Tumor necrosis factor alpha upregulates human microglial cell production of interleukin-10 in vitro.

Interleukin (IL)-10 appears to play an important regulatory role in the systemic inflammatory response; however, production of IL-10 within the human central nervous system has not been described. Using cultures of human fetal microglial cells, the resident macrophages of the brain, we investigated the production and regulation of bioactive IL-10. Lipopolysaccharide stimulated acute release of tumor necrosis factor (TNF)-alpha (peak by 8 h) and delayed production of IL-10 (over a 48-h period) in microglial cell cultures. Treatment of microglial cell cultures with TNF-alpha and IL-6 resulted in a dose-dependent release of IL-10. These cytokines also induced expression of IL-10 mRNA. Treatment of microglial cell cultures with IL-10 markedly inhibited TNF-alpha and IL-6 production. These findings suggest that during inflammation within the brain, acute release of TNF-alpha and IL-6 by activated microglia could promote subsequent release of IL-10, which functions to minimize the potential neurotoxic effects of proinflammatory cytokines.     

Defense mechanisms against bovine herpesvirus: relationship of virus-host cell events to susceptibility to antibody-complement cell lysis.

The interaction of infectious bovine rhinotracheitis virus and susceptible host cells was examined to determine whether an infected cell could be destroyed by humoral immune mechanisms before or after the transmission of virus to susceptible adjacent cells. Viral antigens were detectable on cell membranes at 6 h postinfection, but cells were not susceptible to antibody-complement lysis until 10 h postinfection. Intracellular infectious virus was also detectable at 10 h postinfection, and transmission to adjacent cells by the intracellular route began at this time. Extracellular virus was not detectable until 12 to 13 h postinfection. By the continual addition of antibody and complement, virus dissemination could be reduced more than 50-fold. These results support the hypothesis that the humoral immune mechanism may be involved in the recovery from herpesvirus infections.     

Tumor necrosis factor induces apoptosis (programmed cell death) in normal endothelial cells in vitro.

Tumor necrosis factor (TNF) is cytotoxic for many tumoral cell lines, whereas normal cells generally are considered resistant to this action. This study shows that this cytokine causes massive death of bovine endothelial cells in primary culture in a concentration- and time-dependent manner. Dying cells exhibit all the ultrastructural changes and the inter-nucleosome cleavage of DNA associated with apoptosis or 'programmed cell death.' This is the first report clearly showing a direct toxicity of TNF on endothelial cells and demonstrating that this results from the induction of the program of apoptotic death. Our observation raises the possibility that hemorrhagic necrosis in vivo, after treatment with TNF, might involve a direct cytocidal action on endothelial cells of the tumor neovasculature.     

Rat mesangial cell lysis in vitro is induced by cationic polypeptides.

Immunization and challenge with cationic proteins induces immune complex glomerulonephritis in rodents. Cationic (c) bovine gamma-globulin (BGG), when added to rat mesangial cells in vitro, induced release of lysosomal beta-glucuronidase, an enzyme capable of degrading basement membrane components. Native (n) BGG, alone or in the presence of specific antibody, did not. Morphological changes (cellular swelling) in response to cBGG suggested cell injury; indeed, significant lactate dehydrogenase (LDH) was released into the media, depending on dose, time, calcium, and temperature. Prior trypsinization of cBGG abrogated this response. The synthetic polycation, poly L-lysine > 50 kd, similarly elicited LDH release; 4-kd poly L-lysine or protamine sulfate had little or no effect. Anionic heparin sodium inhibited cBGG-induced morphological changes and, when coincubated with cBGG, significantly reduced LDH release (P < 0.0001) to levels equal to or less than those with the nBGG control. This heparin effect was lost if addition was delayed until 10 minutes after the addition of cBGG, indicating an irreversible effect of cBGG within this time. We conclude that charge alone is not sufficient for polycations to induce LDH release. Moreover, the cellular swelling and rapidity of LDH release suggest that cytotoxicity results from direct plasma membrane destruction, perhaps due to altered sodium ion concentrations.     

Interleukin-6 antagonizes tumor necrosis factor-mediated mycobacteriostatic and mycobactericidal activities in macrophages.

Interleukin-6 (IL-6) is a cytokine produced by a number of cells, including macrophages, and is directly involved in the inflammatory response. The production of IL-6 can be stimulated by monokines such as IL-1 and tumor necrosis factor (TNF). Mycobacterium avium complex organisms frequently cause disseminated disease in patients with AIDS. M. avium is an intracellular bacterium that that mainly infects macrophages. Treatment of M. avium-infected macrophage monolayers with recombinant IL-6 decreased the ability of TNF to activate cultured macrophages to inhibit growth of or kill intracellular M. avium (68% +/- 14% decrease in intracellular killing compared with that in monolayers not treated with IL-6). To further evaluate whether this effect was dependent on the down regulation of membrane receptors to TNF, we examined 125I-TNF binding to macrophages previously exposed to IL-6: the expression of TNF receptors was decreased by 78% +/- 9%. The effect of IL-6 on TNF receptors was observed after 4 h and was reversible. Infection of macrophages with different M. avium serovars was associated with release of IL-6, and IL-6 production peaked at 48 h after infection in concentrations ranging from 328 +/- 87 ng/10(5) cells to 907 +/- 224 ng/10(5) cells. IL-6 did not have any influence on the rate of growth of the tested strains of M. avium within or outside macrophages. These results suggest that release of IL-6 by M. avium-infected macrophages may influence the host's immune response and the outcome of the disease.     

Chlorpromazine protection against interleukin-1 and tumor necrosis factor-mediated activities in vivo.

Interleukin (IL-1) and tumor necrosis factor (TNF) are thought to play a key role in septic shock and inflammation. We had previously shown that chlorpromazine (CPZ) has a protective effect in various models of endotoxic shock and IL-1 toxicity. We have tested the effect of CPZ on several activities of IL-1 in vivo. CPZ (4 mg/kg) inhibited increases in serum corticosterone, triglycerides and serum amyloid A (SAA). Chlorpromazine also antagonized these same effects when they were induced by endotoxin or TNF, suggesting that this activity could be implicated in the protective effect of CPZ in various models of endotoxic shock and IL-1 lethality.     

Mechanical haemolysis in shock wave lithotripsy (SWL): II. In vitro cell lysis due to shear

In this work we report injury to isolated red blood cells (RBCs) due to focused shock waves in a cavitation-free environment. The lithotripter-generated shock wave was refocused by a parabolic reflector. This refocused wave field had a tighter focus (smaller beam width and a higher amplitude) than the lithotripter wave field, as characterized by a membrane hydrophone. Cavitation was eliminated by applying overpressure to the fluid. A novel passive cavitation detector (HP-PCD) operating at high overpressure (up to 7 MPa) was used to measure acoustic emission due to bubble activity. The typical 'double-bang' emission measured in the lithotripter free-field was replaced by a continuum of weak signals when the fluid was enclosed in a pressure chamber. No acoustic emissions were measured above an overpressure of 5.5 MPa. Aluminium foils were used to study shock wave damage and had distinct deformation features corresponding to exposure conditions, i.e. pitting and denting accompanied by wrinkling. Pitting was eliminated by high overpressure and so was due to cavitation bubble collapse, whereas denting and wrinkling were caused by the reflected shock wave refocused by the parabolic reflector. RBCs suspended in phosphate-buffered saline (PBS) were exposed to the reflected wave field from a parabolic reflector and also from a flat reflector. Exposure to the wave field from the parabolic reflector increased haemolysis four-fold compared with untreated controls and was twice that of cell lysis with the flat reflector. Recently we analysed deformation and rupture of RBCs when subjected to a flow field set up by a focused shock. The cell lysis results presented here are in qualitative agreement with our theoretical prediction that haemolysis is directly related to the gradient of shock strength and validates shearing as a cell lysis mechanism in SWL.     

Tumor necrosis factor enhances endothelial cell susceptibility to oxygen toxicity: role of glutathione

The effect of tumor necrosis factor-alpha (TNF) on hyperoxia-induced endothelial injury in vitro was investigated. TNF caused a time- and dose-dependent reduction in the number of viable pulmonary artery endothelial cells. The TNF-mediated endothelial cytotoxicity was more pronounced under hyperoxia (95% O2 and 5% CO2) than under normoxia (95% air and 5% CO2). Pretreatment of endothelial cells with TNF (0.01 micrograms/ml or 240 U/ml) for 18 h at normoxia reduced the intracellular concentration of total glutathione (GSH), whereas the concentration of oxidized GSH was increased. These TNF-treated endothelial cells were more susceptible to hyperoxia- or hydrogen peroxide-mediated cytotoxicity. TNF also induced changes in endothelial morphology and in the distribution and density of actin filaments. Exogenous GSH or L-2-oxothiazolidine-4-carboxylate, which enhanced endothelial GSH concentrations, partially protected endothelial cells against TNF-mediated cytotoxicity, morphologic changes, and actin filament redistribution, especially under the hyperoxic condition. These results suggest an important role of GSH in modulating endothelial response to TNF.     

Tumor necrosis factor production by human macrophages stimulated in vitro by Plasmodium falciparum.

Production of tumor necrosis factor by human macrophages may be induced in vitro by cytoadherent and noncytoadherent strains of Plasmodium falciparum, with an optimal ratio of one to three parasitized erythrocytes per macrophage. Centrifuged and heated crude culture supernatants have the same effect, thus showing the existence of a thermostable soluble factor able to induce this expression. In vitro kinetic experiments have shown that the secretion of tumor necrosis factor appears early, with a maximal peak at 8 h.     

Tumor necrosis factor-mediated inhibition of interleukin-18 in the brain: a clinical and experimental study in head-injured patients and in a murine model of closed head injury.

Tumor necrosis factor (TNF) and interleukin-(IL)-18 are important mediators of neuroinflammation after closed head injury (CHI). Both mediators have been previously found to be significantly elevated in the intracranial compartment after brain injury, both in patients as well as in experimental model systems. However, the interrelation and regulation of these crucial cytokines within the injured brain has not yet been investigated. The present study was designed to assess a potential regulation of intracranial IL-18 levels by TNF based on a clinical study in head-injured patients and an experimental model in mice. In the first part, we investigated the interrelationship between the daily TNF and IL-18 cerebrospinal fluid levels in 10 patients with severe CHI for up to 14 days after trauma. In the second part of the study, the potential TNF-dependent regulation of intracerebral IL-18 levels was further characterized in an experimental set-up in mice: (1) in a standardized model of CHI in TNF/lymphotoxin-alpha gene-deficient mice and wild-type (WT) littermates, and (2) by intracerebro-ventricular injection of mouse recombinant TNF in WT C57BL/6 mice. The results demonstrate an inverse correlation of intrathecal TNF and IL-18 levels in head-injured patients and a TNF-dependent inhibition of IL-18 after intracerebral injection in mice. These findings imply a potential new anti-inflammatory mechanism of TNF by attenuation of IL-18, thus confirming the proposed "dual" function of this cytokine in the pathophysiology of traumatic brain injury.     

Tumor necrosis factor alpha in children with sickle cell disease in stable condition.

Tumor necrosis factor alpha (TNF-alpha) is known to induce wasting in humans and animals. This study was undertaken to determine TNF-alpha concentrations in children with sickle cell disease (SCD) and whether high TNF-alpha levels are more likely to be present in children with growth deficits, infection, or pain crisis. Tumor necrosis factor alpha was measured using enzyme immunoassay in 143 blood samples obtained from 101 children. Mean TNF-alpha levels were higher in patients (50 pg/mL) than in 21 control children (19 pg/mL) and in 26 laboratory employees (20 pg/mL). During the follow-up period, 35%, 38%, and 28% of children with SCD had infection, pain crisis, or a blood transfusion, respectively. Mean TNF-alpha concentrations were higher in children who had an infection than in those who did not. No significant effect of pain crisis or blood transfusion was observed. Tumor necrosis factor alpha concentrations were above normal (> 40 pg/mL) in 15% of controls, 34% of children with SCD, and 52% of children with SCD who had an infection and 33% of those who did not. A higher percentage of children who had elevated TNF-alpha levels had weight (46% versus 31%) or height (50% versus 28.6%) deficits than children who had normal TNF-alpha levels. These results indicate that most children with SCD in stable condition have normal TNF-alpha concentrations and that those with high TNF-alpha levels are more likely to have growth deficits.     

Cell-mediated cell lysis in vitro: genetic control of killer cell production and target specificities in the mouse

The requirements for killer cell production in the course of a mixed leukocyte reaction and the specificity of target cell (PHA-blasts) lysis in the mouse were investigated using inbred strains carrying intra-H-2 recombinant chromosomes. Strong lytic activity was generated in all, and only those, responder-stimulator combinations which differed at either the H-2D or the H-2K, or both regions, even if the MLR incompatibility between responder and stimulator was very weak. Killing activity was specific and directed against determinants controlled by genes in the H-2K and H-2D regions. The slope of the killer dose-response curves is the same for either type of specificity. Quantitative comparison of the lytic activity of a given killer cell population on different targets demonstrated a dose effect of the number of specificities recognized. No significant killing against the Ir or the Ss-Slp regions of the H-2 complex could be detected. Anti? H? 2 sera, if directed against the killer cells, do not inhibit their activity, while they can block killing, if directed against the target. This inhibition is specific in that a serum that blocks killing against the H-2K specificity of a given target does not inhibit the lytic activity directed against the H-2D determinants on the same target.     

Tumor necrosis factor-related apoptosis-inducing ligand and chemotherapy cooperate to induce apoptosis in mesothelioma cell lines

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) can induce apoptosis in certain tumor cells. In addition, TRAIL and chemotherapy can act cooperatively, possibly as a result of chemotherapy-induced increases in expression of a TRAIL receptor, DR5. We used cell lines derived from a highly chemoresistant tumor, malignant mesothelioma, to learn whether TRAIL was effective alone or together with chemotherapy and whether cooperativity depended on increases in DR5 expression. TRAIL (codons 95-285) was expressed in a bacterial expression vector and purified by nickel affinity chromatography. TRAIL alone (25 to 500 ng/ml) had little effect on mesothelioma cells. TRAIL plus chemotherapy (doxorubicin, cis-platinum, etoposide, or gemcitabine) acted cooperatively to induce apoptosis in mesothelioma cells (M28, REN, VAMT, and MS-1). For example, in M28 cells treated for 18 h, apoptosis from TRAIL (100 ng/ml) plus doxorubicin (0.6 microg/ml; 71 +/- 11%) greatly exceeded that from TRAIL alone (21 +/- 8%) or from doxorubicin alone (6 +/- 2%) (means +/- standard deviation; P < 0.03). Mesothelioma cells treated with chemotherapy showed no change in DR5 protein by Western analysis or by immunocytochemistry. TRAIL plus chemotherapy was associated with an increase in mitochondrial cytochrome c release and mitochondrial depolarization. We conclude that TRAIL and chemotherapy act cooperatively to kill mesothelioma cell lines, not by increases in DR5 receptor but in association with mitochondrial amplification of apoptotic signals.     

Tumor necrosis factor-alpha induces cell type and tissue-specific expression of chemoattractant cytokines in vivo.

Recombinant murine tumor necrosis factor-alpha (TNF-alpha) was shown to be a strong, systemic stimulus in vivo for members of the chemoattractant cytokine gene families (JE, KC, IP-10). The three genes showed differential sensitivity to TNF-alpha, and their expression demonstrated differential tissue specificity. IP-10 was the most strongly induced messenger RNA and was seen in the liver, kidney, and spleen but very poorly in the lung or skin. JE exhibited a similar pattern, though the magnitude of expression was markedly lower. KC expression was seen only in the liver of TNF-alpha-treated mice. The time course of expression for IP-10 was rapid and transient and showed strong dose dependence. In mice treated with TNF-alpha intravenously, messenger RNA was localized in the splenic stroma but not in adherent macrophages or nonadherent lymphocytes. In situ hybridization found the majority of intercrime expression in the splenic red pulp with little or no expression seen in the white pulp. In vitro, TNF-alpha was a potent stimulus of chemoattractant messenger RNA expression in fibroblasts but not in inflammatory peritoneal macrophages. These results indicate that TNF-alpha may be an important stimulus for chemoattractant cytokine gene expression in vivo, and the primary cell types responsible may be either stromal fibroblasts, microvascular endothelium, and/or a subset of anchored mononuclear phagocytes.     

JNK and p53 stress signaling cascades are altered in MCF-7 cells resistant to tumor necrosis factor-mediated apoptosis.

Tumor necrosis factor (TNF) signal transduction is a complex process involving activation of receptor-linked and stress-sensitive signaling cascades that stimulate apoptosis in some tumor cell lines. Initial studies suggested that these signaling events cooperatively induced TNF responses, but recent studies suggest that some of these signals antagonize the apoptotic response or play no discernible role in cell death. As TNF induces cellular stress and activates several stress-sensitive cascades that may play a role in apoptosis, TNF-induced stress signaling was examined in MCF-7 cells and compared with a variant MCF-7 cell line resistant to TNF-mediated apoptosis (MCF-7/3E9). TNF rapidly stimulated both NF-kappaB and JNK activation in MCF-7 and MCF-7/3E9 cells, but JNK activation was significantly reduced (threefold) in apoptotically resistant cells. TNF also stimulated p53, p21WAF1, and Bax accumulation with subsequent PARP cleavage and nucleosomal DNA laddering in MCF-7 cells but did not stimulate these processes in MCF-7/3E9 cells. Importantly, 3E9 cells retained wild-type p53 function, induced p21WAF1 in response to DNA damage, and expressed almost equal sensitivity to other stress stimuli (gamma-radiation, chemotherapeutic agents) as parental MCF-7 cells. These results suggest that selective defects in TNF-activated stress cascades are associated with reduced sensitivity to TNF but not other cell death stimuli. Loss of potent TNF-mediated activation of JNK and p53 cascades may permit tumor cells to evade receptor-mediated apoptosis but have only limited influence on cellular sensitivity to other agents that effectively engage these stress pathways.     

Tumor necrosis factor and interferon-gamma induce distinct patterns of endothelial activation and associated leukocyte accumulation in skin of Papio anubis.

Recombinant human interferon (IFN)-gamma (2 X 10(4) or 2 X 10(5) U), tumor necrosis factor (TNF, 10(4) or 10(5) U), or both were injected intracutaneously into baboons (Papio anubis), and biopsies were examined at various intervals for evidence of altered endothelial cell antigen expression, endothelial morphology, and leukocyte infiltration. IFN-gamma induced increased binding of anti-HLA-DP mAb by 24 hours and a mild-to-moderate accumulation of mononuclear cells. TNF induced increased binding of anti-endothelial leukocyte adhesion molecule (ELAM)-1 mAb by 2 hours that was associated with polymorphonuclear leukocyte accumulation, and increased binding of anti-intercellular adhesion molecule (ICAM)-1 mAb by 9 hours that was associated with the onset of progressive mononuclear leukocyte accumulation. TNF also caused endothelial cell hypertrophy and increased vascular permeability. The combination of IFN-gamma and TNF induced a set of changes that qualitatively resemble those of a delayed hypersensitivity reaction to simian agent 8 envelope antigen. These findings are consistent with the concept that cytokine-activated endothelium plays an important role in the adhesion and subsequent extravasation of leukocytes during immune inflammation.     

Tumor necrosis factor alpha and interleukin 1 alpha enhance lipopolysaccharide-mediated bovine endothelial cell injury.

Alveolar macrophages (AMs) are important in the host response to aerogenous pulmonary bacterial infections, such as Pasteurella haemolytica-induced pneumonia in cattle. Previous work has shown that AMs enhance P. haemolytica-mediated pulmonary endothelial cell (EC) damage in vitro. The purpose of this study was to determine the mechanism of AM-enhanced EC damage using an in vitro AM-EC coculture system consisting of AMs cultured on culture plate insert membranes and ECs in the underlying chamber. The addition of lipopolysaccharide (LPS) to the culture plate insert chamber resulted in EC damage indicated by 51Cr release, which was enhanced in the presence of AMs. To determine the role of AM-secreted cytokines, recombinant human interleukin 1 alpha (IL-1) or tumor necrosis factor alpha (TNF) was added to ECs simultaneously with varying concentrations of LPS. Although TNF and IL-1 alone had only marginal toxic effects on ECs, the simultaneous treatment of TNF or IL-1 with LPS greatly increased the LPS cytotoxic effect on ECs. In addition, IL-1 receptor antagonist eliminated the IL-1 enhancement of LPS-mediated EC toxicity. These results suggest that macrophage-secreted cytokines synergistically enhance LPS-mediated pulmonary EC damage.