Interleukin-12-mediated resistance to Trypanosoma cruzi is dependent on tumor necrosis factor alpha and gamma interferon.

The aim of this study was to determine if interleukin-12 (IL-12) has a role in the immune response to Trypanosoma cruzi. Infection of BALB/c mice with the virulent Tulahuen strain of T. cruzi is characterized by a high-level parasitemia, pathology in the heart associated with the presence of amastigotes, and death during the acute phase of the disease. Administration of IL-12 to BALB/c mice infected with T. cruzi resulted in a reduced parasitemia and a significant delay in the time to death compared with those for infected controls. This protective effect was correlated with increased levels of gamma interferon (IFN-gamma) and tumor necrosis factor alpha (TNF-alpha) in serum. To determine if these cytokines were involved in the protective effects of IL-12, we treated infected mice with IL-12 alone or in combination with monoclonal antibodies specific for IFN-gamma or TNF-alpha. These antibodies antagonized the protective effect of exogenous IL-12. Treatment of infected mice with a polygonal antibody specific for IL-12 resulted in a significant increase in parasitemia but did not affect the time to death. These latter studies demonstrate a role for endogenous IL-12 in resistance to T. cruzi. Together, our data identify an IL-12-mediated mechanism of resistance to T. cruzi, which is dependent on IFN-gamma and TNF-alpha.     

Anti-tumor necrosis factor receptor and tumor necrosis factor agonist activity by an anti-idiotypic antibody.

Tumor necrosis factor (TNF) is a cytokine which, among other properties, is a principle mediator of inflammation and septic shock. It acts upon target cells by binding to specific cell surface receptors. A10G10 is a murine monoclonal antibody which recognizes human TNF and neutralizes its activity. A rabbit polyclonal antibody directed at the antigen-binding site of A10G10 was raised and affinity purified over an A10G10 column. The resultant anti-idiotypic antibody recognized not only A10G10 but also both TNF receptors. It showed TNF agonist activity in two different TNF bioassays, and competed with several anti-TNF receptor monoclonal antibodies and TNF itself for binding to cells. These results represent an example of a method for obtaining antibodies to a ligand-specific receptor in the absence of the receptor itself.     

Exfoliation of the epidermal cells and defecation by amphibian larvae in response to coelomic fluid and lysenin from the earthworm Eisenia foetida

Coelomic fluid (CF) and lysenin from the earthworm Eisenia foetida induced heavy epidermal exfoliation in the larvae of Bufo japonicus formosus at developmental stages from hatching (stage 22) to operculum completion (stage 34). In experiments with Xenopus laevis, we observed that exfoliated cells were not stained by trypan blue. Thus, it appeared that these cells were still alive. It is likely, therefore, that both CF and lysenin might disrupt the adhesion between epidermal cells of larvae prior to stage 34. Since it is known that lysenin exerts its toxic effects through its specific binding to sphingomyelin (SM), SM might be involved in such adhesion. This hypothesis was supported by the observations that CF and lysenin which had been incubated with SM-liposomes lost their exfoliative activity. In larvae after stage 34, the mechanism of adhesion between epidermal cells seemed to change and the adhesion was no longer disrupted by CF and lysenin. In larvae at around stage 34, a collagen layer started to form beneath the basement membrane of the epidermis. Furthermore, larvae at around this stage started to eat solid food. The developing collagen layer and food intake might be related indirectly to the chemical change in epidermal adhesion. The induction of exfoliation by CF and lysenin was also observed in other amphibian species. In Bufo larvae, defecation was induced both by CF and by lysenin but this effect was independent of exfoliation.     

Tumor necrosis factor induces tumor necrosis via tumor necrosis factor receptor type 1-expressing endothelial cells of the tumor vasculature

Activation of endothelial cells, fibrin deposition, and coagulation within the tumor vasculature has been shown in vivo to correlate with the occurrence of tumor necrosis factor (TNF)-induced tumor necrosis in mice. In the present study we investigated which target cells mediate the TNF-induced necrosis in fibrosarcomas grown in wild type (wt), TNF receptor type 1-deficient (TNFRp55-/-), and TNF receptor type 2-deficient (TNFRp75-/-) mice. TNF administration resulted in tumor necrosis exclusively in wt and TNFRp75-/-, but not in TNFRp55-/- mice, indicating a dependence of TNF-mediated tumor necrosis on the expression of TNF receptor type 1. However, using wt and TNFRp55-/- fibrosarcomas in wt mice, we found that TNF-mediated tumor necrosis was completely independent of TNF receptor type 1 expression in tumor cells. Thus we could exclude any direct tumoricidal effect of TNF in this model. Soluble TNF induced leukostasis in wt and TNFRp75-/- mice but not in TNFRp55-/- mice. TNF-induced leukostasis in TNFRp55-/- mice was restored by adoptive bone marrow transplantation of wt hematopoietic cells, but TNF failed to induce tumor necrosis in these chimeric mice. Because TNF administration resulted in both activation and focal damage of tumor endothelium, TNF receptor type 1-expressing cells of the tumor vasculature, likely to be endothelial cells, appear to be target cells for mediating TNF-induced tumor necrosis.     

Comparative analysis of hemolytic and hemagglutinating activities in the coelomic fluid of Eisenia foetida and Lumbricus terrestris (Annelida, Lumbricidae)

The CF of Lumbricus terrestris and Eisenia foetida was analyzed with regard to the protein patterns by isoelectric focusing. The CF of E. foetida results in a variety of protein bands; after separation: seven of these with isoelectric points at pH 5.1, 5.2, 5.4, 5.6, 5.8, 6.0 and 6.2 cause the hemolysis of red blood cells. The protein pattern of normal CF of L. terrestris is relatively poor. Most of the protein bands are located at the top of the separation (pH less than 5.1) near the anode. However, the number of protein bands is drastically enhanced within 24 hours after an intracoelomic injection of foreign material. Some of the induced proteins show a similar pattern at comparable pH ranges to the CF of E. foetida. The induced proteins with isoelectric points at pH 4.7, 5.0, 5.1, 5.2, 5.4, 5.8, 6.0 and 6.2 effect the hemolysis of vertebrate erythrocytes. After stimulation with rabbit erythrocytes an enhanced bactericidal and bacteriostatic activity against Proteus vulgaris can be proven and seems to be related to the induced hemolytic proteins. The antibacterial activity can be completely adsorbed by incubation of the CF with rabbit erythrocytes, which indicates its binding ability to marker molecules on the erythrocytes surface.     

MIF-driven activation of macrophages induces killing of intracellular Trypanosoma cruzi dependent on endogenous production of tumor necrosis factor,nitric oxide and reactive oxygen species

The proinflammatory cytokine macrophage migration inhibitory factor (MIF) is a key player in innate immunity. MIF has been considered critical for controlling acute infection by the protozoan Trypanosoma cruzi, but the underlying mechanisms are poorly understood. Our study aimed to analyze whether MIF could favor microbicidal activity of the macrophage, a site where T. cruzi grows and the initial effector cell against this parasite. Using murine macrophages infected in vitro, we examined the effect of MIF on their parasiticidal ability and attempted to identify inflammatory agents involved in MIF-induced protection. Our findings show that MIF is readily secreted from peritoneal macrophages upon T. cruzi infection. MIF activates both primary and J774 phagocytes boosting the endogenous production of tumor necrosis factor-alpha via mitogen-activated protein kinase p38 signaling, as well as the release of nitric oxide and reactive oxygen species, leading to enhanced pathogen elimination. MIF can also potentiate the effect of interferon-gamma on T. cruzi killing by J774 and mouse peritoneal macrophages, rendering these cells more competent in reducing intracellular parasite burden. The present results unveil a novel innate immune pathway that contributes to host defense and broaden our understanding of the regulation of inflammatory mediators implicated in early parasite containment that is decisive for resistance to T. cruzi infection.     

Induction of hyporesponsiveness to an early post-binding effect of tumor necrosis factor by tumor necrosis factor itself and interleukin 1.

Modulation of cellular responsiveness to tumor necrosis factor (TNF) was studied in the human SV-80 cells. A marked cytocidal effect is exhibited by these cells at about 4 to 8 h after application of TNF together with protein synthesis inhibitors. Sensitivity of the cells to TNF toxicity was shown to be markedly decreased following their pretreatment with TNF itself or with interleukin (IL) 1 in the absence of protein synthesis inhibitors. The SV-80 cells respond to TNF also with enhanced phosphorylation of the small heat-shock protein, HSP27. This TNF effect is much more rapid than the cytocidal effect; it is observed within minutes of TNF application. The response to this effect, just like the response to the cytocidal effect, is markedly decreased following preexposure of the cells to either TNF or IL 1. Responsiveness to both effects of TNF is regained at the same time, about 15 to 20 h following removal of TNF or IL 1. The decrease in responsiveness after pretreatment with TNF or IL 1 does not reflect an inability of the pretreated cells to bind TNF. Although there is an initial decrease in TNF binding after such pretreatment, it is fully reversed already about 5 h following removal of the cytokines. The rate of uptake of TNF by the pretreated cells is also normal. In view of the rapidity of the effect of TNF on the phosphorylation of HSP27, it seems likely that the observed hyporesponsiveness reflects impairment of an early step in a signaling pathway, perhaps common to both the stimulation of phosphorylation and the induction of cell death by TNF. By restricting the duration of the effects of TNF this desensitization mechanism may safeguard against harmful consequences of these effects.     

Serum concentrations of insulin-like growth factor-1, soluble tumor necrosis factor receptor-1 and angiogenin in endometriosis patients

PROBLEM: Many soluble factors contributing to the pathophysiology of endometriosis are found at abnormal levels in patients suffering from the disease. We postulated that levels of these factors could also be altered in the serum of patients. We compared levels of insulin-like growth factor-1 (IGF-1), soluble form of tumor necrosis factor-alpha (TNF-alpha) receptor-1 (sTNFR-1) and angiogenin in the serum of patients with endometriosis and controls. METHOD OF STUDY: Levels of IGF-1, sTNFR-1 and angiogenin were measured by enzyme-linked immunosorbent assay in samples from 148 patients (77 cases and 71 controls) with diagnostic confirmed by laparoscopy. Correlations with demographic data and stage of the disease were evaluated and potential confounders in the study population were controlled. RESULTS: A significant increase in sTNFR-1 and angiogenin serum levels was observed in cases in comparison with controls, but only for patients in the follicular phase of the cycle. No significant difference was found in serum levels of IGF-1, sTNFR-1 and angiogenin between cases and controls in the luteal phase of the cycle. Correlations between levels of angiogenin and stage of the disease could also be observed. CONCLUSION: sTNFR-1 and angiogenin represent potential blood markers for endometriosis.     

Taxol, a microtubule-stabilizing antineoplastic agent, induces expression of tumor necrosis factor alpha and interleukin-1 in macrophages.

Taxol, a naturally occurring diterpene with antitumor activity, induces tubulin polymerization to generate abnormally stable and nonfunctional microtubules. Previously, we showed that taxol has lipopolysaccharide (LPS)-like effects on macrophages. As LPS is a potent inducer of macrophage cytokine production, we investigated whether a similar effect is exerted by taxol. In a dose-dependent manner, LPS-free taxol induced release of biologically active tumor necrosis factor alpha (TNF) by inflammatory murine macrophages. Taxol-induced production of TNF was inhibitable by interleukin-10. By Northern blot, taxol (10 and 1 microM) induced TNF mRNA expression to an extent similar to LPS. Induction of TNF mRNA by 10 microM taxol was detectable at 45 min of stimulation, maximal at 90 min, and evident for at least 8 h. The same low concentration of taxol also induced interleukin 1 (IL-1) alpha and beta mRNA expression. We conclude that taxol triggers macrophages for TNF and IL-1 production. These LPS-like effects of taxol might contribute to its antitumor activity.     

Augmentation of GG2EE macrophage cell line-mediated anti-Candida activity by gamma interferon, tumor necrosis factor, and interleukin-1.

The expression of anti-Candida activity in the GG2EE macrophage cell line, generated by immortalization of fresh bone marrow with v-raf and v-myc oncogenes, was studied. GG2EE cells spontaneously inhibited the growth of an agerminative mutant of Candida albicans in vitro. The anti-Candida activity was maximal after 8 h of coculture and was proportional to the effector-to-target ratio. Gamma interferon (IFN-gamma), interleukin-1 (IL-1), and tumor necrosis factor (TNF) all significantly enhanced the anti-Candida activity of GG2EE cells. In contrast, IL-3, IL-4, and colony-stimulating factor 1 were ineffective. The augmentation of anti-Candida activity was not always concomitant with enhancement of phagocytosis, since IFN-gamma and colony-stimulating factor 1, but not IL-1 or TNF, augmented the phagocytic ability of GG2EE cells. Furthermore, the augmentation of anti-Candida activity in GG2EE cells did not correlate with the acquisition of antitumor activity. In fact, none of the cytokines alone were able to induce antitumor activity in GG2EE cells, which, however, could be activated to a tumoricidal stage by IFN-gamma plus heat-killed Listeria monocytogenes. These findings demonstrate that GG2EE cells exhibit spontaneous anti-Candida activity and that such activity is enhanced by TNF, IL-1, and IFN-gamma.     

Amyloid-beta peptide induced inflammatory reaction is mediated by the cytokines tumor necrosis factor and interleukin-1

A chronic inflammatory response possibly mediated by amyloid-beta (A beta) is believed to be a major factor in the pathology of Alzheimer's disease (AD). Recently, we demonstrated that in vivo administration of A beta produces an inflammatory response and vascular disruption as seen in the brains of AD patients. In an inflammatory response, leukocyte activation and extravasation involves cytokine production. Previous studies have indicated that immune interactions exist between the central nervous system and the peripheral immune mechanisms in AD. Increased levels of interleukin-1 beta (IL-1 beta) have been detected in brain tissue, cerebrospinal fluid, and blood/serum from AD patients. In addition, A beta stimulated the production of tumor necrosis factor-alpha (TNF-alpha) in brain astrocytes and murine monocytes. Using an animal model we investigated the role of the cytokines, TNF-alpha and IL-1 beta, in the A beta-induced inflammatory response. Adult male rats were perfused via an intra-aortic cannula with either A beta alone, interleukin-1 receptor antagonist (IL-1 ra) plus A beta, tumor necrosis factor-binding protein (TNF-bp) plus A beta or sterile saline. Serum analysis for TNF-alpha, IL-1 beta, A beta and NO showed a significant increase in TNF-alpha and A beta but not in IL-1 beta or NO after the injection of A beta. Control values for serum A beta averaged 1.6 ng/ml and in rats injected with A beta, 99.6% of this peptide was removed from the blood within 30 min. The mesenteric arterioles and venules were video recorded for 1-2 h and then processed for electron microscopy (EM). In rats given A beta alone there was extensive vascular disruption, including endothelial and smooth muscle damage with leukocyte adhesion and migration. Animals receiving either IL-1 ra or TNF-bp before A beta showed no in vivo leukocyte extravasation or vascular damage under EM. Therefore, the cytokines TNF-alpha and IL-1 beta seem to mediate the vascular disruption and inflammatory response initiated by A beta. Antagonism of these pro-inflammatory cytokines may offer new avenues for AD therapy.     

Subcutaneous perfusion of tumor necrosis factor induces local proliferation of fibroblasts, capillaries, and epidermal cells, or massive tissue necrosis.

Mouse recombinant tumor necrosis factor (TNF) (or its solvent alone as a control) was administered subcutaneously to mice by a cannula connected to an osmotic minipump. Perfusion at a rate of 35 ng/hr for seven days induced the formation of a tissue mass composed mainly of fibroblasts, collagen, and capillaries. Necrosis (apoptosis) of isolated fibroblasts was observed. Polymorphonuclear leukocytes were abundant after three to four days of perfusion but were absent later. The covering epidermis showed a hyperplastic reaction associated with necrosis of isolated keratinocytes. Perfusion at a rate of 170 ng/hr led, after four to five days, to a massive necrosis. Necrosis was completely prevented by rabbit anti-TNF IgG but not by anti-LPS IgG, irradiation, or administration of indomethacin.     

Carvedilol, a nonselective beta-blocker, suppresses the production of tumor necrosis factor and tissue factor by inhibiting early growth response factor-1 expression in human monocytes in vitro

Tumor necrosis factor (TNF) and tissue factor (TF) produced by monocytes and macrophages have been shown to be among the aggravating factors for chronic heart failure (CHF), because they induce cardiac dysfunction and thrombotic complications, respectively. Carvedilol, a nonselective beta-adrenoceptor antagonist with alpha(1)- adrenoceptor blockade action, has been demonstrated to improve the outcome of patients with severe CHF, suggesting that carvedilol might inhibit the production of TNF and TF. In this study, this possibility is examined using isolated human monocytes stimulated with lipopolysaccharide (LPS) in vitro. Carvedilol (10 muM) significantly inhibited LPS-induced production of TNF and TF by monocytes, whereas prazosin (a selective alpha(1)-adrenoceptor antagonist), bisoprolol (a selective beta(1)-adrenoceptor antagonist), ICI-118,551 (a selective beta(2)-adrenoceptor antagonist), and arotinolol (a nonselective beta-adrenoceptor antagonist with alpha(1)-adrenoceptor blockade action) did not. Carvedilol inhibited both expression of early growth response factor-1 (Egr-1) and phosphorylation of extracellular signal-regulated kinase (ERK) 1/2, but it did not inhibit activation of either nuclear factor-kappaB or activator protein-1 in monocytes stimulated with LPS. These results suggest that carvedilol inhibits LPS-induced production of TNF and TF by inhibiting activation of the ERK1/2-Egr-1 pathway independent of its adrenoceptor inhibitory activities in monocytes.     

Natural cytotoxic activity is not necessarily mediated by the release of tumour necrosis factor.

Natural cell-mediated cytotoxicity is mediated by a family of effector cells that express cytolytic activities distinct from those generally attributed to B cells, T cells and macrophages; it includes both natural killer (NK) and natural cytotoxic (NC) activities. There is now convincing evidence to show that NC activity, but not NK activity, is mediated by tumour necrosis factor (TNF). Further, it has been argued that it is the release of TNF, as a freely diffusible factor, that causes NC-mediated target lysis. Here, we present evidence that the admixture of NC-sensitive target cells and spleen cells, under conditions that result in NC-mediated target cell lysis, does not necessarily result in the release of freely diffusible TNF into the culture medium. Also, it is demonstrated that the procedures used do not result in inactivation or loss of significant amounts of TNF during the assay period, which might account for our failure to detect free TNF. These results suggest that NC activity is mediated by either a membrane-associated TNF activity, similar to that described for some of the lytic activity of activated macrophages, or by the release of TNF that is capable of acting only over a very short distance.     

Microparticles of human atherosclerotic plaques enhance the shedding of the tumor necrosis factor-alpha converting enzyme/ADAM17 substrates, tumor necrosis factor and tumor necrosis factor receptor-1

Human atherosclerotic plaques express the metalloprotease tumor necrosis factor (TNF)-alpha converting enzyme (TACE/ADAM-17), which cleaves several transmembrane proteins including TNF and its receptors (TNFR-1 and TNFR-2). Plaques also harbor submicron vesicles (microparticles, MPs) released from plasma membranes after cell activation or apoptosis. We sought to examine whether TACE/ADAM17 is present on human plaque MPs and whether these MPs would affect TNF and TNFR-1 cellular shedding. Flow cytometry analysis detected 12,867 +/- 2007 TACE/ADAM17(+) MPs/mg of plaques isolated from 25 patients undergoing endarterectomy but none in healthy human internal mammary arteries. Plaque MPs harbored mainly mature active TACE/ADAM17 and dose dependently cleaved a pro-TNF mimetic peptide, whereas a preferential TACE/ADAM17 inhibitor (TMI-2) and recombinant TIMP-3 prevented this cleavage. Plaque MPs increased TNF shedding from the human cell line ECV-304 overexpressing TNF (ECV-304(TNF)), as well as TNFR-1 shedding from activated human umbilical vein endothelial cells or ECV-304(TNF) cells, without affecting TNF or TNFR-1 synthesis. MPs also activated the shedding of the endothelial protein C receptor from human umbilical vein endothelial cells. All these effects were inhibited by TMI-2. The present study shows that human plaque MPs carry catalytically active TACE/ADAM17 and significantly enhance the cell surface processing of the TACE/ADAM17 substrates TNF, TNFR-1, and endothelial protein C receptor, suggesting that TACE/ADAM17(+) MPs could regulate the inflammatory balance in the culprit lesion.     

Activation of murine macrophages by tumor necrosis factor, interleukin-1, interferon-gamma and cisplatin

Murine peritoneal macrophages were rendered tumoricidal to Dalton's lymphoma (DL) cells on incubation with recombinant tumor necrosis factor alpha (rTNF-alpha), recombinant interleukin-1 (rIL-1) and cisplatin in vitro. Simultaneous treatment of macrophages with suboptimal doses of rTNF-alpha and rIL-1 had additive effect on the activation of macrophages. Priming of macrophages with recombinant interferon gamma (rIFN-gamma) significantly enhanced the rTNF-alpha and rIL-1-induced macrophage cytotoxicity. Cisplatin was found to up-regulate rIL-1-induced macrophage activation but inhibited the activation of macrophages with rTNF-alpha. These studies indicate the potential of appropriate combination of these Biological Response Modifiers (BRMs) against neoplasia.     

Shiga toxin 1 induces apoptosis in the human myelogenous leukemia cell line THP-1 by a caspase-8-dependent, tumor necrosis factor receptor-independent mechanism

Shiga toxins (Stxs) induce apoptosis in a variety of cell types. Here, we show that Stx1 induces apoptosis in the undifferentiated myelogenous leukemia cell line THP-1 in the absence of tumor necrosis factor alpha (TNF-alpha) or death receptor (TNF receptor or Fas) expression. Caspase-8 and -3 inhibitors blocked, and caspase-6 and -9 inhibitors partially blocked, Stx1-induced apoptosis. Stx1 induced the mitochondrial pathway of apoptosis, as activation of caspase-8 triggered the (i) cleavage of Bid, (ii) disruption of mitochondrial membrane potential, and (iii) release of cytochrome c into the cytoplasm. Caspase-8, -9, and -3 cleavage and functional activities began 4 h after toxin exposure and peaked after 8 h of treatment. Caspase-6 may also contribute to Stx1-induced apoptosis by directly acting on caspase-8. It appears that functional Stx1 holotoxins must be transported to the endoplasmic reticulum to initiate apoptotic signaling through the ribotoxic stress response. These data suggest that Stxs may activate monocyte apoptosis via a novel caspase-8-dependent, death receptor-independent mechanism.     

Inhibition of growth of Chlamydia trachomatis by tumor necrosis factor is accompanied by increased prostaglandin synthesis.

Development of Chlamydia trachomatis (L2/434/Bu) in HEp-2 cells was inhibited by treatment of the cells with recombinant human alpha tumor necrosis factor (TNF). In the infected cultures that were treated with TNF, high concentrations of prostaglandin E2(PGE2) were detected, exceeding by far the concentrations found in TNF-treated but uninfected cells or in infected cells that were not treated with TNF. PGE2 levels increased gradually for 2 days after infection. Raising the tryptophan concentration in the culture medium, which reversed the inhibition of chlamydial replication by TNF, also blocked the increase in PGE2 formation. However, neutralizing antibodies to beta interferon, which also interfered with the antichlamydial effect of TNF, did not decrease PGE2 formation. Excessive formation of PGE2 by cells infected with chlamydiae and treated by TNF might be related to some of the complications associated with chlamydial infection.