Administration Of TNF-alpha Research Articles

Cellular targets of exogenous tumour necrosis factor-alpha (TNF alpha) in human gliomas.

To identify the cellular targets of TNF alpha in human gliomas, a total of 30 surgical specimens (12 glioblastomas, 4 anaplastic astrocytomas, 3 astrocytomas, 7 brains adjacent to tumour (BAT), 4 histologically normal-appearing brains) were examined by in vitro binding technique using biotinylated TNF alpha. The TNF-binding sites (TNF-BS) were recognized in the tumour cells in 8 of the 12 glioblastomas, 3 of the 4 anaplastic astrocytomas and in all the 3 astrocytomas. The TNF-BS were also recognized in the vascular endothelial cells in all these cases. The presence of TNF-BS in blood vessels ranged from 7.7 to 74.4% of the background vessels. This wide range of variation in the presence of TNF-BS within the tumour cells and tumour blood vessels may be relevant to the variable response of individual tumours to TNF alpha therapy. Since the tissue of normal brain, which lacks TNF-BS, might hardly be affected by this cytokine, administration of TNF alpha may be considered as an adjuvant therapy in selected groups of patients.

Effects of isolated limb perfusion with tumour necrosis factor-alpha on the function of monocytes and T lymphocytes in patients with cancer.

The objective of the present study was to investigate the effects of isolated limb perfusion (ILP) with tumour necrosis factor alpha (TNF-alpha) and melphalan in patients with cancer on, first, plasma levels of cytokines, second, systemic monocyte and T-lymphocyte distribution and, third, the ability of mononuclear cells to produce cytokines upon stimulation in vitro. Six patients undergoing an ILP were entered into the study (group 1). In addition, patients undergoing a major surgical operation (group 2) minor operation (group 3) as well as healthy volunteers (group 4) were included as control groups. Sensitive enzyme-linked immunosorbent assays (ELISAs) were used to measure TNF-alpha and interleukin-6 (IL-6) plasma levels at various time points during and after operation. Furthermore, the percentage of monocytes and T lymphocytes was determined in all studied groups using a FACScan. In addition, cytokine production upon stimulation with lipopolysaccharide (LPS) and a combination of anti-CD3/anti-CD28 monoclonal antibodies in whole-blood cultures was investigated. Increased plasma levels of TNF-alpha and IL-6 in patients undergoing ILP was observed, but only IL-6 appeared to be increased in patients treated with a major operation. No significant fluctuations were found in the other groups studied. Concerning the number of monocytes, a significant decrease was observed only in patients treated with ILP. Furthermore, a decreased production of TNF-alpha, IL-6 and IL-8 upon various types of stimulation in vitro was found in those patients, but also after a major operation. In conclusion, the results of the present study show increased plasma levels of cytokines in patients treated with ILP and major operation. Furthermore, a decrease in numbers of monocytes in the circulation and the ability of mononuclear cells to produce cytokines in vitro may be induced by administration of TNF-alpha in ILP. Although similar results were found in patients treated with major operation, the underlying mechanisms of this phenomenon remain to be elucidated.

The preliminary research on the relationship between TNF- and egg-induced granuloma and hepatic fibrosis of schistosomiasis japonica.

The amount and distribution of tumor necrosis factor-alpha (TNF-alpha) in liver at various stages of Schistosomiasis japonicum-infected mice were immunohistochemically determined by SABC method. The results showed that the amount of TNF-alpha in liver began to increase at the 8th-12th week after infection, and reached a peak (9 mice reached 2+ grade or higher) 16 weeks after infection, which was higher than the levels 8 weeks after infection (P = 0.001) and was mainly scattered in and around egg granuloma. At the chronic stage of the infection, the amount of TNF-alpha in liver decrease not increase with an increased secretion of collagen in liver and development of hepatic fibrosis. On the contrary, FN, LN, type I and III collagen in liver began to rise 8-12 weeks after infection, and were linearly scattered around egg granuloma, reaching a peak (more than 70% of infected mice reached 3+(-)4+ grade) at the 20th and 24th week and they became wide, thick and retiform, and deposited around and in egg granuloma. After administration of recombinant TNF-alpha to the infected mice, the amount of FN and LN, the size and the inflammatory response of granuloma in liver showed no significant changes as compared with the untreated group at the same stage. However, the amount of type I collagen in liver increased, among which the amount of type I collagen was significantly higher than that in the untreated group at the same stage (P = 0.01), while similar changes were not observed when recombinant TNF-alpha was administrated to the healthy mice. It is suggested that TNF may play a role in stimulation of fibroblast in liver to proliferate and secrete collagen, but may need to cooperate with some factors.

Tumor necrosis factor-alpha (TNF alpha) enhances cisplatin cytotoxicity to ovarian carcinoma xenografts

Antitumor effect was compared between administration schedule of once a day dosing of cisplatin (DDP) for 2 consecutive days (q40d) with or without TNF alpha. In two controls, TNF alpha or dilutor alone was administered. Against the ovarian carcinoma 2008 cells, DDP given at dose level of daily x 2 (3.5 mg/kg/day q40d) combined with TNF alpha (50 mu g/kg/day) schedule showed 6.08-fold tumor growth delay (TGD) (17.10+/-1.65; P<0.01), produced 3.17-fold greater cell kill [ratio of tumor volume of treated and control groups (T:C)=0.148; P<0.01] and resulted in longer survival [median survival (MS)=166; P<0.01] than DDP alone. These are superior to even high dose DDP (7.0 mg/kg/day x 2d) without TNF alpha administration schedule showing TGD=11.38 days T:C=0.271 and MS=97 days. High dose DDP (7.0 mg/kg/day q40d) with TNF alpha showed further DDP antitumor potency (TGD=29.74+/-2.08, P<0.01), however, this schedule showed only 2.56-fold TGD extension and no improvement was found in survival because of its severe toxicity. TNF alpha did not alter DDP induced systemic toxicity. These data indicate that optimal antitumor activity, tolerance and survival improvement occured on a schedule of low dose DDP combined with TNF alpha, and this has prompted the clinical evaluation of this administration schedule.

Lack of tolerance development to tumor necrosis factor alpha inside the central nervous system.

Tumor necrosis factor alpha (TNF alpha) was repeatedly microinfused into the lateral ventricle of guinea pig brains at a dose of 200 ng, 4 times within 150 min, at intervals of 3 days. In comparison to guinea pigs infused with solvent according to the same time schedule. the animals responded to TNF alpha with pronounced fevers. The quantity of the fever response was the same after each of the 4 microinfusions of TNF alpha. Three days after the last infusion of TNF alpha or solvent all animals received an intramuscular injection of bacterial lipopolysaccharide (LPS). The fever in response to LPS was the same in both groups. Thus, the reported development of tolerance to repeated systemic administration of TNF alpha 1-3 does not develop inside the blood-brain barrier. Also, the febrile response to LPS is not influenced by repeated central pre-treatment with TNF alpha, whereas repeated peripheral treatment does have an effect.

Canventol Inhibits HIV-1 Replication by a Tat-Induced Tar-Independent Mechanism

Canventol (2-isopropyl-4-isopropyldencyclohex-2-ene-l-ol), a blocker of tumor necrosis factor alpha (TNF-alpha) release, inhibits human immunodeficiency virus type (HIV-1) production in chronically and acutely infected cells. This effect of Canventol on virus replication could be correlated with its inhibitory influence on necrosis factor (NF)-kappa B activation and HIV-1 long terminal repeat (LTR)-driven reporter gene expression in Jurkat cells and these could be overcome by the administration of TNF-alpha. Canventol inhibits activation of the promoter by the viral protein Tat through a TAR-independent mechanism. The HIV-1 promoter is synergistically upregulated when both the TAR-independent and the TAR-dependent modes of Tat action are in operation. Tat-induced downstream events, such as the production of cytokines like TNF-alpha and NF-kappa B activation, are central for this upregulation. Inhibitors of the respective modes of action of Tat downregulate HIV-1 LTR activation and virus replication.

Possible roles of nonparenchymal cells in hepatocyte proliferation induced by lead nitrate and by tumor necrosis factor alpha.

A single intravenous injection of lead nitrate (LN) to rats induces liver cell proliferation without causing cell necrosis (direct hyperplasia). We suggested that liver cell proliferation in this model may be triggered by the induction of liver tumor necrosis factor alpha (TNF-alpha). Because administration of TNF-alpha in vivo has been shown to induce proliferation of both parenchymal and nonparenchymal cells of the liver, we analyzed the temporal sequences of DNA synthesis in both cell populations following LN and recombinant TNF-alpha treatment by 5-bromo-2-deoxyuridine (BrdU) immunohistochemistry. The patterns of cell proliferation induced by these agents were further compared with those induced by a single dose of nafenopin (NAF), a direct mitogen which does not induce liver TNF-alpha messenger RNA (mRNA). In male Wistar rats given a single dose of LN (100 micromol/kg), BrdU incorporation of hepatocytes and nonparenchymal cells (Kupffer cells, endothelial cells and periportal nondescript cells) became evident 12 hours after the treatment. The labeling of all cell types reached a peak after 36 hours and declined thereafter. Rats given a single intravenous injection of human recombinant TNF-alpha (46 microg/rat) showed an increase of BrdU labeling in nonparenchymal cells after 24 hours, whereas the labeling of hepatocytes became evident at 36 hours. A single intragastric administration of NAF resulted in a rapid increase in the number of labeled hepatocytes with no substantial labeling of nonparenchymal cells. These results add further support to the notion that LN-induced liver cell proliferation is mediated by TNF-alpha, and suggest that different cell populations are involved in the initial proliferative response of the liver to mitogens, depending on the capacity of the mitogens to stimulate TNF-alpha production.

Tumor necrosis factor-alpha stimulates adrenal glucocorticoid secretion in cholestatic rats.

Tumor necrosis factor-alpha (TNF-alpha ) is capable of activating the hypothalamic-pituitary-adrenal (HPA) axis. We have recently documented altered activation of this axis by endotoxin and interleukin-1 in cholestatic rats. Therefore, in this study, we examined TNF-alpha-induced activation of the HPA axis, in rats with cholestasis due to bile duct resection, with the use of sham-resected rats as controls. Administration of TNF-alpha to bile duct- and sham-resected rats in vivo resulted in a similar increase in plasma adrenocorticotropic hormone levels in both groups of animals but significantly higher corticosterone levels in cholestatic rats, suggesting a direct steroidogenic effect of TNF-alpha in cholestatic rats. This suggestion was confirmed in further experiments by the demonstration of TNF-alpha-induced corticosterone secretion in hypophysectomized cholestatic but not control rats. Furthermore, a direct stimulatory effect of TNF-alpha on adrenal corticosterone secretion in vitro was noted only for cholestatic rats, possibly via augmented adrenal prostaglandin E2 (PGE2) production. These results indicate that TNF-alpha has a direct stimulatory effect on adrenal corticosterone secretion in cholestatic rats, possibly due to augmented adrenal PGE2 release.

Mechanism of hepatocellular dysfunction during hyperdynamic sepsis.

Because of its central role in metabolism and host defense mechanisms, the liver is thought to be a major organ responsible for the initiation of multiple organ failure during sepsis. It is, therefore, important to discuss whether hepatocellular dysfunction occurs during early sepsis and, if so, whether this occurs prior to hepatocellular damage as evidenced by elevation in serum enzyme levels. Because indocyanine green clearance has been demonstrated to be an early and extremely sensitive measure of active hepatocyte transport function, a technique for repeated measurement of hepatocellular function by in vivo indocyanine green clearance was developed in small animals, such as the rat. Studies have indicated that hepatocellular function is markedly depressed during early stages of polymicrobial sepsis despite the increased cardiac output and hepatic blood flow and decreased peripheral vascular resistance. The depression in hepatocellular function in early, hyperdynamic stages of sepsis does not appear to be due to any reduction in hepatic profusion but is associated with elevated levels of circulating proinflammatory cytokines such as tumor necrosis factor (TNF) and interleukin (IL)-6. Furthermore, administration of recombinant murine TNF-alpha at a dose that does not reduce cardiac output and hepatic perfusion produces hepatocellular dysfunction and increases plasma levels of IL-6. Thus upregulation of TNF and/or IL-6 may be responsible for producing hepatocellular dysfunction during early, hyperdynamic stages of sepsis.

Effect of cyclo‐oxygenase inhibitors and modulators of cyclic AMP formation on lipopolysaccharide‐induced neutrophil infiltration in mouse lung

1. The adult respiratory distress syndrome (ARDS) is an acute lung inflammation developed after direct or indirect contact with pathogenic agents. In the present study, a mouse model was developed to mimic this condition using aerosolized bacterial lipopolysaccharide (LPS) and to investigate the mechanisms involved in the lung inflammatory response. 2. Inhalation of LPS led to a time and dose-dependent increase in tumour necrosis factor-alpha (TNF-alpha) production and neutrophil recruitment into the bronchoalveolar lavage fluid (BALF) of Balb/c mice. Under the same conditions, neutrophil infiltration was also found in the BALF of the LPS-sensitive mouse strain C3H/HeN, but was absent in the LPS-resistant strain C3H/HeJ. Intranasal administration of murine recombinant TNF-alpha also triggered neutrophil recruitment. 3. One hour after inhalation of LPS, half of the maximal level of TNF-alpha was measured in the BALF, but only a few neutrophils were detected at this time. The peak TNF-alpha concentration was reached at 3 h, when the neutrophil amount started to increase. At 24 h, maximal neutrophil number was found in the BALF and TNF-alpha was no longer present. 4. Pretreatment of mice under different experimental conditions demonstrated that: (a) cycloheximide almost completely blocks both neutrophil recruitment and TNF-alpha production; (b) anti TNF-alpha antibodies block neutrophil recruitment; (c) indomethacin or aspirin enhance by two fold neutrophil recruitment; (d) indomethacin significantly increases TNF-alpha production 1 h after inhalation of LPS; (e) dibutyryl cyclic AMP and prostaglandin E2 (PGE2) block both neutrophil recruitment and TNF-alpha production. 5. It is concluded that aerosolized LPS in mice triggers an acute lung inflammation which can be used as a potential model of inhalational ARDS and that, strategies leading to the elevation of cyclic AMP levels in vivo can be effective in modulating LPS-induced TNF-alpha synthesis and neutrophil recruitment.

Retroviral Vector-Mediated Transfer of the Tumor Necrosis Factor α Gene Into Human Cancer Cells Restores an Apoptotic Cell Death Program and Induces a Bystander-Killing Effect

Tumor necrosis factor alpha (TNFalpha) may induce tumor cell death by apoptosis, the physiologic program of cell death usually lost during neoplastic progression. However, many tumor cells are resistant to its effect unless high doses are administered. By retroviral vector-mediated gene transfer, we have transduced the TNFalpha gene into the DNA of human tumor cells to investigate whether the indefinite neoplastic cell proliferation could be blocked and the lost physiologic program of cell death restored. Evidence is provided that high-TNFalpha-producing clones generated from a human lymphoma T-cell line (ST4) can undergo apoptosis following transduction of the TNFalpha gene. Internucleosomal DNA cleavage was documented by May-Grünwald-Giemsa and by propidium iodide staining, as well as by gel electrophoresis. The induced apoptotic phenomenon is TNFalpha-mediated, since it can be reverted following incubation with anti-TNFalpha monoclonal antibodies (MoAbs), and it occurs with cytokine levels released in the supernatant by the engineered cells much lower(>100 times) than those required to promote the same effect on parental ST4 cells following administration of exogenous recombinant TNFalpha. The process is associated with a downregulation of the apoptosis-preventing gene, bcl-2, while the expression of bax and p53, genes usually involved in promoting apoptosis, persists. Mixed-culture experiments performed coincubating TNFalpha-transduced and untransduced ST4 cells allowed documentation of a bystander-killing effect on the parental cells. This phenomenon still occurred at transduced to parental cell ratios as low as 1:20 and was blocked in the presence of an anti-TNFalpha MoAb. These findings indicated that TNFalpha may play a regulatory role in the proliferation of human tumor cells, and suggest potential new antitumor therapeutic strategies based on the direct delivery of the TNFalpha gene into cancer cells.

Acute stimulation of the hypothalamic-pituitary-adrenal axis by IL-1 beta, TNF alpha and IL-6: a dose response study.

We investigated the effects of i.v. and intracerebroventricular (i.c.v) administration of increasing doses of recombinant human IL-1 beta, TNF alpha and IL-6 on plasma corticosterone (B) levels in rats. Rats were equipped with a jugular cannula for repeated blood sampling anda subgroup of rats also received an i.c.v implanted cannula. I.v. administration of IL-1 beta, TNF alpha or IL-6 and i.c.v administration of IL-1 beta and IL-6 induced a significant dose-dependent increase in plasma B levels, whereas i.c.v injection of TNF alpha in doses up to 1000 ng/rat was not effective. I.v. pretreatment of rats with anti-CRH antiserum had no significant overall effect on the plasma B response to i.v. administered IL-1 beta (500 and 3000 ng/rat), whereas the plasma B response to i.v. TNF alpha or IL-6 administration (3000 ng/rat) were significantly reduced. I.v. pretreatment of the animals with recombinant human IL-1 receptor antagonist (IL-1ra) significantly blocked the plasma B response to i.v. treatment with IL-1 beta, whereas the TNF alpha- and IL-6-induced increases in plasma B levels were not affected. Our data show that 1) i.v. administration of IL-beta, TNF alpha or IL-6 and i.c.v administration of IL-1 beta or IL-6 dose-dependently stimulate the HPA axis; 2) when given i.v. or i.c.v, IL-1 beta is more powerful than TNF alpha and IL-6 in activating the HPA axis; 3) endogenous CRH is involved in the activation of the HPA axis by acute i.v. administration of TNF alpha and IL-6. It is most likely that in case of i.v. treatment with IL-1 beta a CRH-independent mechanism is involved. This study provides no arguments for the involvement of endogenous IL-1 in TNF alpha- or IL-6-induced activation of the HPA axis.

Morphological effects of tumor necrosis factor-alpha on the blood vessels in rat experimental brain tumors.

The morphological changes in the vascular endothelium caused by the administration of tumor necrosis factor-alpha (TNF-alpha) were studied in an experimental model of rat brain tumors. Wistar rats bearing implanted C6 glioma received human natural-type TNF-alpha (1.7 x 10(5) U/m2) through the carotid artery and were sacrificed 3 or 24 hours later. The endothelial cells of the tumor blood vessels, demonstrated by the immunoreaction to factor VIII-related antigen, were enlarged after TNF-alpha administration. Morphometry demonstrated that the nuclei of these endothelial cells were also increased in size. The endothelial cells in the brain remote to the tumor were not affected. An in vitro binding study demonstrated that TNF-alpha binding sites were distributed in the vascular endothelial cells within the tumor but not in the brain remote to the tumor. The selective effect of TNF-alpha on the tumor blood vessels in experimental brain tumors may be related to the selective distribution of the TNF-alpha binding site.

Reactive oxidants mediate TNF-alpha-induced leukocyte adhesion to rat mesenteric venular endothelium.

We investigated the role of reactive oxygen metabolites (ROMs) as potential mediators of tumor necrosis factor-alpha (TNF-alpha)-stimulated neutrophil adhesion to rat mesenteric venules in vivo, using intravital microscopy and fixed whole mount preparations of mesentery. Intraperitoneal injection of TNF-alpha significantly increased leukocyte rolling, adhesion, and emigration in a dose- and time-dependent manner. Leukocyte adhesion and emigration, but not rolling, were significantly attenuated by prior intravenous administration of monoclonal anti-intercellular adhesion molecule-1 (ICAM-1). Rolling leukocyte flux was significantly attenuated by intravenous preadministration of superoxide dismutase (SOD), catalase, or both. Only catalase or SOD plus catalase significantly inhibited leukocyte adhesion. Catalase alone inhibited emigration. Moreover, postadhesive treatment with catalase but not SOD, 4 h after TNF-alpha administration reduced the flux of rolling (but not adherent) leukocytes that had previously increased in response to TNF-alpha. Intragastric allopurinol (50 mg/kg at 3 and 18 h before TNF-alpha administration) or 3 wk of a tungsten-enriched diet substantially inhibited xanthine oxidase activity but had no significant effects on the above parameters of neutrophil dynamics. In parallel experiments using fixed whole mount preparations of the mesoappendix stained specifically for neutrophil esterase, neutrophil adhesion 2 h after TNF-alpha administration was also inhibited by continuous intravenous administration of catalase but not by SOD, intragastric allopurinol, or tungsten diet. These findings suggest that ROMs, apparently not from xanthine oxidase, are important mediators of TNF-alpha-induced upregulation of neutrophil adhesion in rat mesenteric venules.

TUMOR NECROSIS FACTOR-α ADMINISTRATION INCREASES KUPFFER CELL CYCLIC ADENOSINE MONOPHOSPHATE LEVELS

Although studies have indicated that both Kupffer cell cyclic adenosine monophosphate (cAMP) and circulating TNF levels increase following trauma-hemorrhage and resuscitation, it remains unknown whether the elevated TNF levels are responsible for the increased Kupffer cell cAMP levels. To determine this, recombinant murine TNF-alpha (1.2 x 10(7) U/mg) was infused intravenously (.25 mg/kg body wt) over 30 min in normal rats. At 1 h after TNF-alpha or vehicle infusion, Kupffer cells and hepatocytes were isolated and cAMP levels were determined by radioimmunoassay. The levels of cAMP in the spleen and kidney were also measured. In addition, the maximal binding capacity and affinity of beta-adrenergic receptors were determined in Kupffer cells and hepatocytes by using [125I]iodopindolol. To determine whether there is any correlation between Kupffer cell cAMP and prostaglandin E2 (PGE2) or epinephrine, plasma levels of catecholamines and PGE2 were measured. The results indicated that TNF-alpha infusion significantly increased Kupffer cell cAMP levels while hepatocyte cAMP levels were not altered. Moreover, cAMP levels also increased in the macrophage/lymphocyte-rich spleen but were not altered in the kidney. Kupffer cell beta-receptor binding characteristics were not significantly affected by TNF-alpha infusion. In contrast, TNF-alpha administration markedly increased plasma levels of PGE2 and epinephrine. Thus, the elevated Kupffer cell cAMP levels induced by TNF-alpha are not due to upregulation of beta-adrenergic receptors, but may be associated with the elevated levels of circulating PGE2 and/or epinephrine.

In vivo regulation of replicative Legionella pneumophila lung infection by endogenous tumor necrosis factor alpha and nitric oxide

The in vivo role of endogenous tumor necrosis factor alpha (TNF-alpha) and reactive nitrogen intermediates (RNIs) in modulation of growth of Legionella pneumophila in the lung was assessed using a murine model of replicative L. pneumophila lung infection. Intratracheal inoculation of mice with L. pneumophila resulted in induction of endogenous TNF-alpha, which preceded clearance of L. pneumophila from the lung. Inhibition of endogenous TNF-alpha activity, via in vivo administration of TNF-alpha neutralizing antibody, or inhibition of endogenous RNIs, via administration of the nitric oxide (NO) synthetase inhibitor N-monomethyl-L-arginine (NMMA), resulted in enhanced growth of L. pneumophila in the lung at > or = 3 days postinfection (when compared with untreated L. pneumophila-infected mice). Because of the similar kinetics of enhanced pulmonary growth of L. pneumophila in mice treated in vivo with either anti-TNF-alpha antibody or NMMA, the immunomodulatory effect of NO on endogenous TNF-alpha activity in the lung was assessed. Administration of NMMA to L. pneumophila-infected mice resulted in a significant decrease in endogenous TNF-alpha activity in the lung during replicative L. pneumophila infections in vivo. However, administration of exogenous TNF-alpha to NMMA-treated mice failed to significantly enhance clearance of L. pneumophila from the lung. Results of these studies indicate that both endogenous NO and TNF-alpha facilitate resolution of replicative L. pneumophila lung infections and that regulation of L. pneumophila replication by TNF-alpha is mediated, at least in part, by NO.

Tumor necrosis factor alpha stimulates gluconeogenesis from alanine in vivo.

An increase in gluconeogenesis contributes to the cachexia seen in severe injury, sepsis, and malignancy by converting amino acids from skeletal muscle to glucose. Since tumor necrosis factor alpha (TNF alpha) may mediate this cachexia, we examined the effect of this cytokine on gluconeogenesis. Twenty-eight male Fischer rats were injected intraperitoneally with TNF alpha (250 micrograms/kg) or saline, and after 4 hours, isolated hepatocytes were obtained by in situ collagenase liver perfusion. Hepatocytes were incubated with alanine (10 mM), and rates of gluconeogenesis were determined. Plasma lactate, glucose, insulin, glucagon, cortisol, and amino acids were measured. TNF alpha administration resulted in a 50% increase in gluconeogenesis from alanine (P < 0.05) and a three-fold increase in plasma glucagon (P = 0.01). Total and glucogenic plasma amino acids decreased with TNF alpha injection (P < 0.05). In vivo TNF alpha causes an increase in hepatic gluconeogenesis associated with increased plasma glucagon.

Thermal enhancement of both tumour necrosis factor alpha-induced systemic toxicity and tumour cure in rats

In vitro and in vivo studies have suggested synergistic anti-tumour activity of combined hyperthermia and tumour necrosis factor alpha (TNF-alpha). However, some studies indicated an increased systemic toxicity of TNF by additional hyperthermia. The aim of this study was to obtain starting dosages for a clinical phase I study on the application of deep local hyperthermia and systemic TNF. We investigated the effect of local hyperthermia on the toxicity and efficacy of systemic TNF. Rats (Wag/Rij) carrying a subcutaneously transplanted osteosarcoma in the hind leg received a single intravenous dose of recombinant human (rh) TNF-alpha, either at normothermia or at hyperthermia, by positioning the tumour bearing hind leg in a water bath of 43 degrees C. Dose-effect curves for lethality and tumour cure were established and LD50 and TCD50 values were calculated. Systemic toxicity was increased by local hyperthermia. The LD50 values (+/- s.e.) were 1088 (+/- 61) micrograms kg-1 at normothermia and 205 (+/- 23) micrograms kg-1 at hyperthermia, resulting in a thermal enhancement ratio (TER) of 5.3. Following normothermia, tumour cures were observed at TNF concentrations of 1000-1300 micrograms kg-1, while this was observed at doses of 50-300 micrograms kg-1 when combined with hyperthermia (TCD50 values of 1211 and 188 micrograms kg-1 respectively), resulting in a TER of 6.4. Systemic toxicity and anti-tumour activity of TNF are both increased by local hyperthermia. A safe starting dose for the combined clinical treatment would be 10% of the dose of TNF-alpha that has been recommended for phase II studies on intravenous bolus administration of TNF-alpha at normothermia. In view of the large variability in tumour sensitivity for TNF-alpha, the clinical usefulness of this combined treatment modality has to be determined.

C5a- and Tumor Necrosis Factor-α-Induced Leukocytosis Occurs Independently of β2 Integrins and L-Selectin: Differential Effects on Neutrophil Adhesion Molecule Expression In Vivo

We previously demonstrated in rabbits that various neutrophil chemotactic factors share an ability to induce recruitment of polymorphonuclear leukocytes (PMN) from bone marrow when administered intravenously (Jagels and Hugli, J Immunol 148:1119, 1992). In the study reported here, we investigated the effects of chemotactic factors on the expression of beta 2 integrins and L-selectin in vivo and the roles of these adhesionmolecules in the recruitment process. Leukocytosis was induced by infusion of either C5a (5 micrograms/kg), N-formyl-methionyl-leucyl-phenylalanine (f-MLP; 2.5 micrograms/kg), or tumor necrosis factor alpha (TNF-alpha; 100 ng/kg). C5a increased the expression of CD18 (the common subunit of beta 2 integrins) on PMN by nearly twofold and decreased levels of L-selectin by 50% within 15 minutes after administration. Levels of beta 2 integrins returned to baseline 2 to 3 hours after induction of leukocytosis. L-selectin remained depressed for more than 5 hours, demonstrating that shedding was induced in the recruited bone marrow leukocytes as well as in circulating PMN. In contrast to the response to C5a, TNF-alpha did not cause upregulation of CD18 or shedding of L-selectin. Levels of L-selectin were consistently increased 60 minutes after administration of TNF-alpha, coinciding with a rapid rise in the number of band-form PMN in the circulation. Intact IgG and F(ab)2 forms of the anti-CD18 monoclonal antibody IB4 or the anti-L-selectin antibody DREG-200 were administered intravenously 15 minutes before induction of leukocytosis by the chemotactic factors. Neither IB4 nor its F(ab)2 fragments blocked leukocytosis induced by C5a, f-MLP, or TNF-alpha. DREG-200 also did not block leukocytosis induced by f-MLP, C5a, or TNF-alpha. These results suggest that leukocyte emigration from the bone marrow into the circulation proceeds through interactions distinct from those involved in neutrophil chemotaxis and diapedesis. Shedding of L-selectin from C5a-recruited bone marrow leukocytes demonstrates activation of these cells in the recruitment process and may reflect a potential mechanism for their release. The dissimilar effects of C5a and TNF-alpha on expression of adhesion molecules may result from distinct stimulatory pathways and suggests differential activation states for cellular recruitment by these inflammatory factors.

Potential involvement of IL-10 in suppressing tumor-associated macrophages. Colon-26-derived prostaglandin E2 inhibits TNF-alpha release via a mechanism involving IL-10.

The administration of murine TNF-alpha to colon (C)-26 bearing mice, significantly protects the host against the catabolic effects of the tumor. This effect of exogenous TNF-alpha can be primarily attributed to tumor lysis rather than to a direct anticachectic action. Murine peritoneal macrophages cultured with the C-26 line or with C-26 culture supernatant do not release TNF-alpha in response to LPS stimulation. The reduction in TNF-alpha levels is associated with a significant increase in IL-10 levels. Single cell suspension of freshly disaggregated C-26 tumor (which contains host macrophages), do not produce TNF-alpha but contain significant levels of PGE2 and IL-10. In contrast, PGE2 but not TNF-alpha or IL-10 can be detected in the C-26.IVX cell line that is used to generate tumors in vivo. Neutralizing anti IL-10 Ab but not isotype-matched Ab, significantly reverses the inhibitory effect of the tumor cells and their culture supernatant on macrophage TNF-alpha release. Additional evidence is presented to indicate that the C-26-derived inhibitory activity is related to PGE2. Taken together, these results support the hypothesis that tumor-derived PGE2 prevents tumor-infiltrating macrophages from producing TNF-alpha, in part by augmenting macrophage IL-10 synthesis.