Birgit Störr

Neutralization of pyrogen-induced tumour necrosis factor by its type 1 soluble receptor in guinea-pigs: Effects on fever and interleukin-6 release

1 A soluble form of the tumour necrosis factor (TNF) type 1 receptor (referred to as TNF binding protein, TNF‐bp) at a dose of 1 mg per animal, or an equivalent volume of solvent, was injected together with 10 μg kg−1 lipopolysaccharide (LPS) or 50 μg kg−1 muramyl‐dipeptide (MDP) directly into the arterial circulation of guinea‐pigs and the effects on circulating TNF or interleukin‐6 (IL‐6) and on abdominal temperature were studied. 2 At 15 or 60 min after injection, LPS‐induced and MDP‐induced circulating TNF was below the detection limit of the assay and thus completely neutralized in animals treated with TNF‐bp. In the control group, TNF was still below the limit of detection in most animals 15 min after LPS was injected; in some animals small traces of TNF could already be detected at that time. However, 60 min after administration of LPS, large amounts of TNF (19508 ± 4682 pg ml−1) were measured in the control group. MDP‐induced TNF in plasma was below the limit of detection 15 min after MDP was injected, and rose to 10862 ± 3029 pg ml−1 60 min after injection. 3 Low levels of circulating IL‐6 (20‐40 international units (IU) ml−1) were measured in all groups of animals 15 min after injection of LPS or MDP. This value corresponds to the baseline activity of IL‐6 in plasma of guinea‐pigs. One hour after administration of LPS, IL‐6 rose to 5442 ± 1662 IU ml−1 in the control group and to a significantly lower value of 1485 ± 179 IU ml−1 in guinea‐pigs treated with TNF‐bp. One hour after injection of MDP, circulating IL‐6 was 2614 ± 506 IU ml−1 in the control group, while the corresponding value in animals treated with TNF‐bp again was significantly lower (873 ± 312 IU ml−1). 4 The second phase of the characteristic biphasic LPS fever in guinea‐pigs was significantly attenuated in animals treated with TNF‐bp. The shorter first phase of the febrile response to LPS was identical in both groups of animals. 5 The late phase of MDP‐induced fever (7‐22 h after injection) was depressed by treatment with TNF‐bp, while the first phase of MDP‐induced fever (0‐7 h after injection) was significantly enhanced by the neutralization of TNF by TNF‐bp.

Lack of cross tolerance between LPS and muramyl dipeptide in induction of circulating TNF-α and IL-6 in guinea pigs

In guinea pigs, lipopolysaccharide (LPS) from gram-negative bacteria and muramyl dipeptide (MDP) from gram-positive bacteria are potent inducers of systemic production of proinflammatory cytokines and fever. However, there is a striking difference between these two bacterial pyrogens in so far as repeated administration of LPS, but not of MDP, in short-term intervals induces tolerance by a progressive downregulation of the systemic cytokine network. In the present study, we investigated MDP-induced fever and the systemic release of tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) in LPS-tolerant guinea pigs in comparison with naive animals. Endotoxin tolerance was induced by repeated intramuscular injections of 20 μg/kg LPS at intervals of 3 days. In response to the last of five injections with LPS, systemic production of TNF-α and IL-6 as well as the development of a febrile response was abrogated almost completely. Those guinea pigs that had developed an LPS tolerance could, however, produce the same amounts of TNF-α and IL-6 as naive animals in response to a challenge with MDP. Also, MDP-induced fever was identical in LPS-tolerant and naive guinea pigs. These results provide evidence for a lack of cross tolerance between LPS and MDP in induction of circulating cytokines and fever in guinea pigs.In guinea pigs, lipopolysaccharide (LPS) from gram-negative bacteria and muramyl dipeptide (MDP) from gram-positive bacteria are potent inducers of systemic production of proinflammatory cytokines and fever. However, there is a striking difference between these two bacterial pyrogens in so far as repeated administration of LPS, but not of MDP, in short-term intervals induces tolerance by a progressive downregulation of the systemic cytokine network. In the present study, we investigated MDP-induced fever and the systemic release of tumor necrosis factor-alpha (TNF-alpha) and interleukin-6 (IL-6) in LPS-tolerant guinea pigs in comparison with naive animals. Endotoxin tolerance was induced by repeated intramuscular injections of 20 microg/kg LPS at intervals of 3 days. In response to the last of five injections with LPS, systemic production of TNF-alpha and IL-6 as well as the development of a febrile response was abrogated almost completely. Those guinea pigs that had developed an LPS tolerance could, however, produce the same amounts of TNF-alpha and IL-6 as naive animals in response to a challenge with MDP. Also, MDP-induced fever was identical in LPS-tolerant and naive guinea pigs. These results provide evidence for a lack of cross tolerance between LPS and MDP in induction of circulating cytokines and fever in guinea pigs.

Afferent nerves are involved in the febrile response to injection of LPS into artificial subcutaneous chambers in guinea pigs

In guinea pigs, fever was induced by injections of 100 or 10 microgram/kg lipopolysaccharide (LPS) into artificial subcutaneous chambers and analysed under the influence of the local anesthetic, ropivacaine (ROPI), which was administered into the chamber at a dose of 10 mg/kg 30 min prior to LPS. In response to injections of 100 microgram/kg LPS into the subcutaneous chambers, fever was not modified by pretreatment with ROPI. High amounts of bioactive tumor necrosis factor (TNF) alpha and interleukin-6 (IL-6) were measured in the lavage of the chambers after administration of LPS. Comparatively low concentrations of both cytokines (0.5-4% of the concentrations in the lavage fluid) were detected in blood plasma simultaneously. In response to injections of 10 microgram/kg LPS into the subcutaneous chambers, fever was significantly reduced by pretreatment with ROPI to about 60% of the febrile response of control animals. Levels of TNF and IL-6 were lower in response to the reduced dose of LPS. TNF in plasma was even below the limit of detection. The suppression of fever by the local anesthetic was not observed when ROPI was subcutaneously injected into the contralateral site of the chamber position so that a systemic effect of ROPI in the reduction of fever can be excluded. The results indicate a participation of afferent neural signals in the manifestation of fever. This effect becomes obvious only if the dose of the applied inflammatory stimulus (LPS) is not high enough to activate a systemic generalised inflammatory response.

Inhibition of nitric oxide synthase attenuates lipopolysaccharide-induced fever without reduction of circulating cytokines in guinea-pigs

Abstract It was recently demonstrated that the diffusible messenger molecule nitric oxide (NO) is involved in the febrile response of rats and rabbits to exogenous or endogenous pyrogens. In this study we have investigated the effects of systemic administration of the NO-synthase inhibitor N-nitro-l-arginine-methylester (l-NAME) on abdominal temperature and on lipopolysaccharide- (LPS-) induced fever in guinea-pigs. We further studied the effects of l-NAME on the LPS-induced circulating cytokine network by measurement of tumor necrosis factor α (TNF) and interleukin-6 (IL-6) in blood plasma during the time course of fever. At a dose of 10 mg/kg, intra-arterial injection of l-NAME per se had no influence on the abdominal temperature of guinea-pigs, while administration of 50 mg/kg l-NAME evoked a pronounced fall of body temperature which lasted about 12 h. When injected simultaneously with 10 µg/kg LPS into the arterial circulation, the lower dose of l-NAME that did not decrease abdominal temperature per se caused a significant attenuation of LPS-induced fever due to suppression of the second phase of the biphasic febrile response. The LPS-induced cytokine network remained unimpaired by the treatment with l-NAME. Peak activity of TNF in plasma (measured 60 min after LPS injection) was 20,596±2368 pg/ml in control animals and 18,900±4778 pg/ml in guinea-pigs treated with l-NAME. In addition, circulating levels of IL-6 were not statistically different between both groups of animals 60 min or 180 min after administration of LPS along with l-NAME or vehicle. The results confirm that endogenous NO formation has a role in the generation of LPS-induced fever and demonstrate that the attenuation of fever by inhibition of NO-synthase is independent of the circulating LPS-induced cytokine network.

Inhibition of nitric oxide synthase results in a suppression of interleukin-1β-induced fever in rats

Pro-inflammatory cytokines such as interleukin-1beta (IL-1beta) induce nitric oxide synthase. The purpose of this study was to investigate the role of endogenous nitric oxide in IL-1beta-induced fever in rats. At a dose of 2.5 microg per animal intraperitoneal (i.p.) injections of rat recombinant IL-1beta evoked a febrile response with a duration of 8 hours. Simultaneous i.p. injection of 50 mg/kg N-nitro-L-arginine methyl ester hydrochloride (L-NAME) resulted in a complete suppression of IL-1beta-induced fever in rats. I.p. injection of 50 mg/kg L-NAME alone had no apparent influence on body core temperature. Endogenous formation of IL-6 in response to IL-1beta was not suppressed but rather enhanced by treatment with L-NAME during the early stage of IL-1beta-induced fever. This result indicates that activation of nitric oxide synthase and thereby endogenous NO-formation is essential for the generation of an IL-1beta-induced febrile response in rats and that the suppression of IL-1beta-induced fever by treatment with L-NAME seems not to be caused by an inhibition of IL-6 production.

Fever and production of cytokines in response to repeated injections of muramyl dipeptide in guinea-pigs

Abstract Fever and systemic plasma levels of the cytokines tumour necrosis factor α (TNF) and interleukin-6 (IL-6) were measured in guinea-pigs in response to single or repeated intramuscular injections of 100 μg/kg muramyl-dipeptide (MDP). In a pilot study (experiment 1), MDP-induced fever was monitored for 8 h. The first fever phase 90–360 min after injection of MDP was followed by the second phase which continued beyond the duration of this experiment. High circulating levels of TNF and IL-6 were detected just before body core temperature started to rise. Within the next 90 min TNF declined again by more than 90% while IL-6 remained elevated. In experiment 2, the effects of repeated injections of MDP (5 times at intervals of 3 days) on the same parameters were investigated. In this paradigm, the febrile response started earlier (60 min after injection) and the first phase of fever remained manifest until 360 min after injection, while the late phase, measured 360–720 min after injection, was attenuated. Circulating, bioactive TNF and IL-6, measured 60 and 180 min after MDP was administered, were the same in response to the first, third, and fifth injection. In experiment 3, the influence of five repeated MDP injections on the abdominal temperature was measured for 22 h, and circulating cytokines were analysed before (360 min after injection) and during (480 min after injection) the late phase of MDP-induced fever. The late phase of MDP-induced fever 7–22 h after injection was attenuated in response to the second and further administrations of this pyrogen. At 6 h after the first, third, and fifth administration of MDP, only traces of TNFα were measured, 2 h later no bioactive TNF was detected at all. At these times also IL-6 declined again, compared with the activity of this cytokine measured during the early phase of MDP fever, but was still present in elevated amounts. Compared with the values measured in response to the third and fifth injections of MDP, circulating IL-6 was higher 360 min and 480 min after the first injection. It remains speculative whether the longer duration of elevated IL-6 in plasma is related to the development of the long-lasting, late phase of MDP-induced fever, which was only observed after the first of five repeated injections of MDP at intervals of 3 days.

Dose-dependent attenuation of lipopolysaccharide-fever by inhibitors of inducible nitric oxide-synthase in guinea pigs

Different doses of aminoguanidine or S-methylisothiourea, both predominantly inhibitors of the inducible form of nitric oxide (NO)-synthase, were injected into the arterial circulation of guinea pigs alone or along with 10 microg/kg bacterial lipopolysaccharide. Doses of 10 mg/kg, 50 mg/kg or 250 mg/kg aminoguanidine per se had no influence on abdominal temperature of guinea pigs. Only the highest dose of aminoguanidine (250 mg/kg) completely suppressed the first phase of the biphasic febrile response to lipopolysaccharide-injections. Lipopolysaccharide-fever was not modulated by administration of 10 mg/kg or 50 mg/kg aminoguanidine, when compared to fever in response to injections of lipopolysaccharide along with solvent. Doses of 10 mg/kg or 50 mg/kg S-methylisothiourea did not alter abdominal temperature while a dose of 250 mg/kg S-methylisothiourea had a lethal effect in guinea pigs. The febrile response to lipopolysaccharide was unimpaired by administration of 10 mg/kg S-methylisothiourea, while a dose of 50 mg/kg again attenuated fever predominantly by a suppression of the first fever phase. None of the applied doses of aminoguanidine or S-methylisothiourea resulted in a significant attenuation of the lipopolysaccharide-induced circulating cytokines tumor necrosis factor-alpha (TNF-alpha) and interleukin-6. The drugs themselves, without lipopolysaccharide-injections, did not enhance or reduce circulating levels of the investigated cytokines. The results indicate that endogenous NO may participate in the induction of lipopolysaccharide-fever and that fever suppression by systemic administration of NO-synthase inhibitors occurs independently from the lipopolysaccharide-induced circulating cytokines.

Influence of pentoxifylline on fevers induced by bacterial lipopolysaccharide and tumor necrosis factor-α in guinea pigs

In guinea pigs intraperitoneal (i.p.) injections of 50 mg/kg pentoxifylline had no influence on abdominal temperature while higher doses of pentoxifylline caused a hypothermic response lasting for 2-3 h. Administration of 50 mg/kg pentoxifylline 1 h before intramuscular (i.m.) injections of 20 micrograms/kg bacterial lipopolysaccharide reduced the lipopolysaccharide-induced production of endogenous tumor necrosis factor-alpha (TNF-alpha) by 68%. The second phase of lipopolysaccharide-induced fever was significantly attenuated by pretreatment with 50 mg/kg pentoxifylline, a dose which had, per se, no influence on core temperature of guinea pigs. The thermal response of guinea pigs to administration of exogenous TNF-alpha was not modulated by pretreatment with pentoxifylline. Intra-arterial infusions with 5 micrograms/kg TNF-alpha, a dose which yielded the same circulating TNF bioactivity as i.m. injections of 20 micrograms/kg lipopolysaccharide, induced a biphasic febrile response. The magnitude and duration of TNF-induced fever were the same whether guinea pigs were pretreated with pentoxifylline or with 0.9% saline. The results indicate that endogenous formation of TNF-alpha may contribute to the development of fever induced by lipopolysaccharide, but is not its only mediator, since the first phase of lipopolysaccharide-induced fever was not altered by the blockade of TNF production.

Changes of abdominal temperature and circulating levels of cortisol and interleukin-6 in response to intra-arterial infusions of tumor necrosis factor-α or tumor necrosis factor-β in guinea pigs

The sister proteins tumor necrosis factor (TNF)-alpha and TNF-beta share 35% of their amino acid sequence and a number, but not all, of their biological properties. In the present study we infused amounts of 5 microg/kg TNF-alpha, TNF-beta (both preparations with identical bioactivities) or of solvent (0.9% sterile saline) into the circulation of guinea pigs and studied the effects on abdominal temperature, on the induction of endogenous formation of interleukin-6 and on levels of cortisol in plasma as a parameter of the activation of the hypothalamic-pituitary-adrenal axis. Infusion of TNF-alpha and TNF-beta both resulted in identical circulating TNF-like-activities corresponding to an amount of about 7000 pg/ml. TNF-alpha induced a biphasic fever lasting for more than 6 h, while in response to TNF-beta just the shorter first phase of fever (duration: 120 min) was measured. Circulating interleukin-6 (baseline level: 12-20 International Units (I.U.)/ml) and cortisol (baseline level: 70-120 ng/ml) increased about 6-fold during the first phase of the febrile response 60 min after the start of infusion with TNF-alpha or TNF-beta. Thereafter interleukin-6 and cortisol declined again in response to TNF-beta, but further increased after infusion with TNF-alpha to peak values measured 3 h after the start of infusion (interleukin-6: 258 +/- 52 I.U./ml; cortisol: 790 +/- 167 ng/ml). In animals infused with solvent abdominal temperature and interleukin-6 remained at the baseline values, just cortisol increased slightly. The results demonstrate that TNF-alpha is a much stronger inducer of fever and interleukin-6 production or of HPA-axis activation than TNF-beta in so far as all the investigated responses can be measured for prolonged time in response to TNF-alpha.

The Role of Local Induction of Tumor Necrosis Factor by LPS within a Subcutaneous Air Pouch in the Development of a Febrile Response in Guinea Pigs

In rats, fever can be induced by injection of bacterial lipopolysaccharide (LPS) into a subcutaneous air pouch. This febrile response is in part dependent on the local action of interleukin-1β within the pouch. In the present study, we tried to find out if this model of fever induction can be applied in guinea pigs and if the local LPS-induced formation of tumor necrosis factor-α (TNF-α) participates in the development of the febrile response. A dose of 100 μg/kg LPS was injected into a subcutaneous air pouch along with solvent (0.9% saline), or 100 mg/kg pentoxifylline, or 1 mg/animal soluble 55-kD TNF receptor (referred to as TNF-binding protein, TNF bp). The mean LPS-induced concentration of TNF in the lavage of the air pouch (measured 60 min after injection) was 17,765 pg/ml in animals injected with LPS and solvent. This value was reduced to 7,631 pg/ml if pentoxifylline was injected along with LPS into the pouch. If LPS was injected along with TNF bp, no bioactive TNF was detected in the lavage of the air pouch. Simultaneously, TNF was measured in blood plasma. Circulating concentrations of TNF were about 5–8% of the values detected in the lavage of the air pouch (mean values: 1,366 pg/ml in response to LPS plus solvent; 377 pg/ml in response to LPS plus pentoxifylline; no circulating TNF at all in response to LPS plus TNF bp). These data indicated a small spill-over of TNF from the air pouch into the circulation. Neither the reduction of local TNF formation in the air pouch by pentoxifylline nor the complete neutralization of TNF within the pouch by TNF bp resulted in a significant attenuation of the febrile response induced by injection of LPS into the subcutaneous air pouch. If there were an activation of cutaneous nerves to induce fever by a local formation of cytokines within the air pouch, TNF would not represent a likely candidate to be responsible for such a neural stimulation.