Role of IFN-gamma and tumor necrosis factor-alpha in herpes simplex virus type 1 infection.

One of the characteristics of herpes simplex virus type 1 (HSV-1) is that recurrent diseases often develop from latent infection established after acute infection. Cytokines have been proposed to play an important role in each stage of HSV-1 infection, but the exact role of cytokines remains unclear. In the present study, we investigated the role of interferon-gamma (IFN-gamma) and tumor necrosis factor-alpha (TNF-alpha) in acute infection and reactivation using IFN-gamma gene knockout (IFN-gamma(-/-)) mice and TNF-alpha gene knockout (TNF-alpha(-/-)) mice. We first examined the survival rate after corneal infection with HSV-1. The survival rates of wild-type C57BL/6 (B6) mice, IFN-gamma(-/-) mice, and TNF-alpha(-/-) mice were 97% (73 of 75), 57% (24 of 42), and 83% (60 of 72), respectively. These results suggest that TNF-alpha and IFN-gamma play a protective role in acute infection with HSV-1. We also examined the rate of reactivation induced by ultraviolet (UV) light in latently infected mice over 60 days postinoculation. The reactivation was confirmed by detecting viral DNA extracted from eyeballs by the polymerase chain reaction (PCR) method at day 2 after the UV light stimulation. The rates of reactivation in IFN-gamma(-/-) mice and TNF-alpha(-/-) mice were significantly higher than that in B6 mice; 16% (4 of 25) showed reactivation in B6 mice, 47% (9 of 19) in IFN-gamma(-/-) mice, and 48% (10 of 21) in TNF-alpha(-/-) mice. These results suggest that IFN-gamma and TNF-alpha play an important role in acute infection and reactivation from latency.     

Mitochondria mediate tumor necrosis factor-alpha/NF-kappaB signaling in skeletal muscle myotubes

Tumor necrosis factor-alpha (TNF-alpha) is implicated in muscle atrophy and weakness associated with a variety of chronic diseases. Recently, we reported that TNF-alpha directly induces muscle protein degradation in differentiated skeletal muscle myotubes, where it rapidly activates nuclear factor kappaB (NF-kappaB). We also have found that protein loss induced by TNF-alpha is NF-kappaB dependent. In the present study, we analyzed the signaling pathway by which TNF-alpha activates NF-kappaB in myotubes differentiated from C2C12 and rat primary myoblasts. We found that activation of NF-kappaB by TNF-alpha was blocked by rotenone or amytal, inhibitors of complex I of the mitochondrial respiratory chain. On the other hand, antimycin A, an inhibitor of complex III, enhanced TNF-alpha activation of NK-kappaB. These results suggest a key role of mitochondria-derived reactive oxygen species (ROS) in mediating NF-kappaB activation in muscle. In addition, we found that TNF-alpha stimulated protein kinase C (PKC) activity. However, other signal transduction mediators including ceramide, Ca2+, phospholipase A2 (PLA2), and nitric oxide (NO) do not appear to be involved in the activation of NF-kappaB.     

Roles of gamma interferon and tumor necrosis factor-alpha in shiga toxin lethality

Shiga toxins (Stxs) have been specifically implicated as a causal factor of hemolytic uremic syndrome and acute encephalopathy. The first step of Stx-induced brain damage is considered to injure endothelial cells cooperating with tumor necrosis factor-alpha (TNF-alpha). Gamma interferon (IFN-gamma) is one of the proinflammatory cytokines as well as TNF-alpha is critical in activation of endothelial cells. Therefore we focused on the possibility of IFN-gamma-mediated lethality of Stx1 or Stx2 in mice. All of mice died within 3-4 days after injection with 400 ng of Stx1 and 37.5% of mice, which had been injected with 133 ng, survived. In contrast, a lethal dose of Stx2 was 40 times lower than that of Stx1. When mice were given 400 ng of Stx1 or 10 ng of Stx2, IFN-gamma mRNA was detected in the spleens 24h after injection. Moreover, when mice were injected with 133 ng of Stx1 or 3.3 ng of Stx2, survival rates of IFN-gamma-deficient mice and TNF-alpha-deficient mice were significantly higher than that of wild-type mice. The present study using cytokine-gene knockout mice directly demonstrated that not only TNF-alpha but also IFN-gamma is involved in lethality of Stx1 and Stx2.     

Receptor binding and activation of polymorphonuclear neutrophils by tumor necrosis factor-alpha

The interaction of highly purified recombinant human tumor necrosis factor-alpha (rTNF-alpha) with human polymorphonuclear neutrophils (PMNs) was investigated. Binding of 125I-rTNF-alpha to PMN reached maximum levels in 30 min at 37 degrees C and in 2 h at 4 degrees C. Scatchard analysis of competitive binding data indicated approximately 6000 receptor sites per cell and a Kd of 1.37 nM. Binding data at 37 degrees C indicated a rapid internalization of rTNF-alpha. Following this receptor-mediated interaction, recombinant TNF-alpha was found to inhibit the migration of PMNs under agarose and to enhance PMN production of superoxide anion (O-2) in a dose-dependent manner. Furthermore, rTNF-alpha-activated PMNs caused a marked disruption of human umbilical-vein-derived endothelial cell monolayers and caused inhibition of their proliferative activities. These data substantiate the role of TNF-alpha as an activator of PMN functions and indicate that PMN/TNF-alpha/endothelial cell interactions may play a major role in inflammatory reactions.     

Phagocytosis enhances murine macrophage activation by interferon-gamma and tumor necrosis factor-alpha

Previously, we reported that exposure of bone marrow-derived macrophages (M phi) to a phagocytic stimulus in the simultaneous presence of interferon-gamma (IFN-gamma) induced these cells to generate nitrite (NO2-). This effect was achieved using both living (i.e. promastigotes of the protozoan parasite Leishmania enriettii) and inert (latex beads) particles. When the phagocytic stimulus was Leishmania, enhanced intracellular killing accompanied elevated NO2- secretion. As shown here, the capacity of phagocytosis to elicit NO2- production by IFN-gamma-treated M phi was inhibited by antibody to murine recombinant tumor necrosis factor-alpha (rTNF-alpha), suggesting that phagocytosis enabled IFN-gamma to activate M phi via the induction of TNF-alpha as an autocrine second signal. M phi NO2- production in response to rIFN-gamma and either exogenous TNF-alpha or Leishmania was strongly enhanced by prostaglandin E2, consistent with such a mechanism. However, addition of either Leishmania promastigotes or latex beads to M phi cultures simultaneously exposed to both IFN-gamma and exogenous murine or human rTNF-alpha further potentiated activation as measured by NO2- release. Furthermore, anti-TNF antibody failed to inhibit M phi responses to rIFN-gamma and bacterial lipopolysaccharide (LPS) in the presence or absence of Leishmania; also exogenous rTNF-alpha did not significantly affect NO2- production by IFN-gamma/LPS cultures despite a strong enhancement by Leishmania. These results suggest that phagocytosis enhances M phi responses by a process more complex than the sole induction of TNF-alpha. Phagocytosis also increased M phi NO2- production elicited by IFN-gamma plus TNF-alpha in L-arginine-deficient media. These results indicate that phagocytosis may be an important mechanism of up-regulating M phi microbicidal activity, and could be particularly relevant upon arginine depletion which occurs during an inflammatory response.     

Influenza A virus infects macrophages and stimulates release of tumor necrosis factor-alpha

The clinical picture of influenza A virus infections indicates that release of tumor necrosis factor-alpha (TNF-alpha) may be involved. In the present study we exposed the murine macrophage line PU5-1.8 to influenza A virus and observed a productive infection which was followed by subsequent cell death. Infection of macrophages was accompanied by TNF-alpha mRNA accumulation and TNF-alpha release. TNF-alpha production could only be induced by live virus whereas interferon release was also stimulated by inactivated virus. When virus-infected macrophages were exposed to low amounts of lipopolysaccharide (LPS; 1-10 ng/ml) TNF-alpha production was strongly potentiated. These data show that low LPS concentrations could readily trigger a high TNF-alpha release from influenza-A-virus-infected macrophages which could, at least partially, explain the serious complications of combined influenza A virus and bacterial infections.     

Indirect and selective down-regulation of serum tumor necrosis factor-alpha release by interleukin-1 beta.

A selective inhibition of LPS-induced tumor necrosis factor-alpha (TNF) response in mice was caused by an injection of recombinant human interleukin-1 (IL-1). The decrease in serum TNF level reached 70 to 80 percent of the controls receiving LPS alone when IL-1 was given simultaneously or prior to the challenge. At the same time serum IL-6 release was more elevated. Ex vivo assays have shown that macrophages from IL-1 treated animals did not respond to LPS when stimulated immediately after harvesting but recovered their normal responsiveness after being cultured for 2 hours and then washed. In vitro with or without addition of IL-1, mouse elicited macrophages responded equally to LPS in releasing TNF. In the absence of a direct and lasting effect on TNF-producing cells, the host reaction responsible for the inhibitory effect of IL-1 could be related to the overproduction of corticosterone that occurred after IL-1 injection, since it was not observed in adrenalectomized animals. Indeed the blockade of corticoid secretion by indomethacin prevented the inhibition of TNF production induced by IL-1 administration before LPS challenge. TNF administration did not result in elevation of corticosterone level and in contrast to IL-1 enhanced the TNF response to LPS injection. In vitro and ex vivo assays have shown this enhanced response to LPS was linked to a direct and prolonged effect of TNF on TNF-producing cells. Muramyl dipeptide (MDP) which was used as a known priming agent for enhanced cytokine release had a similar effect on TNF-producing cells.(ABSTRACT TRUNCATED AT 250 WORDS)     

Inhibitory effect of linomide on lipopolysaccharide-induced proinflammatory cytokine tumor necrosis factor-alpha production in RAW264.7 macrophages through suppression of NF-kappaB, p38, and JNK activation

Linomide is an immunomodulator that can effectively inhibit the development of several autoimmune diseases in animal models. Previously, linomide was shown to influence macrophage function, although the mechanism was elusive. In this study, we investigated the effect of linomide on the macrophage inflammatory cytokine, tumor necrosis factor-alpha (TNF-alpha), production induced by lipopolysaccharide (LPS) in vitro on the murine macrophage cell line, RAW264.7. Linomide exposure reduced LPS-evoked TNF-alpha production in a dose-dependent manner. Gel shift and reporter gene analyses revealed linomide inhibited LPS-induced NF-kappaB binding to the NF-kappaB consensus oligonucleotide and NF-kappaB-mediated reporter gene expression. Immunoblot analysis showed that linomide inhibited phosphorylation of p38 kinase and c-jun N terminal kinase (JNK) in LPS-stimulated RAW264.7 cells. Taken together, these results suggest that linomide inhibits TNF-alpha production by suppressing the activation of NF-kappaB and mitogen-activated protein kinase (MAPK), which might, at least in part, contribute to the beneficial effects of linomide in the treatment of autoimmune diseases.     

Induction of tumor necrosis factor-alpha release by lipopolysaccharide and defined lipopolysaccharide partial structures

We have investigated the release of tumor necrosis factor-alpha (TNF-alpha) by human mononuclear cells (MNC) and isolated human monocytes/macrophages stimulated with S- and R-form lipopolysaccharide (LPS), natural lipid A, and natural and synthetic partial structures thereof. We found that LPS of Salmonella minnesota (S. min.) Rb2, which represents a partial structure of wildtype LPS of Salmonella abortus equi (S.a.e.) lacking the O-chain and parts of the outer core region, was the most active inducer of all substances tested, even more active than the wildtype LPS. Lipid A also induced the production of TNF-alpha by monocytes/macrophages but was less active than wildtype LPS. The natural Escherichia coli (E. coli) type hexaacyl lipid A (compound 506) was more active than the natural S. min. type heptaacyl lipid A (compound 516). The 1- and 4'-monodephospho partial structures (compounds 505 and 504) of E. coli lipid A were less active and represented the smallest structures tested that were able to induce TNF-alpha release by monocytes/macrophages. Synthetic tetraacyl lipid A precursor Ia of E. coli lipid A, lacking non-hydroxylated fatty acids (compound 406), and the monosaccharide precursor lipid X did not induce the release of TNF-alpha in MNC or isolated monocytes/macrophages. This might indicate that the ability of a lipid A structure to induce the release of TNF-alpha is closely connected with the conditions to be at least hexaacylated and/or to contain hydroxylated fatty acids. These results demonstrate a structure-dependent hierarchy of LPS and natural or synthetic partial structures in their capacity of inducing TNF-alpha release by monocytes/macrophages.     

Dual role of tumor necrosis factor-alpha in angiogenesis.

The role of tumor necrosis factor-alpha (TNF; cachectin) in angiogenesis has been controversial. In vitro TNF inhibits proliferation of endothelial cells (EC) whereas in the cornea it appears to stimulate vessel growth. The authors tested TNF in their recently developed disc angiogenesis system (DAS), designed to measure the proliferation of microvessels. The DAS, implanted subcutaneously in mice, was either fitted with a central pellet containing mouse recombinant TNF (mrTNF), or exposed to mrTNF delivered subcutaneously by an osmotic minipump. Low doses of mrTNF (0.01-1 ng) induced angiogenesis, which was maximum at 0.1 ng, whereas high doses (1, and 5 micrograms) inhibited it. Subcutaneous mrTNF delivered at the (high) rate of 15-60 ng/hr for 14 days inhibited angiogenesis. These results indicate bimodal, dose-dependent opposing effects and explain some of the in vitro versus in vivo paradoxical results. TNF (native or exogenous) may have opposing effects on microvessels of neoplasms and inflammatory reactions, depending on its local tissue concentrations.     

Cholera toxin activates dendritic cells through dependence on GM1-ganglioside which is mediated by NF-kappaB translocation

Cholera toxin (CT) is a potent adjuvant; however, the mechanism for its ability to enhance mucosal immunity has not been fully elucidated. We report here that CT exerts its adjuvant properties by signaling through the GM1 ganglioside receptor. When ganglioside-defective mice were given the antigen (Ag) ovalbumin (OVA) with CT by the oral route, CT failed to support either OVA-specific antibody or CD4+ T cell responses. In vitro treatment of murine bone marrow-derived dendritic cells (DC) with CT induced full maturation as evidenced by up-regulation of the costimulatory molecules, as well as by an enhanced ability to effectively present OVA for Ag-specific T cell responses. On the other hand, ganglioside-defective DC failed to differentiate to full function as Ag-presenting cells in response to CT. Since ganglioside-defective DC showed a mature phenotype after stimulation with lipopolysaccharide (LPS), the effects of CT on DC was independent of signal transduction through adjuvant receptor for LPS, the Toll-like receptor 4. Furthermore, CT also induced nuclear translocation of nuclear factor (NF)-kappaB in DC in a GM1-dependent fashion. These results highlight gangliosides expressed by DC for recognition of the non-self protein bacterial enterotoxin, which employ a unique signaling pathway to induce both innate and adaptive immunity.     

Synergism between tumor necrosis factor-alpha and interferon-gamma on macrophage activation for the killing of intracellular Trypanosoma cruzi through a nitric oxide-dependent mechanism.

Intracellular replication of the protozoan parasite Trypanosoma cruzi inside macrophages is essential for the production of the disease and the development of the parasite. Two CD4+ T cell lines, A10 and A28, were established from T. cruzi-infected BALB/c mice which specifically proliferated to parasite antigens. The trypanocidal activity of BALB/c macrophages was induced upon culture with the A10, but not with the A28 T cell line. The cell-free supernatant from this A10 line, as well as from immune spleen cells stimulated with specific antigen or concanavalin A, but not from the A28 T cell line also activated the trypanocidal activity of peritoneal macrophages or of the J774 macrophage-like cell line. when the lymphokine content of the supernatants from both cell lines was analyzed, it was found that the A10 T cell line secreted interferon (IFN)-gamma, tumor necrosis factor (TNF)-alpha and interleukin 2, whereas the A28 line did not secrete IFN-gamma upon stimulation. Furthermore, the trypanocidal-inducing ability of A10 supernatant was completely abrogated by neutralizing anti-IFN-gamma antibodies and partially abrogated by neutralizing anti-TNF-alpha antibodies. When recombinant cytokines were added to J774 cells, IFN-gamma was able to induce significant trypanocidal activity whereas TNF-alpha was almost ineffective. However, TNF-alpha or lipopolysaccharide (LPS) showed a synergistic effect with IFN-gamma on macrophage activation. IFN-gamma triggered nitric oxide (NO) synthesis by J774 cells whereas TNF-alpha was almost ineffective. TNF-alpha and LPS were also synergistic with IFN-gamma in the NO production. Both the NO production and the trypanocidal activity in J774 cells induced by T cell supernatants or lymphokine combinations were inhibited by N-monomethyl-L-arginine, a competitive inhibitor of NO synthase activity. A good correlation between the levels of NO production and trypanocidal activity induced by different lymphokine preparations was found. Those results suggest that IFN-gamma and TNF-alpha, secreted by T. cruzi-immune T cells, are involved in the activation of the trypanocidal activity of mouse macrophages through an NO-dependent mechanism.     

Treatment of cancer patients with recombinant interferon-gamma induces release of endogenous tumor necrosis factor-alpha

Study objective: 1) to investigate serum levels of tumor necrosis factor-alpha in patients treated with recombinant interferon-gamma and 2) to relate changes in TNF serum levels to other biological responses observed during treatment with interferon gamma (IFN-gamma). Patients: Five patients suffering from metastasizing renal cell carcinoma. Intervention: Each patient received three treatment cycles of 10 micrograms, 100 micrograms and 500 micrograms IFN-gamma applied three times at weekly intervals. The treatment cycles were separated by a therapy free interval of two weeks. The order of dose levels was randomly assigned to each patient. Measurements and main results: Tumor necrosis factor alpha (TNF-alpha), IFN-gamma and neopterin serum levels, monocyte counts in the peripheral blood and body temperature were measured immediately before and 4, 24, 48, 72, and 168 h after each application of IFN-gamma. Results indicated that elevated serum levels of TNF-alpha are induced by 100 micrograms and 500 micrograms IFN-gamma. Repeated application of the same dose led to downregulation of TNF release into the serum. Changes in TNF serum levels did not correlate with the magnitude of febrile reactions, neopterin production or IFN-gamma serum levels.     

Localization and post-Golgi trafficking of tumor necrosis factor-alpha in macrophages.

Tumor necrosis factor-alpha (TNF-alpha) is a proinflammatory cytokine secreted by activated macrophages. In this study, we examined the intracellular distribution and trafficking of TNF-alpha. Immunofluorescence and immunogold localization demonstrated that in lipopolysaccharide (LPS)-stimulated RAW264 macrophages, the greatest concentration of TNF-alpha is found in the perinuclear Golgi complex. Staining of the Golgi complex appeared 20 min after activation of cells and persisted for 2-12 h, and TNF-alpha appeared on the cell surface only transiently during this time. The rate of disappearance of Golgi staining correlated with the release of the cleaved, mature TNF-alpha into the medium. Pulse chase labeling and subcellular fractionation studies indicated that both 26-kDa and 17-kDa forms of TNF-alpha may be present at the level of the Golgi complex. Post-Golgi trafficking of TNF-alpha was modulated by agents that disrupt the cytoskeleton. Interferon-gamma (IFN-gamma), which primes macrophages for TNF-alpha-dependent cellular cytotoxicity, potentiated the effect of LPS by sustaining enhanced intracellular pools of TNF-alpha and also promoted redistribution of TNF-alpha into post-Golgi vesicular compartments. We propose that the primary pool of biologically active TNF-alpha in activated macrophages is held in the Golgi complex and that the cytokine is recruited directly from this intracellular pool for release in response to tumor cells or pathogens.