Analysis of endotoxin fever in rabbits by using a monoclonal antibody to tumor necrosis factor (cachectin).

A murine monoclonal antibody to rabbit tumor necrosis factor (TNF; cachectin) was injected intravenously into an endotoxin-treated rabbit to examine the role of endogenous TNF in fever. Both early and late peaks of biphasic fever evoked by the endotoxin injection were suppressed by the antibody. TNF activity was detected in an endotoxin dose-dependent manner in the blood 1 h after the endotoxin injection, which was coincident with the early-peak fever. Although the late-peak fever responded to the antibody, no significant TNF activity was detected in the blood obtained 1 h before the peak response. The blood was found to contain endogenous pyrogen activity, which was stable after heating at 70 degrees C for 30 min and resistant to in vitro treatment with the antibody. Rabbit TNF injection also elicited biphasic fever in rabbits, the second phase of which was found to be mediated by the similar endogenous pyrogen. These results suggest that endogenous TNF plays an important role in eliciting a febrile response to endotoxin.     

Augmentation of endotoxin fever by recombinant human beta interferon in rabbits.

Nonpyrogenic amounts of endotoxin (0.1 to 1 ng/kg), hardly detectable by conventional Limulus amoebocyte lysate tests, could produce a fever of around 1 degree C when injected with a nonpyrogenic dose (6 X 10(5) U/kg) of recombinant human beta interferon (IFN-beta) in rabbits. Release of endogenous IFN and tumor necrosis factor by endotoxin was also dramatically increased by recombinant human IFN-beta, and their levels in the blood were closely correlated with the increase of body temperature. These data suggest, if the synergism between IFN and endotoxin also operates in the homologous system (human IFN-human cells), that contaminating endotoxin in IFNs, even if not detectable by Limulus amoebocyte lysate test, can contribute to IFN fever to a considerable extent in humans.     

Neopterin augmentation of tumor necrosis factor production

Gamma interferon (gamma-IFN), lipopolysaccharide (LPS)-gamma or interleukin-2 (IL-2)-induced tumor necrosis factor alpha (TNF alpha) production by both macrophages and peripheral blood mononuclear cells (PBMC), was increased in the presence of neopterin. Addition of neopterin caused an increased level of TNF alpha, but did not affect the kinetics of the TNF alpha production, which showed peak levels of cytotoxic activity 4 h after stimulatory treatment. Using anticytokine antibodies, we concluded that the neopterin effect was mainly gamma-IFN mediated, and only slightly affected by anti IL-2 receptor antibodies. The neopterin augmented TNF alpha production can be attributed to an immunological role for neopterin in the enhancement of cell-mediated immune (CMI) response.     

Reversion of the antichlamydial effect of tumor necrosis factor by tryptophan and antibodies to beta interferon.

Human recombinant tumor necrosis factor-alpha (TNF-alpha) inhibited the growth of Chlamydia trachomatis (L2/434/Bu) in HEp-2 cells. The effect was synergistic with that of gamma interferon (IFN-gamma). TNF-induced resistance to chlamydiae could be blocked with cycloheximide, suggesting that it involves the function of some induced proteins. Tryptophan degradation was enhanced in the TNF-treated cells and was much further increased when the cells were treated with both TNF and IFN-gamma at concentrations at which IFN-gamma by itself had very little effect. Antibodies to IFN-beta blocked the augmentation of tryptophan degradation by TNF and decreased but did not fully eliminate the antichlamydial effect of TNF. Increased concentration of tryptophan in the growth medium (greater than 100 micrograms/ml) resulted in reversion of the antichlamydial effect of TNF. This study suggests that the inhibition of chlamydial growth by TNF is mediated partly through an autocrine function of IFN-beta which, in synergism with TNF, enhances the activity of a tryptophan-degrading enzyme(s) and partly by some other activities of TNF which can be blocked by tryptophan.     

Detection of circulating tumor necrosis factor after endotoxin administration

Cytokines, products of stimulated macrophages, are thought to mediate many host responses to bacterial infection, but increased circulating cytokine concentrations have not been detected consistently in infected patients. We measured plasma concentrations of circulating tumor necrosis factor alpha (cachectin), interleukin-1 beta, and gamma interferon, together with physiologic and hormonal responses, in 13 healthy men after intravenous administration of Escherichia coli endotoxin (4 ng per kilogram of body weight) and during a control period of saline administration. Eight additional subjects received ibuprofen before receiving endotoxin or saline. Plasma levels of tumor necrosis factor were generally less than 35 pg per milliliter throughout the control period, but increased 90 to 180 minutes after endotoxin administration to mean peak concentrations of 240 +/- 70 pg per milliliter, as compared with 35 +/- 5 pg per milliliter after saline administration. Host responses were temporally associated with the increase in circulating tumor necrosis factor at 90 minutes, and the extent of symptoms, changes in white-cell count, and production of ACTH were temporally related to the peak concentration of tumor necrosis factor. Ibuprofen pretreatment did not prevent the rise in circulating tumor necrosis factor (mean peak plasma level, 170 +/- 70 pg per milliliter) but greatly attenuated the symptoms and other responses after endotoxin administration. Concentrations of circulating interleukin-1 beta and gamma interferon did not change after endotoxin administration. We conclude that the response to endotoxin is associated with a brief pulse of circulating tumor necrosis factor and that the resultant responses are effected through the cyclooxygenase pathway.     

Alterations of bacterial clearance induced by endotoxin and tumor necrosis factor.

The purpose of the study was to investigate the potential influence of endotoxin and tumor necrosis factor (TNF) on immune function in terms of systemic clearance and organ distribution of injected Escherichia coli in a rabbit model. To enable quantification of the clearance process, defined numbers of exogenous E. coli (1.3 x 10(8) CFU) were injected intravenously 60 min after bolus application of TNF (4 x 10(5) U, n = 6), after infusion of endotoxin (40 micrograms/kg of body weight) for 1 h (n = 6) or 4 h (n = 6), or after saline infusion (controls, n = 6). Parameters monitored were arterial pressure, oxygen uptake, and rates of bacterial elimination from the blood. At 180 min after E. coli injection, the animals were sacrificed, and tissue samples of liver, kidney, spleen, and lung were collected for bacterial counts. Endotoxin infusion produced a significant delay in blood clearance compared with saline and TNF pretreatment. The diminished systemic bacterial elimination was associated with significantly higher numbers of E. coli in the organs, thus reflecting reticuloendothelial system dysfunction. TNF had no major influence on the elimination kinetics of bacteria but affected the tissue distribution pattern with increased accumulation of E. coli in the lung (up to 100-fold of control values; P < 0.001).     

Restoration of normal febrile response to endotoxin in pyrogen-tolerant rabbits by injection with human beta interferon.

Early-phase pyrogen tolerance was induced in rabbits by two consecutive daily injections of 125 ng of endotoxin per kg of body weight. The second injection of the same dose of endotoxin evoked only a monophasic fever with a peak response 1.5 h after the injection; no second peak was observed. The rabbits were released from the tolerance to develop a typical biphasic fever by an injection of 125 ng of endotoxin along with human beta interferon (HuIFN-beta), although the tolerance-inducing amount of endotoxin alone could not. The profile of the febrile response of tolerant rabbits injected with both endotoxin and HuIFN-beta could not be distinguished from that of normal rabbits. There was no essential difference between natural and recombinant HuIFN-beta in breaking tolerance. Heat-stable (70 degrees C, 30 min) endogenous pyrogen or tumor necrosis factor was increased significantly in concentration in the serum of tolerance-broken rabbits. These results suggest that HuIFN-beta stimulates the production of tumor necrosis factor in tolerant rabbits to elicit the second peak of febrile response.     

Tumor necrosis factor (cachectin) mediates induction of cachexia by cord factor from mycobacteria.

The mechanism by which cord factor (CF), a toxic glycolipid from mycobacteria, induces cachexia was studied in BALB/c mice. Body weight was markedly reduced 48 h after CF administration; the animals became severely wasted and exhibited hypertriglyceridemia, hypoglycemia, and high levels of tumor necrosis factor (TNF) in plasma. After CF administration, a transferable factor which caused cachexia and hypertriglyceridemia in recipient mice was detected in the blood. Dexamethasone partially inhibited the cachexia-inducing action of CF. Conditioned medium from adherent peritoneal cell cultures incubated with CF produced the same wasting symptoms when inoculated intravenously into mice. These studies also demonstrated that adherent peritoneal cells produced a humoral factor in response to CF which was related to CF-induced cachexia. Antiserum to recombinant TNF-alpha prevented the cachectin action in passive-transfer experiments. Our findings indicate that cachectin (TNF) plays a role as a central mediator of the wasting induced by CF.     

In vivo immunohistochemical identification of tumor necrosis factor/cachectin in human lymphoid tissue.

To find an alternative approach to the in vivo detection of tumor necrosis factor/cachectin (TNF alpha), an immunohistochemical method to identify TNF alpha in histologic sections was developed. This method employs the streptavidin-biotin immunoperoxidase technique, and TNF alpha-specific monoclonal and polyclonal antibodies, on cryostat sections of fresh frozen human lymphoid tissue. Staining was evident in most specimens displaying follicular hyperplasia, but was absent from histologically normal tissue. Both tingible body macrophages and follicular dendritic reticulum cells appeared from phenotype analysis in serial sections and by double staining experiments to constitute the main source of TNF alpha. This technique complements other systemically oriented assays that may fail to detect significant in vivo TNF alpha production and activity at a cellular level.     

Mechanism of inhibition of HSV-1 replication by tumor necrosis factor and interferon γ

Tumor necrosis factor (TNF) synergizes with interferon (IFNγ) in the blockade of HSV-1 replication. Antibodies against IFNβ block this synergism, implying a role of IFNβ in the antiviral activity of TNF plus IFNγ. IFNβ₁ added exogenously to Hep-2 cells shows antiviral activity against HSV-1 only at high concentrations, whereas IFNβ₂ (also known as IL-6) alone has no effect on the replication of VSV or HSV-1 even when 1,000 U/ml are present. Our results are in accordance with the idea that TNF induces IFNβ₁ and that both cytokines must be present in the culture medium to synergize with IFNγ in order to inhibit HSV-1 replication.     

Activation of neutrophils by cachectin/tumor necrosis factor: priming of N-formyl-methionyl-leucyl-phenylalanine-induced oxidative responsiveness via receptor mobilization without degranulation

Human recombinant cachectin/tumor necrosis factor (TNF) was shown to prime neutrophils (PMNs), in a dose-dependent fashion, for subsequent oxidative responsiveness toward n-formyl-methionyl-leucyl-phenylalanine (FMLP). One basis for this phenomenon appeared to be TNF-mediated FMLP receptor mobilization. The maximal observed priming response was associated with a nearly twofold increase in the expression of PMN FMLP surface receptors, without changes in receptor affinity. Priming was not seen following stimulation with phorbol myristate acetate, possibly eliminating a role for the protein kinase C-dependent transductional components of FMLP-induced oxidative activity in the priming process. FMLP receptor mobilization occurred without significant degranulation as evident by an absence of increased granular enzyme release. These data support a potential role of macrophage-derived TNF in the augmentation of PMN host-defense during infectious and inflammatory challenge. TNF-mediated PMN oxidative priming may also promote oxidant tissue injury as seen in septic shock, adult respiratory distress syndrome, and multiple system organ failure.     

Endogenous tumor necrosis factor (cachectin) is essential to host resistance against Listeria monocytogenes infection.

During a sublethal murine infection with Listeria monocytogenes cells, tumor necrosis factor (TNF) activity was detectable in neither sera nor spleen homogenates at any stage of the infection when a bioassay with L-929 cells (less than 4 U/ml) was used. However, injecting the mice with an immunoglobulin fraction obtained from a rabbit hyperimmunized with recombinant murine TNF-alpha resulted in acceleration of listeriosis. When 1 mg of anti-TNF antibody was injected per mouse, all the mice died from listeriosis, even though the infectious dose was sublethal for the untreated controls. The antigen-specific elimination of the bacterium from the spleens and livers of anti-TNF antibody-treated mice was delayed, depending on the dose of the antibody injected. Endogenous TNF seemed to be produced early in infection, because suppression of antilisterial resistance was significant when a single injection of anti-TNF antibody was given between day zero and day 2 of infection. The effect of endogenous TNF on antilisterial resistance was due to neither regulation of alpha interferon (IFN-alpha) and IFN-gamma production nor induction of IFN-beta subtype 1 (IFN-beta 1), because anti-TNF antibody treated-mice produced normal levels of IFN-alpha and IFN-gamma in the bloodstream during infection and administration of monoclonal anti-murine IFN-beta 1 antibody had no effect on the development of listeriosis. Alternatively, the listericidal activity of peritoneal macrophages of L. monocytogenes-infected mice could be abrogated by injection of anti-TNF antibody in vivo. These results suggest that the lower level of TNF is produced endogenously in mice that received L. monocytogenes infection and that it plays an essential role in the host defense against L. monocytogenes infection.     

Toxic effects of recombinant tumor necrosis factor in suckling mice. Comparisons with interferon alpha/beta.

Newborn Swiss and A2G mice were given daily subcutaneous injections for 1 week of highly purified recombinant mouse tumor necrosis factor (TNF) or mouse interferon alpha/beta. Both treatments resulted in inhibition of growth of suckling mice and severe fatty changes and necrosis in the liver. The simultaneous injection of polyclonal antibody to interferon alpha/beta abrogated the effects of interferon but did not block the effects induced by TNF. The kidneys of TNF-treated suckling mice could be distinguished from interferon-treated mice by the absence of glomerular basement membrane abnormalities and the presence of numerous rounded eosinophilic hyaline granules within the cytoplasm of the proximal tubules. Treatment of suckling mice with TNF and interferon alpha/beta induced similar changes in the spleen and thymus. Interferon treatment of suckling A2G mice resulted in the appearance of pulmonary cysts, which were not observed in TNF-treated mice. It is concluded that the pattern of lesions induced in suckling mice by mouse TNF is both similar and different from that induced by mouse interferon alpha/beta.     

Weight loss associated with an endotoxin-induced mediator from peritoneal macrophages: the role of cachectin (tumor necrosis factor)

Endotoxin-induced cells of the reticuloendothelial system were shown to produce mediator(s) that evoke a state of cachexia in recipient animals. The factor(s) responsible were assayed in endotoxin-resistant (C3H/HeJ) mice, which were injected with dialyzed conditioned medium obtained from lipopolysaccharide-induced peritoneal macrophages. The mice exhibited weight loss and anorexia, and they died if sufficient quantities of medium were administered. The syndrome was reversible if injections were discontinued. Endotoxin alone did not produce this effect, and no gross pathologic lesions were discernable in the treated animals. In this model system, cachexia appears to result from the action of soluble macromolecules produced by activated macrophages in vitro. Cachectin (murine tumor necrosis factor) is thought to play a central role in this phenomenon.     

Does interleukin-1 mediate tumour necrosis factor alpha-induced fever in rabbits?

We investigated the humoral mechanisms involved in tumour necrosis factor alpha (TNFα)-induced fever in rabbits. No change in lymphocyte-activating factor activity was detected in serum drawn during TNFα-induced fever. The pyrogenic activity of recombinant rabbit interleukin-1β (IL-1β) was entirely abolished by pre-incubation with anti-IL-1β antiserum from the goat. Fever induced by intravenous (i.v.) injection of IL-1β was significantly diminished by i.v. infusion of the antiserum. However, i.v. infusion of the antiserum for 1 h did not affect fever induced by i.v. injection of TNFα, when the antiserum infusion began either simultaneously with, or 2 h after, the injection of TNFα. Furthermore, intracerebroventricular injection of the antiserum did not affect TNFα-induced fever. The intracerebroventricular administration of naloxone (an opioid receptor antagonist) significantly diminished TNFα-induced fever. The results suggest that IL-1, both in the blood circulation and in the brain, may not be involved in TNFα-induced fever. Similar to the contribution of eicosanoids, the opioid system in the brain seems some-how to contribute to the mechanism of the development of fever induced by TNFα in rabbits.     

Induction of interferon-gamma and tumor necrosis factor by Legionella pneumophila: augmentation of human neutrophil bactericidal activity

We have previously reported that Legionella pneumophila antigens can induce interferon-gamma (IFN-gamma) and tumor necrosis factor (TNF) in vitro and in vivo in mice. Furthermore, treatment of murine polymorphonuclear leukocyte (PMN) cultures with these cytokines resulted in augmented killing of the bacteria in vitro. The purpose of the present study was to determine if these findings could be extended to human responses. Here we report that Legionella antigens induced IFN-gamma and TNF in nonimmune human leukocytes cultures, and that these cytokines were able to stimulate the bactericidal activity of isolated PMN against L. pneumophila in vitro. Furthermore, optimal production of IFN-gamma was found in cultures which were enriched for large granular lymphocytes (LGL). The phenotype of IFN-producing cells was determined to be CD11+, CD16+, CD2+, and negative for CD4, CD8, CD14, and Leu 7. Additionally, Legionella-infected monocytes were found to produce TNF in a dose-dependent response to the number of infecting bacteria, and the addition of recombinant IFN-gamma to infected monocytes resulted in augmented production of TNF in a synergistic manner. Finally, treatment of PMN with recombinant IFN-gamma and recombinant TNF augmented their bactericidal activity against Legionella in a dose-dependent response. Thus, cytokines which can be induced by L. pneumophila antigens are able to stimulate PMN function in vitro, suggesting that resistance to infection results from a complex interaction of cytokines and cell responses.     

A tumor necrosis factor (TNF) receptor fragment attenuates TNF-α- and muramyl dipeptide-induced sleep and fever in rabbits

It is hypothesized that tumour necrosis factor (TNF) is an endogenous substance involved in sleep responses occurring during bacterial infection. If this hypothesis is correct, then blocking endogenous TNF, using a TNF inhibitor, should attenuate the bacterial cell wall-derived, muramyl dipeptide (MDP)-induced sleep. To test this hypothesis, the effects of intracerebroventricular (i.c.v.) injection of a TNF inhibitor, a biologically active fragment of the soluble TNF 55 kDa receptor (TNFRF), on TNF-α- and MDP-induced sleep were determined in rabbits. I.c.v. injection of 250 ng human recombinant TNF-α- or 150 pmol MDP increased non-rapid-eye-movement sleep (NREMS), decreased rapid-eye-movement sleep (REMS), enhanced electroencephalogram slow-wave activity (SWA) during NREMS and induced fever. Pretreatment of rabbits with 25 μg of the TNFRF significantly inhibited TNF-α- and MDP-induced sleep and fever responses. Finally, intravenously (i.v.) injected MDP enhanced NREMS, suppressed REMS, enhanced SWA, and induced fever; pretreatment of animals with the TNFRF injected centrally attenuated i.v. MDP-induced sleep responses but not fever. These results suggest that the TNFRF acts as a TNF-α antagonist in vivo and support the hypothesis that MDP-induced sleep is partially mediated via brain TNF-α.     

Chlamydiae modulate gamma interferon, interleukin-1 beta, and tumor necrosis factor alpha receptor expression in HeLa cells

Chlamydia psittaci was found to modulate receptor expression for the cytokine receptors that are involved in the synergistic induction of indoleamine dioxygenase in epithelial cells. Increases in receptor expression were seen even with inactivated Chlamydia, suggesting that chlamydial antigens and not products of infection are important for up-regulating cytokine receptor expression.     

二甲亚砜对家兔内毒素性发热的影响

目的:观察二甲亚砜(DMSO)对家兔内毒素性发热的影响。方法:以健康封闭群新西兰兔为实验对象,随机分组。经耳缘静脉注射内毒素(ET)和DMSO,用WRY-B型微机热原测温仪测定家兔的结肠温度。结果:静脉注射ET引起家兔结肠温度双相性升高,其发热反应指数明显高于对照组。静脉注射ET10min前注射不同剂量DMSO组,家兔发热反应指数明显低于静脉注射内毒素组,并呈剂量依赖关系。静脉注射ET(0.4μg/mL,1mL/kg)10min后注射DMSO(60%,1mL/kg)组,发热反应指数同样明显低于静脉注射内毒素组。结论:DMSO明显抑制家兔内毒素性发热。