Requirement of endogenous tumor necrosis factor/cachectin for recovery from experimental peritonitis

By intrasplenic immunization we raised a rat mAb (mAb V1q; IgG2a, kappa) with a potent neutralizing activity against natural mouse TNF (1 microgram/ml mAb V1q/100 U/ml TNF). mAb V1q was used to study the role of endogenous TNF in experimental peritonitis induced by sublethal cecal ligation and puncture. mAb V1q persisted for over 5 days in the serum of mice injected with 100 micrograms of the antibody and, therefore, proved useful for in vivo experiments. As little as 20 micrograms mAb V1q/mouse prevented lethal shock of the animals by 400 micrograms LPS/mouse. In sublethal cecal ligation and puncture i.p. injection of mAb V1q directly and up to 8 h after induction of experimental peritonitis lead to death of the animals within 1 to 3 days. The lethal effect of mAb V1q was compensated by injection of recombinant mouse TNF. Similar mAb V1q effects as in immunocompetent mice were shown in severe combined immune deficiency mice deficient of mature functional B and T cells. Taken together, these data suggest that during the early phase of peritonitis endogenous TNF may stimulate nonlymphoid cells such as granulocytes, macrophages, platelets, and fibroblasts to ingest bacteria and to localize inflammation, respectively. These beneficial effects of TNF may determine survival. Thus, our data may have implications for the therapeutic management of a beginning peritonitis.     

Lysates of Sarcocystis cruzi bradyzoites stimulate RAW 264.7 macrophages to produce tumor necrosis factor (cachectin)

Lysate of Sarcocystis cruzi bradyzoites induced RAW 264.7 macrophages to release a factor that killed L 929 cells. Rabbit antibody to murine tumor necrosis factor (TNF) inhibited this killing effect, indicating that the factor causing cell death was similar to TNF (also called cachectin). Based on the present findings we hypothesize that TNF mediates, at least in part, the disease associated with this parasitic infection.     

Crystallization of trimeric recombinant human tumor necrosis factor (cachectin)

Crystals of tumor necrosis factor (TNF) have been obtained in two forms. Rhombohedral crystals grow in 1.8 to 2.0 M ammonium sulfite, pH 7.8 at 21 degrees C, and tetragonal crystals grow in 2.6 M magnesium sulfate, pH 5.5 at 25 degrees C. Analysis of TNF by isoelectric focusing under native and denaturing conditions indicates that TNF molecules exist as trimers in solution. The rhombohedral cachectin crystals belong to space group R3 and have unit cell constants a = b = c = 47.65 A and alpha = beta = gamma = 88.1 degrees. Density determinations and the space group indicate that the unit cell contains one 51,000-dalton trimer. These crystals are stable in the x-ray beam and diffract to at least 1.85 A but are apparently twinned by merohedry. The tetragonal crystals are space group P4(3)2(1)2 or its enantiomorph P4(1)2(1)2 and have unit cell constants a = b = 95.08, c = 117.49. The asymmetric unit contains one trimer; the crystals are stable in the x-ray beam and diffract to beyond 3 A.     

Production of a factor (CIF) from normal fibroblast cells inhibiting tumor necrosis factor/cachectin production

Murine embryonic fibroblast cells produce a factor designated cytotoxin-inhibiting factor (CIF) which inhibits tumor necrosis factor (TNF) and interleukin 1 production as well as tumoricidal activity by lipopolysaccharide-activated macrophages. This study determines the physiologic conditions of CIF production in serum-free medium. CIF production was largely dependent upon the presence of lipopolysaccharide. A quantitative correlation between fibroblast cell number, lipopolysaccharide concentration, and incubation time was established. Evidence is presented that CIF inhibited the production or release of TNF. CIF did not destroy TNF after production and release nor did it sequester secreted TNF. The supernatant fluids which inhibited TNF production did not suppress the capability of resting macrophages to phagocytize opsonized sheep erythrocytes, suggesting that only functions expressed in the activated state are inhibited.     

The effect of tumor necrosis factor/cachectin on follicle-stimulating hormone-induced aromatase activity in cultured rat granulosa cells

We investigated the effects of tumor necrosis factor (TNF)/cachectin on follicle-stimulating hormone (FSH)-induced aromatase activity in cultured rat granulosa cells using the stereospecific transfer of 3H from [1 beta-3H] androstenedione into 3H2O. TNF (10 pg/ml-10 ng/ml) inhibited FSH (250 ng/ml)-induced aromatase activity in a concentration-dependent manner, and 10 ng/ml of TNF completely abolished the FSH-induced aromatase activity. A time course analysis of the effects of TNF showed that TNF had no effect on induced aromatase activity, but inhibited the further induction of the enzyme by FSH. TNF (10 ng/ml) also inhibited the ability of TGF beta (1 ng/ml) to enhance aromatase activity and increase progesterone synthesis. Thus, TNF is a component of the complex array of proteins that modulate ovarian function and, as such, may play a physiological role in the regulation of the granulosa cell. In view of its association with cachexia, it may also play a pathophysiological role in the suppression of reproductive function during chronic illness.     

Regulation of tumor necrosis factor gene expression and protein synthesis in murine macrophages treated with recombinant tumor necrosis factor

The effect of murine rTNF-alpha on c-fos and TNF mRNA accumulation and protein synthesis was investigated in bone marrow-derived macrophages to examine the mechanism(s) by which TNF modulates macrophage activity. A rapid and transient expression of the c-fos gene was induced by murine rTNF. This was blocked by 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine, an inhibitor of protein kinase C, suggesting that the murine rTNF stimulated a protein kinase C-dependent signal transduction pathway. Although LPS induced the accumulation of one TNF mRNA species, murine rTNF induced the synthesis of two distinct TNF mRNA species. Both LPS- and murine rTNF-induced TNF mRNA accumulation was equally enhanced by pretreatment with mouse rIFN-gamma. In contrast, cycloheximide pretreatment had little effect on murine rTNF-induced TNF mRNA accumulation, whereas this treatment increased LPS-induced TNF mRNA by sevenfold. These results argue that TNF mRNA accumulation can be modulated in macrophages by distinct mechanisms. As assessed by Western blot and immunoprecipitation analysis, LPS stimulated the synthesis of both cell-associated and secreted forms of TNF protein. In comparison, newly synthesized TNF protein was not detected when macrophages were treated with murine rTNF alone or in combination with murine rIFN-gamma. This demonstrates that although murine rTNF stimulated the synthesis of two distinct TNF mRNA species, additional signal(s) are necessary for their translation into protein and that such signals are present after LPS stimulation.     

Alternative cleavage of the cachectin/tumor necrosis factor propeptide results in a larger, inactive form of secreted protein

Cachectin/tumor necrosis factor is initially synthesized as a 26 kilodalton prohormone. This molecule is 76 (human) or 79 (mouse) amino acids longer than the mature protein, as a result of additional residues present at the amino terminus. A short hydrophobic stretch of amino acids serves to anchor the prohormone in lipid bilayers; the mature protein, as well as a partially processed form of the hormone, are secreted after cleavage of the propeptide. We have analyzed the cleavage of the murine propeptide as it occurs in RAW 264.7 cells and now report that scission occurs at a site within the propeptide fragment, 10 residues before the amino-terminal leucine of the mature protein. This incompletely processed form of the molecule also begins with a leucine residue. Although secreted as a soluble product, it is biologically inactive in the L-929 cell cytotoxicity assay.     

Sequence of the tumor necrosis factor/cachectin (TNF) gene from Peromyscus leucopus (family Cricetidae)

The nucleotide sequence data reported in this paper have been submitted to the GenBank nucleotide sequence database and have been assigned the accession number M59233.     

Complex regulation of tumor necrosis factor mRNA turnover in lipopolysaccharide-activated macrophages

The turnover of tumor necrosis factor (TNF) mRNA in permanently transfected macrophages of the RAW 264.7 cell line was studied directly (by Northern blot analysis using a probe specific for TNF) and indirectly (through studies of the turnover of various reporter mRNAs, either containing or lacking the TNF 3' untranslated region (UTR)). The TNF mRNA was found to be very unstable in RAW 264.7 cells. Instability appeared to result from two distinguishable nucleolytic processes. The major degradative process involved was not specific for the TNF 3' UTR of reporter mRNAs, and was inhibited by actinomycin D pretreatment. It appeared to be expressed constitutively, in that cell activation by lipopolysaccharide (LPS) did not modify message stability. When cells were treated with actinomycin D, a minor nucleolytic activity was 'uncovered'. This minor activity was noted to increase with time following LPS activation. It also exhibited specificity, in that reporter mRNAs bearing the 3' UTR of TNF were more susceptible to degradation in the presence of actinomycin D than were constructs lacking the 3' UTR of TNF. Thus, TNF mRNA turnover appears complex, and depends upon at least two separable degradative pathways. The TNF 3' UTR apparently contributes only modestly to the instability of this mRNA under normal conditions.     

Disparate effects of tumor necrosis factor-alpha/cachectin and tumor necrosis factor-beta/lymphotoxin on hematopoietic growth factor production and neutrophil adhesion molecule expression by cultured human endothelial cells

Tumor necrosis factor-alpha/cachectin (TNF-alpha) and tumor necrosis factor-beta/lymphotoxin (TNF-beta) are inflammatory mediators with similar spectrums of cytotoxic activity against tumors in vitro and in vivo. We compared the effect of purified recombinant human TNF-alpha and TNF-beta on neutrophil adhesion molecule expression and hematopoietic growth factor production by cultured human umbilical vein endothelial cells. Endothelial cells acquired adhesive properties for neutrophils after a 4-hr incubation with as little as 5 U/ml TNF-alpha. TNF-alpha stimulated a dose-dependent increase in endothelial cell adhesiveness for neutrophils, with a maximal effect at 250 U/ml. In contrast, TNF-beta did not enhance endothelial-dependent neutrophil adherence until a concentration of 600 to 1200 U/ml was reached. Endothelial cells cultured for 24 hr with TNF-alpha, 10 to 1,000 U/ml, released hematopoietic colony-stimulating activity. TNF-beta failed to augment growth factor production by endothelial cells at any concentration tested. Inhibitor assays showed that the absence of detectable colony-stimulating activity was not due to direct inhibition of colony growth by TNF-beta or to release of hematopoietic inhibitors by the TNF-beta-stimulated endothelial cells. Purified natural TNF-beta was similar to recombinant TNF-beta in its effect on neutrophil adhesion molecule expression and growth factor production by endothelial cells. These results indicate that the two immunomodulatory proteins TNF-alpha and TNF-beta differ in their effects on a common target tissue. TNF-beta, which retains tumoricidal properties, shows fewer proinflammatory activities on cultured endothelial cells than TNF-alpha in vitro.     

Effect of tumor necrosis factor/cachectin on the activity of the low density lipoprotein receptor on human skin fibroblasts

We have investigated the effects of recombinant human tumor necrosis factor (TNF)/cachectin on the cellular binding of human low density lipoprotein (LDL) to human skin fibroblasts. When recombinant TNF was added to cultured cells, LDL binding doubled after 24 h of incubation. The effect of TNF was dose-dependent and its maximal effect was observed at concentrations of 1-10 ng/ml. TNF also stimulated the growth of human skin fibroblasts 1.6-fold. These results indicate that TNF increases LDL-receptor activity, which might be related to its stimulatory effect on cell growth.     

单核／巨噬细胞分泌TNF—α的调控及在老年时的改变

肿瘤坏死因子－α（ＴＮＦ－α）是一种主要由单核／巨噬细胞分泌的细胞因子。它不仅有杀伤肿瘤细胞的特性,还在炎症、休克和自身免疫等病理过程中起重要作用。刺激巨噬细胞分泌ＴＮＦ－α的主要物质有内毒素、病毒、某些细胞因子和有丝分裂原以及神经肽等。而前列腺素Ｅ（ＰＧＥ）、ｃＡＭＰ类似物和肾上腺素类等可抑制其分泌。很多文献报道ＴＮＦ－α在老年时单核／巨噬细胞的分泌有改变。     

Lipoproteins of Borrelia burgdorferi and Treponema pallidum activate cachectin/tumor necrosis factor synthesis. Analysis using a CAT reporter construct

Lipoproteins from two pathogenic spirochetes (Borrelia burgdorferi and Treponema pallidum) induced the biosynthesis of TNF in murine macrophages and in permanently transformed macrophages of the cell line RAW 264.7. Induction was studied by measuring the secretion of biologically active TNF and by measuring the activity of the reporter enzyme chloramphenicol acetyltransferase (CAT) produced within macrophages transfected with an endotoxin-responsive CAT construct. Several lines of evidence indicated that the induction of TNF and CAT was attributable to the spirochete lipoproteins rather than to contaminating or endogenous LPS: 1) the dose response curves observed for the lipoproteins were markedly different from those obtained with LPS; 2) lipoprotein-mediated activation was unaffected by amounts of polymyxin B that completely neutralized the induction of TNF and CAT by LPS, 3) low concentrations of the lipoproteins induced TNF in macrophages from endotoxin-unresponsive C3H/HeJ mice as effectively as in macrophages from normal C3H/HeN mice, and 4) isolated spirochete lipoproteins, but not a non-lipoprotein immunogen, were potent inducers of CAT in the transformed macrophages. Moreover, LPS was not detected in the B. burgdorferi lipoprotein mixtures by Limulus amebocyte lysate assay. Proteolytic digestion of the intact bacterial protein preparations only modestly diminished their ability to activate the cells, suggesting that small lipopeptides comprise the biologically active portions of the molecules, as is the case with the murein lipoprotein of Escherichia coli. Through their ability to induce TNF production by macrophages, spirochete lipoproteins may play important roles in the development of the local inflammatory changes and the systemic manifestations that characterize syphilis and Lyme disease.     

Effect of dietary alpha-linolenate/linoleate balance on lipopolysaccharide-induced tumor necrosis factor production in mouse macrophages

We examined the effect of dietary alpha-linolenate (18:3n-3)/linoleate (18:2n-6) balance on lipopolysaccharide (LPS)-induced tumor necrosis factor (TNF) production in mouse macrophages. Resident and casein-induced peritoneal macrophages from mice fed a high alpha-linolenate diet produced a higher amount of TNF than in the high linoleate diet group. However, TNF production was not affected by the dietary alpha-linolenate/linoleate balance when thioglycollate- and complete Freund's adjuvant-induced macrophages were stimulated with LPS. Serum TNF levels of mice intraperitoneally injected with LPS was also higher in the high alpha-linolenate group than in the high linoleate group. These diets affected the n-3/n-6 ratios of 20 and 22 carbon highly unsaturated fatty acids in macrophage lipids. Thus, the dietary enrichment with alpha-linolenate was found to enhance TNF production of macrophages isolated under limited conditions.     

Fc-receptor cross-linking induces rapid secretion of tumor necrosis factor (cachectin) by human peripheral blood monocytes

In this study it was demonstrated that cross-linking of FcR on human monocytes induces the secretion of the cytotoxic and immunoregulatory cytokine TNF. Both soluble and insoluble immune complexes, solid-phase antibody and antibody-coated phagocytizable particles were used to cross-link FcR on monocytes. It was observed that monocytes secreted large amounts of TNF in each of these instances. Kinetic studies performed with soluble immune complexes showed that TNF was secreted very rapidly, e.g., within 2 h after addition of immune complexes to monocytes. These findings are relevant for the understanding of FcR-mediated immune responses by monocytes and macrophages, for example antibody-dependent cellular cytotoxicity and phagocytosis, and for disease states associated with circulating or tissue-fixed immune complexes.