Necrotizing activity of tumor necrosis factor: histopathological investigation using Meth A sarcoma and granulation tissue

The action of tumor necrosis factor (TNF) was investigated histopathologically in mice using methylcholanthrene A (Meth A)-induced sarcomas and granulation tissue induced by autotransplantation of fragments of liver and spleen. Highly purified murine TNF caused hemorrhagic necrosis of both the tumors and the granulation tissue. Proliferation of tumor capillaries, demonstrated microangiographically, occurred 2 h after TNF administration and hyperemia of tumor vessels was obvious after 3-6 h. Hyperemia and capillary leakage were also observed in the granulation tissue 6 h after TNF injection and hemorrhage was noted in the epidermis after 12 h. These results strongly suggest that the in vivo necrotizing action of TNF is mainly related to capillary injury.     

Regulation of tumor necrosis factor (TNF) expression: interferon-gamma enhances the accumulation of mRNA for TNF induced by lipopolysaccharide in murine peritoneal macrophages

The secretion of tumor necrosis factor (TNF) by macrophages is initiated by lipopolysaccharide (LPS); considerable evidence indicates that such secretion can be potentiated by interferon-gamma (IFN-gamma). The present studies show that accumulation of mRNA for tumor necrosis factor, which represents an important regulatory focus for controlling secretion of TNF, is enhanced by physiologic doses of IFN-gamma (20 units/ml of purified recombinant IFN-gamma). mRNA for TNF induced by LPS, which was maximal 2 hr after LPS was applied to the cells, was enhanced 5- to 8-fold by IFN-gamma as determined by Northern blot analysis. Interferon did not change the kinetics of accumulation but did change the dose effects of LPS in that increasing amounts of LPS led to increasing amounts of TNF mRNA in IFN-gamma-treated macrophages. IFN-gamma itself, however, did not induce expression of TNF mRNA. These studies document that IFN-gamma potentiates the cytoplasmic accumulation of mRNA for TNF induced in murine peritoneal macrophages by LPS.     

肿瘤坏死因子(TNF)的制备、纯化及其抗肿瘤活性的实验研究

 本文报道用卡介苗(BCG)和内毒素(LPS)诱导兎产生瘤肿坏死因子(TNF),获得含TNF血清(TNS)。TNS序顺通过DEAE-sephadex、SephadexG-200、Sephadex G-75和con-A-Agarose柱层析纯化,可获得单一电泳纯的TNF(SDS-PAGE和IEF),其分子量为67kD,等电点为pH5.2。抗肿瘤效应的结果表明,人胃癌细胞株:FGC-85和MGC-803对TNS/TNF敏感,SGC-7901不敏感;TNS/TNF可使小鼠移植性实体瘤(S_(180)、S_(37)、L_(Ⅱ)、U_(14)和黑色素瘤)发生出血性坏死,其中S_(180)对TNF最敏感。     

Differential cardiovascular effects of endotoxin derived from Escherichia coli or Pseudomonas aeruginosa.

Sepsis is characterized by various symptoms, signs and underlying pathophysiology. To investigate possible mechanisms underlying this diversity, we compared the cardiovascular effects of lipopolysaccharide (LPS) derived from Escherichia coli (E-LPS) with those of LPS from Pseudomonas aeruginosa (P-LPS) in rats. We also examined the possible roles of tumor necrosis factor-alpha (TNF-alpha) and oxidative stress in LPS-induced cardiovascular damage. E-LPS (10 mg/kg body weight) or P-LPS (2 mg/kg body weight) was administered intravenously to Wistar rats. Echocardiography was serially performed. E-LPS induced an increase in left ventricular fractional shortening that persisted for at least 6 h, whereas P-LPS elicited an initial increase and a subsequent decrease in this parameter. Histological analysis revealed that P-LPS induced interstitial edema, congestion, intramyocardial bleeding, myocardial necrosis, infiltration of inflammatory cells, and formation of fibrin thrombi in the heart, whereas no pathological changes were apparent in the hearts of rats treated with E-LPS. Furthermore, the plasma concentration of TNF-alpha in rats treated with P-LPS was greater than that in rats treated with E-LPS, but the glutathione redox ratio in the heart was not affected by either type of LPS. In conclusion, E-LPS and P-LPS induced distinct patterns of functional and structural responses in the cardiovascular systems of rats. These differential responses may be attributable in part to the difference in the associated increases in the plasma concentration of TNF-alpha. The cardiovascular effects of LPS thus depend on the causative organisms.     

Assessment of the activity of tumor necrosis factor in normal state and in ovarian tumors

The cytotoxic activity of tumor necrosis factor of healthy individuals and patients with cystadenoma and adenocarcinoma of ovaries was investigated. Tumor necrosis factor (TNF) spontaneous activities of healthy donors and patients with cystadenoma of ovaries was the same. The level of TNF spontaneous activity of patients with adenocarcinoma was increased. LPS-induced TNF cytotoxicity in patients with cystadenoma was decreased. Mononuclear cells of patients with adenocarcinoma did not respond to LPS stimulation by increased TNF production.     

Tumor necrosis factor and its induction by bacterial endotoxins

Using suitable immunomodulators (Corynebacterium parvum vaccine, Zymosan or muramyl dipeptide), lipopolysaccharides (LPS) from various members of the family Enterobacteriaceae (Escherichia, Salmonella, Serratia, Shigella) were tested on rabbits in relation to the production of tumor necrosis factor (TNF). TNF was determined by means of the serum titration of L-929 cell cultures in the presence of Actinomycin D, this with resulting titres of 3.2 x 10(3) to 5.1 x 10(4) IU TNF/ml. Analogous titres were noted after the action of denatured LPS (ie LPS subjected to alkaline hydrolysis or H2O2).     

The production of tumor necrosis factor by mouse bone marrow-derived macrophages in response to bacterial lipopolysaccharide and a chemically synthesized monosaccharide precursor

Lipid X, a monosaccharide biosynthetic precursor of lipid A, has been chemically synthesized and was shown to induce bone marrow-derived macrophages to release tumor necrosis factor (TNF) in vitro. However, relatively high amounts of lipid X were necessary for induction, and the levels of TNF were much less than those induced by small amounts of lipid A itself or LPS. Lipid X prepared by extraction of Escherichia coli mutants induced higher levels of TNF than the chemically synthesized material, but this is probably partially due to amounts of impurities in the extracted material. Pretreatment of macrophages with IFN-gamma resulted in the release of higher amounts of TNF on subsequent induction with either LPS or lipid X. In contrast, pretreatment of macrophages with LPS induced hyporesponsiveness for TNF production on subsequent rechallenge with LPS. Lipid X, on the other hand, was incapable of making macrophages hyporesponsive for TNF production.     

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.     

A flow cytometric and immunofluorescence microscopic study of tumor necrosis factor production and localization in human monocytes

The production and localization of tumor necrosis factor (TNF) in human monocytes were investigated by using monoclonal and polyclonal antibodies against recombinant human TNF together with flow cytometry and immunofluorescence microscopy. Lipopolysaccharide (LPS) induced a rapid and transient accumulation of TNF in perinuclear vesicles which was detected 20 min after the addition of LPS. The fluorescence intensity of the vesicles peaked at 40 min of LPS exposure, concomitantly with the release of TNF into the medium. Thus, our results indicate that the secretion of TNF is typical for secretory proteins as it involves passage through the secretory apparatus. Additional studies demonstrated that plasma membrane-associated TNF could not be detected in live monocytes not exposed to LPS. However, after 90 min with LPS, a small population of monocytes expressed membrane-associated TNF, and by 24 hr approximately 50% of the monocytes displayed TNF on the plasma membrane. Furthermore, our results indicate that plasma membrane-associated TNF does not represent released TNF bound back to its own receptor. Thus, our findings support the view that TNF exists as a surface trans-membrane protein in LPS-stimulated monocytes.     

beta2-Adrenoceptor blockade partially restores ex vivo TNF production following hemorrhagic shock

The aim of the study was to assess the mechanisms through which leukocyte deactivation occurs upon hemorrhagic shock. In particular, the influence of beta-adrenergic tone was evaluated. BALB/c mice were hemorrhaged and resuscitated 60 min after hemorrhage. Animals were sacrificed 60 min later by exsanguination. Blood from exsanguination was cultured ex vivo with lipopolysaccharide (LPS) and heat-killed Staphylococcus aureus Cowan I (SAC). Hemorrhage resulted in a major decrease of LPS-induced TNF production whereas IL-10 production was significantly enhanced. Selective beta(2)-adrenoceptor antagonists (ICI 118,551) attenuated the decrease in TNF production and further enhanced IL-10 production. Hemorrhage did not alter SAC-induced TNF production levels whereas IL-10 production was increased. ICI 118,551 further increased the production of both TNF and IL-10. These data suggest that leukocyte deactivation after LPS stimulation is not a generalized phenomenon since TNF production was maintained when another microbial activator was used. IL-10 production was enhanced after hemorrhagic shock, independently of the nature of the triggering agent. Finally, this study demonstrates that beta(2)-adrenoceptor ligands play an important role in blood leukocyte deactivation to LPS after hemorrhagic shock.     

Neurosteroid levels are increased in vivo after LPS treatment and negatively regulate LPS-induced TNF production

Neuroactive steroids such as dehydroepiandrosterone sulfate and pregnenolone inhibit lipopolysaccharide (LPS)-induced tumor necrosis factor (TNF) production. Corticosteroids not only inhibit TNF production but their levels are increased in vivo after endotoxin injection, thus representing a feedback system that limits TNF production. We wondered whether the same could be true for neuroactive steroids. Thus, the possibility that neuroactive steroids might be increased concomitantly to TNF induction in vivo in mice treated with LPS was investigated. Increased plasma and hippocampal levels of allopregnanolone (but not of dehydroepiandrosterone or pregnenolone) were found 90 min after LPS injection. Allopregnanolone and progesterone (IC50 10- 7 and 10- 9 M, respectively) also inhibited TNF production by mouse peritoneal macrophages in vitro at concentrations in the range of those detected in vivo. These findings suggest that neuroactive steroids may act as endogenous inhibitors of cerebral and systemic TNF production.     

Tumor necrosis factor release from lipopolysaccharide-stimulated human monocytes: Lipopolysaccharide tolerance in vitro

Human peripheral blood monocytes secrete tumor necrosis factor (TNF) in response to stimulation with bacterial lipopolysaccharide (LPS). We have shown that isolated human monocytes pretreated with LPS for 24 h secrete lower levels of TNF on a second stimulation with LPS than monocytes that have been stimulated with a single dose of LPS either immediately after isolation or 24 h after isolation. The levels of TNF released by monocytes after the second stimulation with LPS are proportional to the LPS concentration over a range from 1 ng/mL to 10 micrograms/mL. Increasing concentrations of LPS used during the first 24-h stimulation induce greater suppression of TNF release after a second stimulation with LPS. After an initial stimulus of 10 micrograms/mL LPS, a second stimulation of monocytes even with 10 micrograms/mL LPS will result in TNF secretion similar to that of unstimulated cells. This in vitro tolerance apparently can be overcome by stimulating previously activated cells with phorbol myristate acetate. We have also shown that neither prostaglandin E2 nor dexamethasone added during the initial stimulation with LPS had an effect on the subsequent reduction in TNF release on a second stimulation of monocytes with LPS.     

Regulation of lipopolysaccharide-induced tumor necrosis factor production by cyclosporin A in mice primed with muramyl dipeptide

Abstract The effect of cyclosporin A (CsA) on tumor necrosis factor (TNF) or interleukin-6 (IL-6) production was evaluated in vivo in primed or unprimed mice challenged with lipopolysaccharide (LPS). Both pretreatment with BCG infection or with muramyl dipeptide (MDP) prior to LPS challenge resulted in an increase in the cytokine bioactivity level in the blood. CsA administration inhibited the TNF production. In unprimed mice, either normal or sensitized to LPS lethality by galactosamine treatment, a marked decrease in the cytokine level was observed after injection of CsA. After adrenalectomy, the yield of both TNF and IL-6 following LPS injection was markedly elevated but decreased by CsA administration. Ex vivo experiments have shown that the inhibitory effect of CsA could be demonstrated at the level of macrophages from mice previously given the drug. If mice had received MDP, in vitro responses of cells to LPS were enhanced but again CsA decreased the mRNA expression and protein secretion.     

Preventive effect of several drugs againstPseudomonas aeruginosa infection and the toxicity of combined tumor necrosis factor with lipopolysaccharide: Relationship between lethality and the arachidonic cascade

The participation of tumor necrosis factor (TNF) and lipopolysaccharide (LPS) inPseudomonas aeruginosa (Pa) infection was examined. The lethal challenge of Pa or TNF and LPS injection could be prevented by pretreatment with anti-TNF antibody, polymyxin B, ONO 1078, or Shosaiko-to. The combined effects of TNF and LPS may be deeply related to the lethality of Pa infection. The activities of leukotriene(LT) C₄/D₄/E₄ or platelet activating factor (PAF) were also related to the lethality of Pa infection, probably due to the subsequently produced TNF which acts in combination with LPS. Activating the host defence mechanism with biological response modifiers like Chinese medicines was effective against Pa infection. One mechanism could involve an activity as an LT inhibitor or PAF antagonist. Following the administration of TNF and/or LPS, the serum levels of arachidonic cascade products underwent various changes. With a combination of TNF and LPS, there was a synergistic increment of prostaglandins, thromboxane, and LT. Following pretreatment with Shosaiko-to, suppression of LTs was dominant even with the combination of TNF and LPS, which might be related to the lethality of the infection or combined TNF with LPS.     

Nonsteroidal Anti-Inflammatory Drugs Increase Tumor Necrosis Factor Production in the Periphery but Not in the Central Nervous System in Mice and Rats

Abstract: Nonsteroidal anti-inflammatory drugs (NSAIDs), which inhibit prostaglandin (PG) synthesis, augment production of tumor necrosis factor (TNF) in most experimental models. We investigated the effect of two NSAIDs, indomethacin and ibuprofen, on the production of TNF in the CNS induced by intracerebroventricular injection of lipopolysaccharide (LPS). Indomethacin and ibuprofen, administered intraperitoneally, augmented (three- to ninefold) the levels of TNF in serum and peripheral organs of mice injected intraperitoneally with LPS and in rats with adjuvant arthritis (up to a sevenfold increase). However, NSAIDs (intraperitoneally or intracerebroventricularly) did not increase brain TNF production induced by intravenous LPS. In fact, indomethacin decreased (1.4–1.8-fold) TNF levels in the spinal cord of rats with experimental autoimmune encephalomyelitis and in the cortex of rats with focal cerebral ischemia. Systemic administration of iloprost inhibited serum TNF levels after intraperitoneal LPS, whereas intracerebroventricular injection of iloprost or PGE₂ did not inhibit brain TNF induced by intracerebroventricular LPS. Both peripheral and central TNF productions were inhibited by cyclic AMP level-elevating agents or dexamethasone. Thus, a PG-driven negative feedback controls TNF production in the periphery but not in the CNS.     

Antitumor effects of endotoxin against solid murine Meth A tumors of different ages

Mice with 3-, 6-, 9- and 15-day Meth A tumors in the skin were injected intravenously with endotoxin to study age-dependent induction of tumor necrosis and reactive changes in draining lymph nodes. By 24 h after treatment with endotoxin, macroscopic necrosis was seen in 9-day, and to an even greater extent in 15-day tumors; microscopy showed extensive necrosis in 9-day tumors and some necrosis in 6-day tumors. The necrosis was predominantly coagulative, but the 9- and 15-day tumors showed a rim of hemorrhagic necrosis near the skin with surviving tumor tissue located at the lateral and basal margins. All the tumors showed about equal hyperemia by 4 h and mitotic arrest by 4 and 24 h. Depletion of mast cells, which were most numerous in the 9-day tumors, was seen on the dermal aspects by 24 h. Endotoxin did not change the morphology of the lymph nodes and disseminated tumor cells within 24 h. Endotoxin is known to cure 9-day Meth A tumors, and the extent of the rapid necrosis induced is clearly the crucial factor. Necrosis, however, is not a direct consequence of early hyperemia or mitotic arrest and other factors related to the age and site of the tumor, apparently affect whether or not necrosis ensues.     

Tumor necrosis factor inhibits the proliferation of cultured capillary endothelial cells

We have examined the effect of tumor necrosis factor (TNF) on the proliferation of capillary endothelial cells derived from brain or adrenal cortex. In both cell types, TNF inhibits basal as well as basic fibroblast growth factor (bFGF)-stimulated cell proliferation. TNF induces an additional cytotoxic effect in bFGF-stimulated, but not in unstimulated, capillary endothelial cells. These results suggest that TNF could act as a negative regulator of angiogenesis in vivo and further, that TNF might induce selective cytotoxicity of capillary endothelial cells stimulated by tumor-derived bFGF. These results could explain why TNF induces hemorrhagic necrosis of certain, solid tumors.     

Immunostimulating effects of muramyl dipeptide, glucosaminyl muramyl dipeptide and their synthetic derivatives in vitro

The effect of muramyldipeptide (MDP), glucosaminylmuramyldipeptide (GMDP) and their six synthetic derivatives on production of tumor necrosis factor (TNF), interleukin-1 (IL-1) and interleukin-2 (IL-2) by murine spleen cells in vitro was studied. MDP induced insignificant TNF production and did not stimulate production of IL-1 by the murine splenocytes within a 24-hour cultivation period whereas in combination with lipopolysaccharide (LPS) it induced significant production of both the cytokins. GMDP induced marked production of TNF (54 per cent cytotoxic index) and IL-1 (stimulation index 8). Addition of LPS in an amount of 10 ng/ml increased production of TNF by the murine splenocytes under the effect of GMDP but had no effect on production of IL-1. Neither MDP nor GMDP even in combination with LPS induced production of IL-2 by splenocytes of mice DVA/2 and C57B1/6 at activation for 24 hours. All the synthetic derivatives of MDP and GMDP except the MDP polymer activated TNF production by the murine spleen cells. GMDP lysine had the highest effect: 67 per cent cytotoxic index. In combination with LPS its cytotoxic index amounted to 87 per cent. The TNF activity was always higher when LPS in an amount of 10 ng/ml was added to the glycopeptides.     

Lipopolysaccharides indirectly stimulate apoptosis and global induction of apoptotic genes in fibroblasts

Following Gram-negative bacterial infection there is a reduction in matrix-producing cells. The goal of the present study was to examine the apoptotic effects of lipopolysaccharide (LPS) on fibroblastic cells and to investigate the role that the host response plays in this reaction. This was accomplished in vivo by subcutaneous inoculation of LPS in wild type and TNFR1(-/-)R2(-/-) mice. The direct effects of LPS on fibroblast apoptosis was studied in vitro with normal diploid human fibroblasts. The results indicate that LPS in vivo induces apoptosis of fibroblasts. By RNA profiling we demonstrated that LPS stimulates global expression of apoptotic genes and down-regulates anti-apoptotic genes. Fluorometric studies demonstrated that LPS in vivo significantly increased caspase-8 and caspase-3 activity and by use of specific inhibitors, the activation of caspase-3 was shown to be initiated by caspase-8 with no contribution from caspase-9. In vitro studies demonstrated that LPS did not induce apoptosis of fibroblasts, whereas tumor necrosis factor (TNF) did. In addition, the pattern of apoptotic gene expression induced by TNF in vitro was nearly identical to that induced by LPS in vivo, as measured by RNase protection assay. Moreover, pre-treatment of cells with TNF greatly enhanced apoptosis induced by a second stimulation with TNF 24 h later, suggesting that the global induction of pro-apoptotic genes was functionally significant. Thus, LPS acts to modulate the expression of a large number of genes that favor apoptosis of fibroblastic cells that is dependent upon activation of caspase-8 and is largely mediated by TNF.     

Modulation of antibody-mediated glomerular injury in vivo by bacterial lipopolysaccharide, tumor necrosis factor, and IL-1

We have investigated the effects of LPS, human rTNF (hrTNF) and human rIL-1 beta (hrIL-1 beta) pretreatment on the intensity of antibody-mediated injury in vivo by using a passive model of anti-glomerular basement membrane (GBM) antibody-mediated nephritis in rats. The experiments show that all three pretreatments exacerbate injury in this model whether judged by albuminuria or the prevalence of glomerular capillary thrombi. The effect on albuminuria was dose dependent with all three treatments. The lowest effective dose of LPS was 0.025 microgram while those for hrTNF and hrIL-1 beta were 0.4 microgram and 0.5 microgram, respectively. All three pretreatments also increased the prevalence of glomerular capillary thrombi which were rare in rats injected with anti-GBM antibodies without pretreatment. LPS pretreatment appeared to be more effective in causing glomerular capillary thrombi than hrTNF or hrIL-1 beta and this was reflected in the correlations between albuminuria and the proportion of glomeruli with capillary thrombi. This relation was linear for all three pretreatments but the slope was appreciably greater for rats pretreated with LPS (0.37) when compared with results from rats given either hrTNF (0.22) or hrIL-1 beta (0.29). Pretreatment of nephritic rats with both cytokines increased the slope to 0.42 demonstrating a synergistic effect. The synergism of hrTNF with hrIL-1 beta was also demonstrated by the effective doses needed to induce albuminuria which was evident in rats treated with 0.05 microgram of IL-1 beta and 0.4 microgram of TNF. Neither the cytokines nor LPS caused clinical, morphologic, or biochemical evidence of renal toxicity when given alone in the dose used here but they did cause a transient increase in the number of neutrophils marginated in glomerular capillaries. Pretreatment of rats with LPS or cytokines increased the glomerular neutrophil influx after anti-GBM antibodies by roughly sixfold. Our experiments show that TNF and IL-1 can increase the severity of glomerular injury in nephritis; they may be important in modulating glomerular injury clinically.