necrosis factor (TNF) and interleukin-6 (IL-6), were evaluated as putative stimuli for MIP-1alpha expression after bleomycin challenge in CBA/J mice. Elevated levels of bioactive TNF and IL-6 were detected in bronchoalveolar lavage (BAL) fluid and lung homogenates from bleomycin-treated CBA/J mice at time points post-bleomycin challenge, which precede MIP-1alpha protein expression. Treatment of bleomycin-challenged mice with soluble TNF receptor (sTNFr) or anti-IL-6 antibodies significantly decreased MIP-1alpha protein expression in the lungs. Furthermore, normal alveolar macrophages secreted elevated levels of MIP-1alpha protein in response to treatment with TNF plus IL-6 or bleomycin plus IL-6, but not TNF, bleomycin, or IL-6 alone. Finally, leukocytes recovered from the BAL fluid of bleomycin-challenged mice secreted higher levels of MIP-1alpha protein, compared to controls, when treated with TNF alone. Based on the data presented here, we propose that TNF and IL-6 are part of a cytokine network that modulates MIP-1alpha protein expression in the profibrotic inflammatory lesion during the response to intratracheal bleomycin challenge.     

TNF-alpha, unlike other pro- and anti-inflammatory cytokines, induces rapid release of the IL-1 type II decoy receptor in human myelomonocytic cells

The present study was designed to investigate the effect of a series of cytokines on the release of the type II IL-1 decoy receptor, which represents a unique pathway of negative regulation of the IL-1 system. After 20 min, IL-1, IL-2, IL-4, IL-6, IL-10, IL-12, IL-13, IFN-gamma, granulocyte-CSF, macrophage-CSF, and TGF-beta had little or no effect on IL-1 binding by human polymorphonuclear cells. In contrast granulocyte-macrophage-CSF and, to a greater extent, TNF markedly reduced IL-1 binding. The action of TNF was rapid, reaching 50% of its maximum (80%) at 2 min, and plateauing at 20 min with decrease in receptor number and no significant change in affinity. Loss of surface receptor was associated to rapid release of a 45-kDa IL-1-binding molecule identified as the decoy RII. TNF-induced release of the decoy RII was independent of protein synthesis and reactive oxygen intermediates. Monocytes showed a similar response to TNF, except for the size of the released molecule (approximately 60 kDa). TNF induced rapid release of its own receptors. In contrast IL-1beta affected neither its own receptors nor the TNF-R. TNF and, more efficiently, PMA caused release of the decoy RII in fibroblasts transfected with the full-length decoy RII or with a cytoplasmatic deletion mutant. TNF-induced decoy RII release represents an unidirectional pathway of communication in the interplay between the IL-1 and TNF system.     

Prevention of tumor necrosis factor (TNF)-mediated induction of p21WAF1/CIP1 sensitizes MCF-7 carcinoma cells to TNF-induced apoptosis

The MCF-7 breast carcinoma and MRC-5 lung fibroblast cell lines are sensitive and resistant to tumor necrosis factor (TNF)-induced apoptosis, respectively. As the cyclin-dependent kinase inhibitor p21WAF1/CIP1 (p21) is involved in cell cycle regulation and has been implicated in apoptosis, we studied the influence of p21 on growth of MRC-5 cells and on growth and apoptosis in MCF-7 cells. TNF induced p21 mRNA and protein in both cell types. p21 induction by > 0.5 ng/ml TNF in MRC-5 and MCF-7 cells correlated with the inhibition of cell growth. In contrast, < 0.1 ng/ml TNF stimulated MRC-5 (but not MCF-7) cell growth without reduction in p21 levels. TNF-induced apoptosis in MCF-7 cells was first detected after the TNF-mediated increase in p21 and growth arrest had occurred. MCF-7 cells stably transfected with antisense p21 cDNA became more sensitive to TNF-induced apoptosis. Thus, TNF-induced p21 accompanied by growth arrest may counteract or delay TNF cytotoxicity in MCF-7 cells.     

Cytokine-specific activation of distinct mitogen-activated protein kinase subtype cascades in human neutrophils stimulated by granulocyte colony-stimulating factor, granulocyte-macrophage colony-stimulating factor, and tumor necrosis factor-alpha

To clarify the differences of the signaling pathways used by granulocyte colony-stimulating factor (G-CSF), granulocyte-macrophage colony-stimulating factor (GM-CSF), and tumor necrosis factor- (TNF), we investigated activation of mitogen-activated protein kinase (MAPK) subtype cascades in human neutrophils stimulated by these cytokines. G-CSF exclusively tyrosine-phosphorylated extracellular signal-regulated kinase (ERK). GM-CSF tyrosine-phosphorylated ERK strongly and p38 MAPK weakly, whereas TNF tyrosine-phosphorylated p38 MAPK strongly and ERK weakly. Consistent with these findings, MEK, an upstream kinase of ERK, was phosphorylated by G-CSF, GM-CSF, and TNF, whereas MKK3/MKK6, an upstream kinase of p38 MAPK, was phosphorylated by GM-CSF and TNF, but not by G-CSF. The potency of these cytokines to phosphorylate ERK and MEK was GM-CSF > G-CSF > TNF, whereas that to phosphorylate p38 MAPK and MKK3/MKK6 was TNF > GM-CSF. C-Jun amino-terminal kinase (JNK) was not tyrosine-phosphorylated by any cytokine despite the existence of JNK proteins in human neutrophils, whereas it was tyrosine-phosphorylated by TNF in undifferentiated and all-trans retinoic acid-differentiated HL-60 cells. Increased phosphorylation of ERK or p38 MAPK was detected within 1 to 5 minutes after stimulation with each cytokine and was dependent on the concentrations of cytokines used. MEK inhibitor (PD98059) reduced tyrosine phosphorylation of ERK, but not p38 MAPK, induced by G-CSF, GM-CSF, or TNF. GM-CSF- or TNF-induced superoxide (O2-) release was inhibited by p38 MAPK inhibitor (SB203580) in a dose-dependent manner, suggesting the possible involvement of p38 MAPK in GM-CSF- or TNF-induced O2- release. The results indicate that G-CSF, GM-CSF, and TNF activate the overlapping but distinct MAPK subtype cascades in human neutrophils and suggest that the differential activation of ERK and p38 MAPK cascades may explain the differences of the effects of these cytokines on human neutrophil functions.     

Angiographic evaluation of culprit lesions in acute coronary syndrome: relation to the original site on previous coronary angiography

Recent studies suggest that release of cytokines during inflammatory states such as septic shock leads to hypocholesterolemia. To examine whether tumor necrosis factor alpha (TNF), which is the major cytokine in inflammatory disease, causes hypocholesterolemia, we measured serum levels of total (bioactive and receptor-bound) TNF, cholesterol, Apo B, and Apo A1 in seven patients with septic shock over a period of 8 days. Since elevated serum TNF levels are accompanied by the release of soluble TNF receptors, levels of TNF receptors p55 and p75 were also measured. Patients with septic shock had significantly higher serum TNF and TNF receptor levels compared with healthy controls. Increased cytokine levels were accompanied by a significant decline in total serum cholesterol apolipoprotein A1 and B. In vitro studies with cultured human skin fibroblasts, human umbilical vein endothelial cells, and HepG2 hepatoma cells showed that TNF increased the degradation of 125I-labeled low-density lipoprotein in all the cell lines tested. Recombinant soluble TNF receptors inhibited the TNF-induced stimulation of low-density lipoprotein receptor in a concentration-dependent manner. However, the calculated ratio of TNF receptors to total TNF measured in serum of these patients was not able to counteract the stimulatory effect of TNF, possibly due to the higher molar excess of TNF receptors required to achieve this effect in vitro. Our data strengthen the hypothesis that serum values of total TNF determine the extent of hypocholesterolemia during sepsis and septic shock despite the presence of a high concentration of TNF receptors. Studies with recombinant TNF also confirm the role of TNF in hypocholesterolemia in inflammation.     

Deleterious brain cell membrane effects after NMDA receptor antagonist administration to newborn piglets

Heroin administered i.c.v. acts on supraspinal mu opioid receptors in ICR mice but on delta receptors in Swiss Webster mice. The purpose of this study was to determine the degree to which genotype plays a role in the opioid receptor selectivity of heroin across a range of fully inbred strains of mice. Six inbred strains were given heroin i.c.v. 10 min before the tail-flick test. Differences in the descending neurotransmitter systems involved in supraspinal opioid-induced analgesia were evaluated as the first step. Antagonism by bicuculline given intrathecally indicated the involvement of supraspinal delta receptors in activating spinal gamma-aminobutyric acid (GABA) receptors; antagonism by intrathecal methysergide indicated either mu or kappa receptor involvement. Antagonism by intrathecal yohimbine implicated mu and eliminated kappa receptor involvement. Intracerbroventricular opioid antagonists (beta-funaltrexamine, 7-benzylidenenaltrexone, naltriben, or nor-binaltorphimine) provided further differentiation. Based on these initial results, receptor selectivity was determined by more extensive ED50 experiments with i.c.v. administration of heroin with opioid antagonists, beta-funaltrexamine (for mu), naltrindole (for delta), and nor-binaltorphimine (for kappa). The combined results indicated that heroin analgesia was predominantly mediated in C57BL/6J by delta, in DBA/2J and CBA/J by mu, and in BALB/cByJ and AKR/J by kappa receptors. The response in C3H/HeJ appeared to involve mu receptors. The results indicate that the opioid receptor selectivity of heroin is genotype-dependent. Because these genotypes are fully inbred, the genetically determined molecular and neurochemical substrate mediating the different opioid receptor selectivities of heroin can be studied further.     

The effect of pyrimidines, levamisole and prodigiozan on the antigen-specific regulation of delayed hypersensitivity in relation to the age and strain of the mice

The effect of pyrimidine, levamizol, and prodigiozan on the retarded hypersensitivity (RH) was studied on a model of adoptive transfer of the splenocytes from donors of different ages to young syngeneic recipients. Splenocytes from CBA and BALB/c suckers, receiving pyrimidine, activate the RH suppression. Levamizol and prodigiozan exert the same effect on BALB/c mice and inhibit the RH suppression in CBA mice. In mature CBA mice these preparations potentiate HST. The splenocytes from young and aged BALB/c donors receiving the preparations oppositely induce the RH recipients.     

The tumor necrosis factor signaling complex: choosing a path toward cell death or cell proliferation

Signal transduction pathways which are initiated by the tumor necrosis factor (TNF) utilize receptors which are devoid of intrinsic catalytic activity. Recently identified two families of proteins that directly associate with the cytoplasmic domains of the TNF receptor family members, have partially bridged a molecular gap within the TNF-induced signaling pathways. Clearly, there are numerous alternate routes that originate from the TNF ligand-receptor assembly and terminate on the diverse cellular responses, including proliferation, differentiation, or death. This review focuses on recent advances characterizing the TNF ligand-receptor signaling network, which allow to better understand its participation in a life-death balance within the target cell.