Molecular weight of recombinant human tumor necrosis factor-alpha

Recombinant DNA-derived human tumor necrosis factor-alpha from Escherichia coli was examined by equilibrium ultracentrifugation under conditions similar to those where gel filtration experiments suggested an oligomeric structure. Short-column equilibrium experiments at concentrations in the range 0.015-0.12% at pH 8.5 in 0.04 M Tris/Tris-HCl gave molecular weights corresponding to 3 times the sequence molecular weight both in the presence and absence of 0.1 M NaCl. Long (2.6 mm)-column experiments under the same solvent conditions indicated molecular weights of 51,900 +/- 900 in the absence of added NaCl and 52,600 +/- 700 in the presence of added 0.1 M NaCl. No evidence of any species other than the trimer was found.     

Role of single disulfide in recombinant human tumor necrosis factor-alpha

Two analogs of tumor necrosis factor-alpha (TNF-alpha) were produced by in vitro site-directed mutagenesis. In these analogs, cysteine residues at positions 69 and 101, which form a disulfide bond, were changed to alanine or leucine. CD spectra showed that the analogs are apparently similar in secondary and tertiary structure to the natural sequence TNF-alpha. In addition, the molecular size of the analogs was identical to that of the natural sequence TNF-alpha as determined by gel filtration. However, fluorescence spectra and quenching indicated that the removal of the disulfide bond alters the local conformation around tryptophan residues. The cytolytic, macrophage activation, and lipogenic activities decreased in the order of the natural sequence TNF-alpha greater than the alanine analog greater than the leucine analog, suggesting that the surface involving the disulfide bond plays a role in these biological functions and the introduced modifications decrease the activity. Differential effect of the modifications was suggested in the antiviral activity, since in this assay only the leucine analog showed significantly lower activity.     

Predominant suppression of neutrophil colony growth by recombinant human tumor necrosis factor

To investigate the suppressive effect of recombinant human tumor necrosis factor (rH-TNF) on colony growth of human granulocyte-macrophage progenitor cells (CFU-GM), cytochemical examinations of CFU-GM colonies were performed by a triple staining method. Each colony was classified into five subtypes, and the effects of rH-TNF on each subtype were analyzed. Neutrophil colony growth was inhibited by rH-TNF in a dose-dependent manner, and it was almost completely suppressed at 100 U/ml. In contrast, no significant suppressive effect of rH-TNF was found on the growth of monocyte-macrophage and eosinophil colonies at 100 U/ml or less. When recombinant human granulocyte colony-stimulating factor which almost exclusively stimulates neutrophil colony formation was used as a source of colony-stimulating activity, the total colony growth was almost completely suppressed by 100 U/ml of rH-TNF. These results indicate predominant inhibition of neutrophil colony growth by rH-TNF.     

Enhancement of neutrophil superoxide production by preincubation with recombinant human tumor necrosis factor

Tumor necrosis factor (TNF) is a 17,000-Da protein which is produced by mononuclear cells upon exposure to endotoxin. Increases in adherence, phagocytosis, hydrogen peroxide release, and lysozyme secretion have been demonstrated after prolonged incubation of human neutrophils with TNF. In this study, the ability of highly purified recombinant human TNF to modulate neutrophil responses to soluble stimuli was evaluated. Tumor necrosis factor alone (0.1 to 10,000 units/ml) failed to induce neutrophil superoxide anion (O2-) production, granule release, or aggregation when incubated for up to 25 min at 37 degrees C. TNF did, however, stimulate a significant time-, dose-, and temperature-dependent increase in neutrophil F-actin content. Although exposure of neutrophils to TNF alone caused no superoxide anion production, it enhanced the O2- production in response to the chemotactic peptide, f-methionyl-leucyl-phenylalanine (FMLP) or the tumor promotor, phorbol myristate acetate, by as much as 278%. The enhancement was time-, dose-, and temperature-dependent and was due to a more rapid initial rate of O2- production. The TNF enhancement of FMLP-induced O2- production was blocked when an anti-TNF monoclonal antibody 241-1H11, is present during the preincubation period. TNF preincubation also enhanced FMLP-induced lysozyme release, but had no effect on aggregation and actin polymerization by FMLP. The kinetics of NADPH oxidase activation by arachidonic acid was unaltered by TNF. These results indicate that brief exposures to recombinant human TNF are able to enhance or prime the neutrophil oxidative burst in response to a second stimulus.     

Differential roles of tumor necrosis factor-alpha and interferon-gamma in mouse hypermetabolic and anorectic responses induced by LPS

Lipopolysaccharide (LPS)-induced effects on energy balance are characterized by alterations in energy expenditure (hypermetabolism) and food intake (anorexia). To study the role of tumour necrosis factor alpha (TNF-alpha) on some of these metabolic responses to endotoxin, we have used transgenic mice expressing soluble tumour necrosis factor receptor-1 IgG fusion protein (TNFR1-IgG) as well as TNF-alpha knockout (KO), lymphotoxin-alpha (LT-alpha) KO, and interferon-gamma receptor (IFN-gamma R) KO mice. The results from TNFR1-IgG transgenic mice suggest that the hypermetabolic and anorectic responses induced by LPS are independently regulated since, in the absence of TNF-alpha or LT-alpha, the LPS-induced hypermetabolism is almost prevented but not the anorexia. The anorectic response shows the strongest association with IFN-gamma since both IFN-gamma R KO mice and mice treated with anti-IFN-gamma antibody showed marked reduction in the LPS-induced anorexia compared to other mice. IFN-gamma R KO mice also have an attenuated thermogenic response to endotoxin. Anti-Asialo GM1 antibody treatment attenuated both the hypermetabolic and anorectic responses to LPS, to an extent comparable to that observed in IFN-gamma R KO mice. This finding suggests that natural killer cells (lymphocytic subsets) may be involved in IFN-gamma production and play an important role in the metabolic alterations induced by LPS. We also showed that the hypermetabolic response of control mice is associated with an upregulation of cytokine expression within the brain and an increase in permeability of the blood brain barrier. LPS-induced anorexia appears to involve peripheral cytokine expression.     

Serotonin inhibition of tumor necrosis factor-alpha synthesis by human monocytes

Serotonin inhibited in a concentration dependent way (10(-3) M to 10(-10) M) the LPS induced Tumor Necrosis Factor-alpha synthesis both, when added to the monocyte cultures from the beginning and when added together with the activating stimulus 8 hours before the end of the culture. The inhibitory effect was specifically blocked by the 5-HT1 and 5-HT2 serotonin antagonist methysergide and the 5-HT2 receptor antagonist ketanserin. This indicates that only the 5-HT2 receptor family (5-HT2 or 5-HT1C) may be involved in the inhibitory effect. Serotonin seems to play an important immunomodulatory role in macrophage functions.     

Protective effect of recombinant tumor necrosis factor-alpha in murine salmonellosis

The enhancement of resistance by i.p. administration of murine rTNF-alpha into mice against i.p. challenge with virulent Salmonella typhimurium was studied. Administration of TNF-alpha (5 x 10(4) U/mouse) into mice at 6 or 12 h before the challenge with S. typhimurium organisms enhanced the bactericidal capacity in the peritoneal cavities of the mice. The diminution of the infecting organism in the peritoneal cavities of the TNF-alpha-treated mice was not due to the systemic spread of the organism inasmuch as few organism were recovered from other areas such as the spleen and liver. The TNF-alpha treatment effected a slight increase of neutrophils in the peritoneal cavity, but did not effect an increase of macrophages, including Ia-bearing macrophages. The survival rate of mice infected with Salmonella was improved by the i.p. injection of TNF-alpha before infection. Co-administration of smaller doses of TNF-alpha (5 x 10(3) U) and murine rIFN-gamma (10(2) U) at 6 h before the challenge also effectively enhanced bactericidal activity and protectivity. The cooperative effect of TNF-alpha and IFN-gamma was only seen when these recombinant cytokines were injected together at the proper time before the challenge. Injection of rabbit anti-TNF-alpha serum abolished the effects of TNF-alpha and the cooperative effect of TNF-alpha and IFN-gamma. Furthermore, the serum could abolish the cooperative effect of IFN-gamma and LPS on bactericidal activity, suggesting participation of LPS-induced TNF-alpha in the cooperation.     

Phase I study of recombinant human tumor necrosis factor-alpha in patients with advanced malignancies

A clinical phase I trial with recombinant human tumor necrosis factor-alpha (rTNF-alpha) was performed in 30 patients with advanced malignancies. The maximal tolerated dose (MTD) by 3 times weekly intramuscular (i.m.) application was 150 micrograms m-2. Main subjective toxicities including chills, fever, hypotension, fatigue, and anorexia were dose-related. In addition, transient changes in hematologic parameters and lipid metabolism were noted. Two out of 25 evaluated patients showed a minor tumor response after eight weeks of therapy. There was evidence for an improvement of in vivo immuneresponsiveness as revealed from positive delayed type hypersensitivity (DTH) skin tests of 3 out of 6 pretherapeutically anergic patients. We conclude from this phase I trial that rTNF-alpha can be safely administered at doses up to 150 micrograms m-2 i.m., 3 times weekly, without evidence of cumulative toxicity in long-term treatment.     

Effects of recombinant murine tumor necrosis factor-alpha on immune function

TNF-alpha is a macrophage-derived cytokine with diverse biologic activities, including potent immunomodulatory effects. In vitro studies have implied that TNF-alpha has predominantly proinflammatory and immunostimulatory effects, but paradoxically in vivo studies have demonstrated that administration of TNF-alpha suppresses murine lupus. To assess the effects of TNF-alpha on immune function in normal mice, we treated C57BL/6 mice with recombinant murine TNF-alpha (10 micrograms i.p.) or PBS on alternate days for up to 8 wk. Administration of TNF-alpha decreased the percentage of splenic T and B cells and increased the percentage of splenic macrophages without significantly altering the total number of mononuclear cells. Administration of TNF-alpha also caused progressive inhibition of splenic lymphocyte function, out of proportion to the quantitative reduction in B and T cells. After 8 wk of therapy, the proliferative responses of splenic lymphocytes to Con A, PHA, and LPS were reduced by 100, 90, and 60%, respectively, in treated mice compared with control mice. The reduction in T cell proliferation was due primarily to alteration of accessory cell function rather than direct inhibition of T cell function. Treatment with TNF-alpha markedly inhibited T cell cytotoxicity induced by immunization with allogenic target cells, and it virtually ablated NK cell activity. Inhibition of these in vitro tests of lymphocyte function correlated with inhibition of delayed type hypersensitivity in vivo. In contrast, treatment with TNF-alpha did not impair humoral immunity. These findings imply that TNF-alpha may affect cell-mediated immunity more profoundly than humoral immunity. This observation may be relevant to the mechanism whereby TNF-alpha suppresses murine lupus.     

Transforming growth factor-beta 1 enhances the suppression of human hematopoiesis by tumor necrosis factor-alpha or recombinant interferon-alpha

The effects of transforming growth factor-beta 1 (TGF-beta 1) on human hematopoiesis were evaluated in combination with two other regulatory cytokines, namely, recombinant human tumor necrosis factor-alpha (TNF-alpha) and recombinant human interferon-alpha (rIFN-alpha). Combinations of TNF-alpha and TGF-beta 1 resulted in a synergistic suppression of colony formation by erythroid progenitor cells (BFU-E) and an additive suppression of granulocyte-macrophage (CFU-GM) and multipotential (CFU-GEMM) progenitor cells. In addition, TGF-beta 1 synergized with rIFN-alpha to suppress CFU-GM formation, while the combined suppressive effects of both cytokines on CFU-GEMM and BFU-E were additive. When TGF-beta 1 was tested with TNF-alpha or IFN-alpha on granulocyte/macrophage colony-stimulating factor (GM-CSF)-stimulated bone marrow cells in a 5-day proliferation assay, the antiproliferative effects of TGF-beta 1 and TNF-alpha were additive, while those with TGF-beta 1 and rIFN-alpha were synergistic. A similar pattern was seen in the suppression of the myeloblastic cell line KG-1 where TGF-beta 1 in combination with TNF-alpha resulted in an additive suppression while inhibition by TGF-beta 1 and IFN-alpha was synergistic. These results demonstrate for the first time the cooperative effects between TGF-beta and TNF-alpha and IFN-alpha in the suppression of hematopoietic cell growth, raising the possibility that TGF-beta might be used in concert with TNF-alpha or IFN-alpha in the treatment of various myeloproliferative disorders.     

Tumor-stimulated release of tumor necrosis factor-alpha by human monocyte-derived macrophages.

Tumor necrosis factor-alpha (TNF) release by monocytes and macrophages may be an important determinant of the physiologic response of the host to neoplastic disease; however, the mechanisms which regulate TNF release by macrophages in hosts with neoplastic diseases are poorly understood. The purpose of this study was to determine if cell membranes and growth medium from human leukemia cell lines and solid tumor cell lines induced TNF release by cultured human blood monocyte-derived macrophages. The capacity for TNF release and direct tumor killing was highest in monocytes cultured for 7 to 11 days. Cell membranes and culture media from K562 erythroleukemia and several small cell lung carcinoma cell lines, including H82, induced the release of up to 1500 TNF units per 10(6) macrophages over 24 hr. By contrast, allogeneic peripheral blood lymphocytes, cell membranes from normal mixed donor peripheral blood leukocytes, or growth medium from normal embryonic lung fibroblasts induced the release of little or no TNF during culture up to 24 hr, suggesting that this macrophage response was specific for tumor cells. Release of TNF by tumor-stimulated macrophages was gradual, peaking 24 hr following the addition of stimuli. Induction of macrophage TNF release was concentration dependent, with half-maximal TNF levels induced by 12.5 and 25 micrograms/ml cell membranes prepared from K562 and H82, respectively. Pretreatment of tumor cell membranes with polymixin B, which inhibits many of the actions of endotoxin, failed to neutralize tumor induction of TNF, suggesting that endotoxin was not responsible for this activity. Depletion of macrophages by treatment with 3C10 monoclonal antibody and complement abrogated tumor-induced TNF release, indicating that macrophages were the source of the secreted TNF. HPLC analysis of H82 growth medium demonstrated a single peak of macrophage activating activity with approximate 40-kDa molecular weight. We have demonstrated that cell membranes and growth medium from some human leukemia and solid tumor cell lines, but not from normal human cells, induce human peripheral blood monocytes and monocyte-derived macrophages to release functionally active TNF. This process may contribute to the host response to some neoplastic diseases.     

Role of tyrosyl phosphorylation in neutrophil priming by tumor necrosis factor-alpha and granulocyte colony stimulating factor.

The ability of human tumor necrosis factor-alpha (TNF-alpha) and human granulocyte colony stimulating factor (G-CSF) to induce phosphorylation of protein tyrosyl residues in human peripheral neutrophils (PMN) was investigated by Western blot analysis with antiphosphotyrosine antibody. Both TNF-alpha and G-CSF increased the tyrosyl phosphorylation of various proteins, such as species of 54-, 63-, 72-, 83-, 98-, 108-, and 115-kDa proteins. The ligand-stimulated tyrosyl phosphorylation of the 115-kDa protein was time- and concentration-dependent. When the 115-kDa protein was phosphorylated, it was recovered from membrane fractions. The phosphorylation of the 115-kDa protein was inhibited by genistein and alpha-cyano-3-ethoxy-4-hydroxy-5-phenylthiomethylcinnamamide (ST 638), inhibitors of tyrosine kinase (TK), and was enhanced by 1-(5-isoquinoline-sulfonyl) methyl-piperazine dihydrochloride (H-7) and staurosporine, inhibitors of Ca(2+)- and phospholipid-dependent protein kinase (PKC). Similar inhibition by the TK inhibitors and stimulation by the PKC inhibitors were also observed with formylmethionyl-leucyl-phenylalanine (FMLP)-induced superoxide (O2.-) generation by TNF-alpha- or G-CSF-primed PMN. Phosphorylation of the 115-kDa protein occurred in parallel with the ligand-dependent generation of O2.-. These and other observations suggested that substrate proteins for tyrosine kinase, such as the 115-kDa protein, might play critical roles in the mechanism for priming of neutrophils. This is the first report describing that tyrosyl phosphorylation is involved in the priming of neutrophils by G-CSF and TNF-alpha.     

Enhancement of antigen- and mitogen-induced human T lymphocyte proliferation by tumor necrosis factor-alpha

The capacity of human recombinant tumor necrosis factor-alpha (rTNF alpha) to modulate human T cell proliferation was examined. To examine the effect of rTNF alpha on the responding T cell directly, T cell activation was studied in the absence of viable accessory cells (AC). Highly purified AC-depleted peripheral blood T4 or T8 cells were stimulated with immobilized monoclonal antibodies to the cluster of differentiation (CD)3 molecular complex, an AC-independent stimulus. rTNF alpha augmented anti-CD3-stimulated T4 and T8 cell proliferation. The capacity of rTNF alpha to enhance T cell proliferation varied inversely with the density of immobilized anti-CD3 and the number of responding cells in each culture. The capacity of rTNF alpha to enhance antigen-induced T4 cell proliferation was also examined. Antigen-bearing paraformaldehyde-fixed antigen-presenting cells induced modest T4 cell proliferation when cultured in flat-bottomed wells; this response was enhanced by rTNF alpha. The results demonstrate that rTNF alpha has direct effects on T cells, facilitating their capacity to proliferate in response to mitogens and antigens. These data indicate that rTNF alpha may play an immunoregulatory role, enhancing the proliferation of T lymphocytes.     

Differential priming for endotoxin-induced circulating cytokine production by tumor necrosis factor-alpha and interleukin 1 beta

In unprimed mice, a single injection of a non-lethal dose of lipopolysaccharide (LPS) produced a rise in tumor necrosis factor (TNF) and interleukin 6 (IL 6) activities. Peak serum concentrations were attained, respectively, 1.5 hr and 2.5 hr after the challenge. Pretreatment with recombinant human TNF-alpha (rHuTNF) had a priming effect for enhanced production of both serum cytokines without any change in kinetics. The enhancement was more pronounced in the TNF (15-fold) than in the IL 6 (4-fold) response. Recombinant murine TNF caused a comparable increase in LPS-induced cytokine release. In contrast, comparable pretreatment with another macrophage-derived cytokine, recombinant human interleukin 1 beta (HuIL1-beta), revealed a negative effect on LPS-induced TNF release whereas IL 6 in the blood reached levels similar to those found after priming with rTNF. Moreover, when administered in combination with rHuTNF, rHuIL1-beta inhibited the priming effect on TNF autocrine production.     

Effects of neutrophil adherence on the characteristics of receptors for tumor necrosis factor-alpha.

Human recombinant [125I]TNF-alpha was incubated with non-adherent human neutrophils, cells adherent to fibronectin-coated plastic, or adherent cells scraped into suspension (post-adherent). Binding of TNF to all cells increased with doses of added TNF but adherent cells bound little TNF. Binding of TNF by post-adherent cells was greater than when adherent, but still significantly less than that of non-adhered neutrophils, suggesting that TNF receptors were relocated on the adherent surface of neutrophils. Scatchard analysis showed that adherent cells expressed significantly fewer TNF receptors, but of higher affinity, than non-adherent cells. The results suggest that altered expression of TNF receptors might contribute to the differential effects of TNF on adherent and non-adherent neutrophils.     

Enhancement of release of granulocyte- and granulocyte-macrophage colony-stimulating factors from phytohemagglutinin-stimulated sorted subsets of human T lymphocytes by recombinant human tumor necrosis factor-alpha. Synergism with recombinant human IFN-gamma

The influence of purified recombinant human TNF-alpha (rhuTNF-alpha) was assessed, alone and in combination with purified recombinant human IFN-gamma (rhuIFN-gamma), for its effects on enhancing release from human T lymphocytes of activities that stimulate colony formation by granulocyte-macrophage, erythroid, and multipotential progenitor cells. rhuTNF-alpha or rhuIFN-gamma enhanced the release of CSF, which were determined to be granulocyte-CSF and granulocyte-macrophage-CSF by human bone marrow colony assays, morphologic assessment of colony types, and neutralization studies with rabbit anti-human granulocyte-CSF and monoclonal mouse anti-human granulocyte-macrophage-CSF. The CSF were released only when PHA was used, whether or not rhuTNF-alpha and/or rhuIFN-gamma were present while the lymphocytes conditioned the medium. T lymphocytes were sorted into subsets by using three-color immunofluorescence and a dye laser flow cytometry system with cells incubated with biotin anti-Leu-4 labeled with Texas Red, FITC-conjugated anti-Leu-3a, and phycoerythrin-conjugated anti-Leu-2a. Both the Leu-4+3a+2a- and the Leu-4+2a+3a- cells released CSF in response to PHA, but the release of CSF from PHA-stimulated lymphocytes was enhanced by rhuTNF-alpha and rhuIFN-gamma only from the Leu-4+3a+2a- subset of cells. Use of the three-color cell sorting made it highly unlikely that NK cells were involved, because both sorted subsets were positive for Leu-4. rhuTNF-alpha and rhuIFN-gamma synergized to enhance release of CSF such that low concentrations of each molecule, which were inactive when used alone, were active when the two molecules were used together. These studies suggest a role, at least in vitro, for TNF-alpha and IFN-gamma in the release of CSF from subsets of T lymphocytes stimulated with PHA.     

Trafficking of ganglioside GD3 to mitochondria by tumor necrosis factor-alpha

The interaction of mitochondria with proapoptotic proteins activates apoptosis pathways. Previous findings have identified ganglioside GD3 (GD3) as an emerging apoptotic lipid intermediate that targets mitochondria in response to death signals. Using immunoelectron and laser scanning confocal microscopy, we characterize the trafficking of GD3 to mitochondria in response to tumor necrosis factor-alpha (TNF-alpha) in rat hepatocytes. In control hepatocytes, GD3 is present predominantly at the plasma membrane as well as in the endosomal/Golgi network, as verified by its colocalization with the asialoglycoprotein receptor. Following TNF-alpha exposure, GD3 undergoes a rapid cellular redistribution with a gradual loss from the plasma membrane before its colocalization with mitochondria. This process is mimicked by acidic sphingomyelinase and ionizing radiation but not by neutral sphingomyelinase or staurosporin. TNF-alpha stimulated the colocalization of GD3 with early and late endosomal markers, Rab 5 and Rab 7, whereas perturbation of plasma membrane cholesterol or actin cytoskeleton or inhibition of glucosylceramide synthase prevented the trafficking of GD3 to mitochondria. Finally, prevention of the TNF-alpha-stimulated neosynthesis of GD3, cyclosporin A, and latrunculin A or filipin protected sensitized hepatocytes from TNF-alpha-mediated cell death. Thus, the intracellular redistribution and mitochondrial targeting of GD3 during TNF-alpha signaling occurs through actin cytoskeleton vesicular trafficking and contributes to TNF-alpha-mediated hepatocellular cell death.