Serum Tumor Necrosis Factor Activity Research Articles

Importance of cytokines, nitric oxide, and apoptosis in the pathological process of necrotizing pancreatitis in rats.

Ischemia-reperfusion injury can be involved in the pathophysiology of acute necrotizing pancreatitis. The aim of our study was to determine the production of cytokines, tumor necrosis factor (TNF) and interleukin-6 (IL-6), the activation of the inducible nitric oxide synthase (iNOS), and the development of apoptosis during this pathologic process. Acute pancreatitis was produced in male Wistar rats by injection of 200 microL of 6% taurocholic acid into the main pancreatic duct in combination with the temporary (15 minutes) occlusion of the inferior splenic artery. Six and 24 hours later, the histologic damage was evaluated, and serum amylase, TNF, IL-6 levels, and iNOS and apoptotic activity from pancreatic and pulmonary tissues were determined. Twenty-four hours after the induction of pancreatitis, the mortality rate was 63%. During this period, the serum TNF and IL-6 levels were permanently high (50 +/- 12 and 58 +/- 10 U/mL and 7083 +/- 1610 and 6790 +/- 850 U/mL after 6 and 24 hours, respectively). The iNOS activity showed an increasing tendency in the pancreas, and a decrease following an initial increase in the lung (from 4.2 +/- 0.6 to 5 +/- 0.4 and from 6.8 +/- 0.6 to 3.8 +/- 0.5 pmol/min/mg protein after 6 and 24 hours, respectively). Histologic examination confirmed severe necrotizing pancreatitis. In the pancreas, the apoptotic activity increased significantly (from 4 +/- 4 to 27 +/- 5/mm at 6 and 24 hours), while in the lungs, following an initial increase it declined during the course of necrotizing pancreatitis (from 49 +/- 4 to 11 +/- 6/mm at 6 and 24 hours). Our results indicate that intraductal taurocholic acid and ischemia-reperfusion provokes severe acute necrotizing pancreatitis with a high mortality rate and leads to systemic inflammatory reaction, which appears to be the consequence of the activation of the cytokine cascade and iNOS. The degree of NO overproduction by iNOS corresponds with the apoptotic process in the pancreas and the lung.

Effect of 3-fluorothalidomide and 3-methylthalidomide enantiomers on tumor necrosis factor production and antitumor responses to the antivascular agent 5,6-dimethylxanthenone-4-acetic acid (DMXAA).

5,6-Dimethylxanthenone-4-acetic acid (DMXAA) is an antivascular drug that induces tumor necrosis factor (TNF) in mice. Thalidomide inhibits TNF induction by DMXAA and also potentiates its antitumor activity. We investigated whether these effects were enantiomer specific, using the R- or S-enantiomers of two nonracemizable thalidomide analogues. Racemic 3-fluorothalidomide (3FThal) and racemic 3-methylthalidomide (3MeThal) were separated into enantiomers of greater than 98% optical purity using preparative chiral column chromatography. C57Bl/6 mice implanted with subcutaneous Colon 38 tumors were treated with DMXAA (25 mg/kg) alone or together with the pure R- or S-enantiomers by a single i.p. injection. TNF levels in the serum or tumor tissues 3 h after treatment were measured using ELISAs and tumor growth was also measured. 3FThal and 3MeThal, at their respective single maximum tolerated doses (MTD) of 15 and 50 mg/kg, were more toxic in mice than thalidomide (100 mg/kg). The R- and S-enantiomers of either 3FThal or 3MeThal, at their respective MTD, inhibited DMXAA-induced TNF activity in serum and tumor tissue, but no significant differences were observed between the enantiomers. Coadministration of racemic or enantiomers of 3FThal or 3MeThal at their respective MTD did not potentiate the antitumor responses above that obtained with DMXAA alone, and no enantioselectivity was apparent. We conclude that there is no advantage in using the nonracemizable thalidomide analogues to improve the antitumor activity of DMXAA.

Role of Submandibular Salivary Glands in LPS-Induced Lung Inflammation in Rats

Objective: Literature data suggest that rodent salivary glands can exert a neuroimmunomodulatory influence on distant inflammatory events. The release of regulatory factors by salivary glands appears to be influenced by time-dependent factors. In this paper we examined this possibility directly by studying the role of submandibular salivary glands in the temporal profile of lypopolysaccharide (LPS)-induced lung inflammation in rats. Methods: The submandibular glands were removed (SMGx) or not (sham) and, 4 days later, the animals received an intravenous LPS injection (Salmonella abortus equi, 1 mg/kg). Cells in peripheral blood and in bronchoalveolar and bone marrow lavages were quantified after 90 min, 1, 3 and 5 days. Tumor necrosis factor (TNF) activity and corticosterone concentrations in serum were also determined. Baseline values were determined in a group of naïve rats. Results: One day after the LPS injection, neutrophil counts in lungs and blood in both animal groups were elevated, but the SMGx rats presented a significantly lower response in comparison to the sham-operated controls. Five days after LPS treatment, however, SMGx rats had higher neutrophil counts in the lungs than did sham animals, but numbers of blood neutrophils were equal. Ninety minutes after LPS injection, a peak of serum TNF activity was detected in both groups compared with naïve levels. At this time point, TNF activity was about 135% higher in the serum of the SMGx group than in controls. Corticosterone levels of sham-operated controls rose only on the 5th day after LPS, whereas SMGx rats had significant peaks of corticosterone both on the 1st and the 5th day, but not on the 3rd day. Conclusion: Our data indicate that submandibular glands have a dual effect on inflammatory pulmonary response by differentially modulating the profile of lung neutrophil influx.

Relationship between serum TNF activity and insulin resistance in dairy cows affected with naturally occurring fatty liver.

To clarity the relationship between tumor necrosis factor (TNF) and insulin resistance in dairy cows affected with fatty liver, naturally occurring cases were investigated. The affected cows were classified into following three groups according to histopathologic findings of the liver: mild fat droplet deposition (group 1; n=11), severe fat droplet deposition (group 2; n=10), and cloudy swelling (group 3; n=8). Serum TNF activities in Group 2 (8.67 +/- 2.16 U/ml) and Group 3 (11.65 +/- 1.92 U/ml) were significantly higher than that in Group 1 (3.57 +/- 0.81 U/ml) (p<0.05). The insulin-tolerance tests showed that the insulin-stimulated glucose disposal rates (GDR) in Group 2 (27.6 +/- 7.8%) and Group 3 (15.8 +/- 9.1%) were significantly lower than that in Group 1 (41.7 +/- 9.8%). There was a significant negative correlation between serum TNF activity and GDR in affected cows (r=-0.56, p<0.01). These results indicate that serum TNF activity is correlated with insulin resistance in cows with fatty liver.

Experimental study of Tong Xia purgative method in ameliorating lung injury in acute necrotizing pancreatitis.

AIM:To investigate the role of tumor necrosis factor (TNF) in lung injury during acute necrotizing pancreatitis (ANP), and the therapeutic effect of Tong Xia purgative method in minimizing the severity of lung injury.METHODS:Fourteen canines were randomly divided into 3 groups:the Tong Xia treatment group (n = 5) using Dachengqitang; saline control group (n = 5), and the sham operation group (n =4). TNF activity in serum and in bronchoalveolar lavage fluid (BALF), the serum endotoxin levels were measured,and the severity of lung injury evaluated.RESULTS:Elevation of TNF activity was more prominent in BALF than in serum. TNF activity in serum at 6 and 12 hours and in BALF was significantly decreased in the Tong Xia treatment group than in the saline control one (q = 21.11, q = 12.07, q = 9.03, respectively, P <0.01) and the lung injury was significantly alleviated at 12 hours as compared with that in the saline group, manifested as amelioration of the lung wet/dry weight ratio, decrease in protein concentration and neutrophils count in BALF, and improvement of pulmonary inflammatory changes. A positive correlation was demonstrated between serum TNF activity and endotoxin level.CONCLUSION:Hypersecretion of TNF is shown to be one of the major causes of lung injury during ANP; Tong Xia purgative method could alleviate the degree of lung injury mediated by TNF.

Pathogenicity ofSerpulina hyodysenteriae:in vivoinduction of tumor necrosis factor and interleukin-6 by a serpulinal butanol/water extract (endotoxin)

Lipopolysaccharide (LPS) of Gram-negative bacteria is a classic inducer of inflammatory cytokines. In the present experiments, LPS-like (phenol/water extract) or endotoxin-like (butanol/water extract) preparations fromSerpulina hyodysenteriaewere examined for their ability to induce serum tumor necrosis factor (TNF) or interleukin (IL)-6 bioactivity in mice and swine.Serpulina hyodysenteriaeendotoxin (butanol/water extract) elicited increased serum TNF activity in mice, although serpulinal endotoxin was at least 10 times less potent than the LPS preparations ofE. coliorS. typhimuriumon a weight basis for induction of TNF bioactivity.S. hyodysenteriaeLPS induced lower levels of serum TNF in mice thanS. hyodysenteriaeendotoxin. In contrast, pigs injected withS. hyodysenteriaeendotoxin demonstrated no increase in serum TNF activity. However, an induction of IL-6 bioactivity was observed in serum samples from pigs injected with serpulinal endotoxin. In pigs, the serpulinal preparations were five times less potent (on a weight basis) thanE. coliorS. typhimuriumLPS for induction of IL-6 bioactivity. These data indicate that serpulinal endotoxin, although less bioactive thanE. coliorS. typhimuriumLPS, is nonetheless capable of inducing thein vivoproduction of specific pro-inflammatory cytokines.

Dimethylthiourea protects rats against gram-negative sepsis and decreases tumor necrosis factor and nuclear factor κB activity

The thiol-containing compound dimethylthiourea (DMTU) is a known protectant in various models of oxidant-mediated tissue damage. Protective effects of DMTU have also been reported in studies on endotoxin-induced (LPS-induced) tissue injury. DMTU may exert this protective effect by reducing oxidative stress. In this study we investigated the effect of DMTU on survival, oxidative stress, and tumor necrosis factor (TNF) activity in two rat models of gram-negative bacterial sepsis. Intraperitoneal injection of 500 mg DMTU/kg protected against the lethal effects of intraperitoneally injected LPS (5 mg/kg) and live Salmonella typhimurium (3.3 x 10 10 CFU/kg). LPS injection resulted in oxidative stress, as indicated by an elevated concentration of hydrogen peroxide (H 2O 2) in normal and carbon monoxide-treated deproteinized blood. We also observed increased H 2O 2 levels in animals injected with live Salmonella typhimurium. Although DMTU improved survival in both models, H 2O 2 concentrations were not affected by it. This is consistent with our in vitro observation that DMTU is a weak H 2O 2 scavenger. Serum TNF activity, however, was substantially decreased by DMTU, and this was associated with a reduced activation of nuclear factor κB in the peritoneal cells of LPS-treated rats. In addition, LPS-induced TNF production in vitro by rat peritoneal macrophages was inhibited by DMTU ( p < 0.05). These results suggest that the protective effect of DMTU in gram-negative bacterial sepsis may be the result of a reduction in TNF activity. DMTU does not exert this effect by H 2O 2 scavenging but may inactivate toxic H 2O 2 metabolites.

Cytokine mRNA expression after transient and prolonged hypotension.

The role of cytokines in hemorrhagic shock remains controversial, with some studies showing an elevation of cytokines, whereas others do not. We thus analyzed the changes in tumor necrosis factor (TNF) activity and in mRNA of TNF alpha, interleukin-1beta (IL-1beta), and interleukin-6 (IL-6) after transient and prolonged hypotension. In the transient hypotension group (TH group), chronically cannulated rats were bled (20 ml/kg x 3 min) without fluid resuscitation. They showed transient hypotension, but their blood pressure (BP) quickly stabilized. In the prolonged hypotension group (PH group), the rats were bled and maintained at a mean BP of 40 mmHg for 60 min, and then were resuscitated with the shed blood and an equal volume of saline over 60 min. The serum TNF activity was measured by cytotoxicity against the L929 tumorigenic murine fibroblast. Messenger RNA of TNF alpha, IL-1beta, and IL-6 in liver was measured semi-quantitatively by high-performance liquid chromatography after reverse transcription and polymerase chain reaction. The increases in the TNF activity were not significant in either of the groups above the prehemorrhage levels, whereas mRNA of TNF alpha and IL-1beta showed a transient elevation in the TH group and a persistent elevation in the PH group. IL-6 mRNA did not increase significantly in the TH group, but did increase in the PH group. These results show that a large hemorrhage induces cytokine mRNA in the liver and also show the differences in the changes of cytokine mRNA after transient and prolonged hypotension.

Cross-tolerance between acute alcohol intoxication and endotoxemia.

This study tests two hypotheses: (1) prior exposure to LPS induces cross-tolerance for the hepatic effects of subsequent short-term alcohol intoxication; and (2) short-term alcohol intoxication renders the liver resistant to the effects of acute endotoxemia, resulting in reduced production of superoxide and tumor necrosis factor. In the first group of experiments, male Sprague-Dawley rats were treated intravenously with E. coli lipopolysaccharide (LPS) (0.5 mg/kg) 48 hr before they were given an intravenous bolus of ethanol (1.75 g/kg), followed by 250-300 mg/kg/hr) for 3-5 hr. Superoxide release in the perfused liver was measured after 3-hr ethanol infusion. Pretreatment with LPS attenuated ethanol-mediated superoxide anion release by the perfused liver. The stimulatory effect of phorbol myristate acetate on hepatic release of superoxide was also decreased. In the second group of experiments, rats previously treated with ethanol for 5 hr, received an intravenous injection of LPS (1 mg/kg). At 90 min after LPS, sera were collected for tumor necrosis factor alpha assay. Hepatic release of superoxide anion was determined 3 hr after LPS. Acute ethanol intoxication for 5 hr significantly reduced LPS-induced serum tumor necrosis factor activity and free radical release by the perfused liver. LPS-induced mortality was also decreased. In both groups of experiments serum corticosteroid levels were reduced during cross-tolerance. These results demonstrate that cross-tolerance develops between acute alcohol intoxication and endotoxemia manifesting in reduced hepatic production of cytotoxic cytokines and superoxide anions.

Systemic anti-tumor necrosis factor antibody treatment exacerbates endotoxin-induced uveitis in the rat

Tumor necrosis factor is released in the circulation and aqueous humor during endotoxin-induced uveitis, and induces acute uveitis when injected intraocularly in rats. To elucidate the role of tumor necrosis factor in the development of endotoxin-induced uveitis we analysed the effect of neutralizing anti-tumor necrosis factor antibodies and of pentoxifylline, a drug that inhibits tumor necrosis factor synthesis. Lewis rats were treated with: (a) a single intracardial injection of polyclonal rabbit anti-murine tumor necrosis factor antiserum prior to foot pad injection of 200 micrograms lipopolysaccharide; (b) an intraperitoneal injection of 10 mg pentoxifylline 1 hr before, at the time of, and 3 hr after foot pad injection of lipopolysaccharide; or (c) an intravitreal injection of 20 to 500 micrograms pentoxifylline together with 1 microgram lipopolysaccharide. The ocular inflammation was examined by slit-lamp and evaluated for the presence of hyperemia, flare, miosis, infiltrating cells or hypopyon. Levels of tumor necrosis factor in serum and aqueous samples were determined using a bioassay. Systemic treatment with either anti-tumor necrosis factor antibodies or pentoxifylline resulted in a significant inhibition, 90 and 70% respectively, of serum tumor necrosis factor activity at 3 to 4 hr after lipopolysaccharide injection. Systemic pentoxifylline treatment had no influence on the severity of uveitis. Anti-tumor necrosis factor antibody treatment, in contrast, caused an exacerbation of endotoxin-induced uveitis at t = 20 hr; mean uveitis score 3.9 vs. 1.4 in controls; P < 0.01. Intraocular administration of pentoxifylline together with lipopolysaccharide also had an aggravating effect on uveitis, that was associated with increased levels of intraocular tumor necrosis factor. The results show that inhibition of serum tumor necrosis factor activity does not block the development of endotoxin-induced uveitis. In fact, anti-tumor necrosis factor antibody treatment exacerbates the intraocular inflammation. These findings suggest that tumor necrosis factor may have other than proinflammatory properties in this uveitis model.

Digital Starling forces and hemodynamics during early laminitis induced by an aqueous extract of black walnut (Juglans nigra) in horses

SUMMARY Starling forces and hemodynamics in the digits of 5 horses were studied during early laminitis induced by oral administration of an aqueous extract of black walnut (Juglans nigra). The black walnut extract was prepared from heartwood shavings and was administered by nasogastric tube. Heart and respiratory rates, rectal temperature, central venous and arterial pressures, digital pulses, and signs of lameness were monitored. Blood samples were collected for determination of wbc count, hemoglobin concentration, and pcv and for endotoxin and tumor necrosis factor assays. Total wbc count and central venous pressure were monitored until they decreased by 30 or 20%, respectively. These decreases in wbc count and central venous pressure were observed 2 to 3 hours after dosing with black walnut extract. Respiratory and heart rates, body temperature, systolic and diastolic blood pressures, pcv, and hemoglobin concentration did not change significantly. Anesthesia was induced, heparin (500 IU/kg of body weight) was administered iv, and a pump-perfused extracorporeal digital preparation was established. Digital arterial and venous pressures were maintained at 100 and 30 mm of Hg, respectively. Blood flow, capillary pressure, lymph and plasma protein concentrations, and weight of the isolated digit during rapid increase in venous pressure were measured. Isogravimetric capillary filtration coefficient, vascular compliance, vascular and tissue oncotic pressures, tissue pressure, osmotic reflection coefficient, and precapillary and postcapillary resistances were calculated. Mean digital blood flow was 14 ml/min/100 g, capillary pressure was 52 mm of Hg, and vascular compliance was 0.06 ml/mm of Hg. The vascular and tissue oncotic pressures were 21.49 and 4.93 mm of Hg, respectively. The osmotic reflection coefficient was 0.71, and tissue pressure was 41 mm of Hg. The precapillary and postcapillary resistances were 7 and 2 mm of Hg/ml, respectively. Capillary permeability to proteins was not significantly different from that previously measured in healthy horses, suggesting that the increased capillary filtration coefficient reflected increased capillary hydrostatic pressure and perfusion of previously nonperfused capillaries. Neither endotoxin nor serum tumor necrosis factor activity was detected in any samples. The hemodynamic and Starling forces observed in this study were similar to those observed after laminitis was induced by administration of a carbohydrate gruel. Significant differences between the 2 models were detected for total vascular resistance, postcapillary resistance, and capillary filtration coefficient. It is likely that these differences were identified because the horses administered the black walnut extract were at an earlier stage in the disease process. The findings of this study suggest that the increase in capillary pressure causes transvascular fluid movement, resulting in increased tissue pressure and edema. We hypothesize that further increases in tissue pressure may collapse capillary beds and lead to tissue ischemia.

Serum TNF activity had no influence on megakaryocytic emperipolesis in the rat bone marrow.

The relationship between megakaryocytic emperipolesis in the bone marrow and lipopolysac-charide (LPS)-induced tumor necrosis factor (TNF) was investigated in adrenalectomized or dexameth-asone (Dex)-treated rats. Adrenalectomized rats were injected LPS intravenously at a single dose of 0.5μg/kg body weight 48 h after the operation, and Dex-treated rats were given LSP at a dose of 0.5mg/kg body weight 1 h after the Dex-treatment. Serum TNF activity markedly increased in the adrenalectomized rats (3279.3units/ml), but reduced in the Dex-treated rats (227.3units/ml) I h after LPS-treatment. Megakaryocytic emperipolesis was not influenced by these treatments 24 h after LPS-administration. Thus, LPS-induced serum TNF activity could not increase megakaryocytic emperipolesis.

FK506 inhibits renal glomerular thrombosis induced in rats by nephrotoxic serum and lipopolysaccharide

We investigated the effect of the potent immunosuppressive agent, FK506, on experimental glomerular thrombosis in rats by combined injections of nephrotoxic serum (NTS) and lipopolysaccharide (LPS). Either FK506 or placebo was administered intramuscularly three hours prior to injection of NTS that was followed one hour later by LPS. Rats were killed five hours after the LPS injection. Compared with placebo, FK506 pretreatment significantly reduced thrombosis formation, in a dose-dependent manner. FK506 also reduced proteinuria and the rise of serum creatinine level. Early infiltration of polymorphonuclear leukocytes into the glomeruli after LPS injection was significantly suppressed in the FK506 group compared with the placebo group. We also measured serum tumor necrosis factor (TNF) activity by using an L929 fibroblast cytotoxicity assay. Peak serum TNF activity was observed one hour after LPS injection, and FK506 significantly suppressed the elevation. Thrombosis was also developed in athymic nude rats, suggesting thrombosis formation is T cell independent. These data suggest that the FK506 has inhibitory effects on non-lymphocytes and possesses an anti-inflammatory effect in vivo.

Elimination of macrophages by liposome-encapsulated dichloromethylene diphosphonate suppresses the endotoxin-induced priming of Kupffer cells.

Abstract This study was performed to elucidate the role of Kupffer cells during endotoxemia by assessing the consequences of macrophage depletion by liposome-encapsulated dichloromethylene diphosphonate (L-DMDP) following lipopolysaccharide (LPS) treatment. Results show that more than 90% of the largest Kupffer cells, arbitrarily termed KC3, were eliminated, while only 50% of the smaller Kupffer cells were depleted. The selective elimination of a subpopulation of Kupffer cells was accompanied by a significant attenuation of endotoxin (LPS)-induced serum tumor necrosis factor activity by almost 90%. Hepatic sequestration of neutrophils into the liver after LPS injection was not altered by L-DMDP. The priming action of LPS on superoxide release by neutrophils recovered from the liver in response to in vitro PMA or zymosan was not altered by L-DMDP. However, the LPS-induced priming of superoxide formation in vitro by Kupffer cells, particularly KC3, was significantly attenuated. These results indicate that selective elimination of a subpopulation of Kupffer cells by L-DMDP down-regulates the LPS-induced cytokine release in vivo and endotoxin-mediated priming of hepatic macrophages for enhanced formation of toxic oxygen metabolites. However, biological activities of neutrophils (i.e., superoxide release and hepatic sequestration) are not altered by L-DMDP, further emphasizing the specificity of L-DMDP action on Kupffer cells. J. Leukoc. Biol. 55: 321–327; 1994.

Differential effects of anti-tumor necrosis factor monoclonal antibodies on systemic inflammatory responses in experimental endotoxemia in chimpanzees

Tumor necrosis factor (TNF) is considered to be a pivotal mediator of endotoxin-induced lethality. To assess the intermediate role of TNF in specific systemic inflammatory responses known to contribute to tissue injury in endotoxemia, eight healthy adult chimpanzees were intravenously injected with Escherichia coli endotoxin (4 ng/kg). In four of these animals the administration of endotoxin was followed immediately by a bolus intravenous injection of an anti-TNF monoclonal antibody (15 mg/kg). Treatment with anti-TNF completely prevented the endotoxin-induced increase in serum TNF activity, and profoundly reduced the appearance of interleukin-6 and -8 (both P < .05). Neutrophilia and lymphopenia were not affected by anti-TNF, whereas neutrophil degranulation, as measured by the plasma concentrations of elastase-alpha 1-antitrypsin complexes, was only slightly reduced (peak levels after endotoxin alone 31.0 +/- 3.4 ng/mL, versus 25.5 +/- 3.4 ng/mL after endotoxin with anti-TNF; P < .05). Anti-TNF did not influence endotoxin-induced activation of the coagulation system, as reflected by unchanged increases in the plasma concentrations of the prothrombin fragment F1 + 2 and thrombin-antithrombin III complexes. In contrast, anti-TNF strongly attenuated the activation of the fibrinolytic system, ie, peak plasma levels of plasmin-alpha 2-antiplasmin were 33.8 +/- 11.1 nmol/L after endotoxin alone and 17.0 +/- 2.9 nmol/L after endotoxin with anti-TNF (P < .05). These results suggest that TNF is not the common mediator of systemic inflammatory changes in low-grade endotoxemia. Moreover, the finding that in this mild model anti-TNF specifically inhibited fibrinolysis suggests that treatment with anti-TNF potentially may enhance the tendency towards microvascular thrombosis in sepsis.

Relevance of tumor necrosis factor to graft-versus-host disease after small bowel transplantation.

The small bowel (SB), an organ replete with lymphocytes, may provoke graft-versus-host disease (GVHD) after transplantation (Tx). Since tumor necrosis factor (TNF) has been suspected of mediating the tissue lesions of GVHD, we sought to determine whether TNF could be detected in the serum of rats undergoing GVHD after SBTx or lymphocyte transfer. For this purpose, postoperative serum TNF activity was determined in Lewis x Brown Norway (LBNF1) hybrid rats suffering from GVHD after undergoing transplantation of an entire (group 1; n = 8) or a segmental (group 2; n = 4) Lew SB, or after i.p. injection with lethal doses (500 x 10(6)) of Lew lymphocytes (group 3; n = 3). Control LBNF1 received i.p. small doses (50 x 10(6)) of Lew lymphocytes (group 4; n = 4). Serum TNF activity was assessed using the WEHI bioassay. In rats with acute and lethal GVHD after entire SBTx (group 1) or injection with large doses of lymphocytes (group 3), TNF activity gradually increased and reached high levels by the time the rats were agonal. In segmental SBTx rats (group 2), GVHD was less severe than in entire SBTx rats. Similarly, the increase in TNF activity was less intense and only transient since it had returned to control levels by the time the rats had completely recovered from GVHD. In control rats primed with small doses of lymphocytes (group 4), GVHD did not occur and no increase in TNF activity was detected.(ABSTRACT TRUNCATED AT 250 WORDS)

In vitro and in vivo canine mononuclear cell production of tumor necrosis factor induced by muramyl peptides and lipopolysaccharide

Tumor necrosis factor (TNF) is a potent mediator of tumor cell killing by activated monocytes and macrophages. We measured TNF activity induced by muramyl peptides and lipopolysaccharide (LPS) in normal dogs. Canine adherent mononuclear cells were isolated and cultured in either medium alone or medium containing muramyl dipeptide (MDP) or MDP plus LPS. After 18 h, culture supernatants were collected and assayed for TNF activity. Sera from dogs injected with liposome-encapsulated muramyl tripeptide-phosphatidylethanolamine (L-MTP-PE) were also evaluated for TNF activity. TNF activity both in supernatants and in sera was detected in a 18 h WEHI-164 cell cytotoxicity assay and was confirmed by a monoclonal antibody directed against recombinant human TNF-α. Results showed a significant increase in TNF activity from mononuclear cells exposed to MDP or MDP plus LPS of 20% and 88%, respectively; P<0.0005. Serum TNF activity rapidly increased within 2–3 h post L-MTP-PE injection and subsequently declined to pretreatment level at 4 h post administration. This study demonstrates that MDP±LPS can stimulate canine adherent mononuclear cells to release TNF and intravenous injection of L-MTP-PE is capable of rendering the in vivo release of TNF in normal dogs.

Complement component C5 modulates the systemic tumor necrosis factor response in murine endotoxic shock.

Patients with disseminated Neisseria meningitidis infections (meningococcemia) suffer from a fulminant shock syndrome that is accompanied by extraordinarily high concentrations in serum of tumor necrosis factor (TNF). People with homozygous deficiencies of late complement components (C5, C6, C7, and C8) experience a high incidence of disseminated neisserial infections yet suffer from an attenuated form of the disease. The mechanisms that account for this disparity in host response are unclear, but they may in part be related to differences in the systemic TNF response that are modulated by terminal complement components (C5 to C9). The role of C5 in the modulation of the systemic endotoxin-induced TNF response was studied with matched strains of C5-deficient (B10 D2/Osn) and complement-sufficient (B10 D2/Nsn) mice. Following lipopolysaccharide (LPS) administration, complement-sufficient mice exhibited more rapid increases in pulmonary and hepatic vascular permeabilities than did C5-deficient controls. Complement-sufficient mice developed acute passive hepatic congestion, they appeared more ill than C5-deficient mice, and they exhibited a twofold greater rise in serum TNF activity compared with that by C5-deficient mice. C5-deficient mice reconstituted with normal serum before an LPS injection exhibited pulmonary and hepatic vascular permeability increases and serum TNF levels approaching those observed in complement-sufficient mice. Alveolar and peritoneal macrophages isolated from complement-sufficient and C5-deficient mice and incubated in heat-inactivated serum did not exhibit differences in TNF mRNA expression or secreted TNF activity following stimulation with LPS. However, incubation of macrophages in complement-sufficient mouse serum (before LPS stimulation) resulted in increased TNF mRNA expression and TNF activity compared with those in cells incubated in C5-deficient serum. In vitro studies employing human complement components and peripheral blood monocytes revealed that recombinant C5a, in the presence or absence of LPS, can induce increased concentrations of TNF and that C5b to C9 had no additional modulatory effect on the TNF response. These data suggest that C5 modulates the endotoxin-triggered TNF response. The role of complement components distal to C5 (i.e., C5b to C9) in the endotoxin-triggered TNF response remains unclear.

Serum tumor necrosis factor activity in inflammatory bowel disease.

Serum tumor necrosis factor (TNF) levels in 33 patients with inflammatory bowel disease (IBD) were measured by using a sensitive enzyme immunoassay. Four of five Crohn's diseases (CD) and nine of twenty eight ulcerative colitis (UC) had elevated levels of serum TNF. In active CD or UC, a greater fraction of patients studied had significantly increased serum TNF levels (3/3 for CD and 8/11 for UC). Production of TNF by peripheral blood monocytes when stimulated by lipopolysaccharide was also increased in these patients and correlated with their serum TNF levels. These results suggest that TNF may have some pathoetiological meaning in IBD.

Serum tumor necrosis factor activity in horses with colic attributable to gastrointestinal tract disease

SUMMARY Over a 24-month period, serum tumor necrosis factor (tnf) activity was determined in 289 horses with colic attributable to gastrointestinal tract disease. Serum tnf activity was quantitated by use of a modified in vitro cytotoxicity bioassay, using WEHI 164 clone-13 murine fibrosarcoma cells. Causes for colic, determined by clinical and laboratory evaluation, exploratory celiotomy, or necropsy included: gastrointestinal tract rupture (gtr); ileal impaction; small intestinal strangulating obstruction (sio); proximal enteritis (pe); transient small intestinal distention; large-colon displacement; large-colon vovulus; large-colon impaction; colitis; small-colon obstruction; peritonitis; and unknown. Each diagnosis was placed into 1 of 3 lesion categories: inflammatory disorders (gtr, pe, colitis, peritonitis); strangulating intestinal obstruction (sio, large-colon volvulus); and nonstrangulating intestinal obstruction (ileal impaction, transient small intestinal distension, large-colon displacement, large-colon impaction, small-colon obstruction, unknown). The prevalence of high serum tnf activity and/or mortality were evaluated. Differences were tested at significance level of P &lt; 0.05. Approximately 20% of the 289 horses has serum tnf activity greater than that found in clinically normal horses (&gt; 2.5 U/ml). Twenty-three horses (8%) had marked increase in serum tnf activity (≥ 10 U/ml) which was more prevalent among horses with sio and pe than in horses of other diagnostic groups, except those with gtr. Mortality and marked increase in serum tnf activity were greater in horses with intestinal inflammatory disorders or strangulating intestinal obstruction than in horses with nonstrangulating intestinal obstruction. Similarly, a greater proportion of the horses that died had markedly high serum tnf activity than did horses that lived. Mortality of horses with serum tnf ≥ 10 U/ml was greater than that of horses with serum tnf activity &lt; 10 U/ml. Results indicate possible association between colic and serum tnf activity in horses and that high mortality may be associated with horses with markedly increased serum tnf activity.