Interleukin-1-deficient mice exhibit high sensitivity to gut-derived sepsis caused by Pseudomonas aeruginosa

BACKGROUND: The role of interleukin (IL)-1 in infectious diseases is controversial; some investigators indicated an enhancing effect of IL-1 on host resistance whereas others demonstrated the protective role of IL-1 receptor antagonist in infection. We evaluated the role of endogenous IL-1 in gut-derived sepsis caused by Pseudomonas aeruginosa, by comparing IL-1-deficient mice and wild-type (WT) mice. METHODS: Gut-derived sepsis was induced by intraperitoneal injection of cyclophosphamide after colonization of P. aeruginosa strain D4 in the intestine. RESULTS: The survival rate of IL-1-deficient mice was significantly lower than that of WT mice (P<0.01). Bacterial counts in the liver, mesenteric lymph node and blood were significantly higher in IL-1-deficient mice than in WT mice. Tumor necrosis factor alpha and IL-6 in the liver were significantly higher in IL-1-deficient mice than in WT mice. In vitro, phagocytosis and cytokine production by macrophages were impaired in IL-1-deficient mice compared with WT mice. CONCLUSION: Our results indicate a critical role for IL-1 during gut-derived P. aeruginosa sepsis. The results also suggest that both impairment of cytokine production and phagocytosis by macrophages are caused by IL-1 deficiency and lead to impaired host response.     

Mechanisms mediating metabolic abnormalities in the livers of Ehrlich ascites tumor-bearing mice

Previously we reported that intermittent intraperitoneal administration of ornithine decarboxylase-inducing factor (ODC factor), interleukin 1alpha (IL-1alpha), and tumor-necrosis factor-alpha (TNF-alpha) to normal mice induced biological changes in the hosts which included changes in the pattern of expression of pyruvate kinase (PK) isozymes in the liver and hypertrophy of the spleen. In the study reported here, we investigated the chronic and combined effects of these factors on hepatic enzymes using alzet microosmotic pumps implanted in the subcutis of the backs or abdominal cavities of mice. Continuous administration of ODC factor and recombinant human IL-1alpha (rhIL-1alpha) reduced the activity of L-type PK, which is a glycolysis-related enzyme in the liver, and induced the activity of M2-type PK, a known marker of liver dedifferentiation. Serine dehydratase (SDH) and tyrosine aminotransferase (TAT), enzymes associated with amino acid metabolism, were not significantly influenced at the examined concentration. The simultaneous continuous infusion of ODC factor and rhIL-1alpha or rhTNF-alpha caused alterations in the patterns of expression of PK isozyme activity profiles and reduced overall PK activity. SDH and TAT activities were also significantly induced. Moreover, mice treated with these combined factors displayed many other metabolic changes normally associated with cancer cachexia. These findings suggest that the tumor-derived ODC factor and cytokines such as IL-1alpha and TNF-alpha might function synergistically in the metabolic perturbations observed in Ehrlich ascites tumor bearers.     

Dual effect of IL-4 on resistance to systemic gram-negative infection and production of TNF-alpha

To determine the effect of interleukin 4 (IL-4) administration in a live sepsis model characterised by high-level production of tumour necrosis factor a (TNF-alpha), mice infected systemically with lethal or sublethal inocula of Pseudomonas aeruginosa were given the recombinant cytokine at different times before infection. Improved survival and decreased TNF-alpha production were observed in lethally infected mice treated with the cytokine 1 day before challenge. In contrast, increased mortality and overproduction of TNF-alpha were observed in sublethally infected mice given IL-4 at the time of infection.     

In vivo induction of IL-6 by administration of exogenous cytokines and detection of de novo serum levels of IL-6 in tumor-bearing mice

We investigated the capacity of several recombinant cytokines to induce IL-6 in vivo in both normal and tumor-bearing (TB) mice. Intravenous administration of human rhTNF-alpha, rhIL-1, rhIL-2, rhIFN-alpha A/D, and rmIFN-gamma were all capable of inducing circulating IL-6. rhTNF-alpha administration caused the greatest induction of IL-6. TB animals consistently produced more IL-6 in response to rhTNF-alpha than did normal mice (2 h after 4 micrograms rhTNF-alpha, TB = 24,100 HGF U/ml, non-TB = 3600 HGF U/ml of IL-6). A single daily i.v. dose of rhTNF-alpha (4 micrograms/mouse/day) for 5 days led to decreased IL-6 induction in TB animals by day 3 of treatment (peak levels of IL-6, day 1 = 72,800 HGF U/ml, day 3 = 23,400 HGF U/ml, day 5 = 26,400 HGF U/ml). rhIL-1 administration also resulted in considerable IL-6 production, although peak values were less than those resulting from administration of rhTNF-alpha. Administration of rhIL-1 induced similar IL-6 levels (TB = 10,025 and non-TB = 10,600 HGF U/ml) in TB and normal mice. Single high doses of rhIL-2, rhIFN-alpha A/D, and rmIFN-gamma induced lower but consistent levels of circulating IL-6 in mice with and without tumor. In addition, the sera of untreated TB mice contained levels of IL-6 which paralleled the extent of tumor burden (serum IL-6 in day 30 MCA 106 TB mice = 420 HGF U/ml). The detection of de novo IL-6 was also confirmed in animals bearing tumors of different histologies (the MCA 102 sarcoma, MCA 38 adenocarcinoma, and B16 melanoma). At no time was IL-6 measurable in the sera of untreated normal mice. The identification of IL-6 was verified by neutralization studies using specific antimurine IL-6 antibody. Although the exact role of IL-6 in TB animals remains to be elucidated, its known pleotrophic immune and metabolic effects may be important in the host response to malignancy.     

Chemoprotective effects of recombinant human IL-1 alpha in cyclophosphamide-treated normal and tumor-bearing mice. Protection from acute toxicity, hematologic effects, development of late mortality, and enhanced therapeutic efficacy

In this study, recombinant human IL-1 alpha (rhIL-1 alpha) was used to protect normal and tumor-bearing BALB/c mice from the acute toxicity caused by lethal doses of cyclophosphamide (Cy) and 5-fluorouracil. Pretreatment of mice for 7 days with 10,000 U/day of rhIL-1 alpha protected 70 to 100% of mice from the acute death induced by lethal doses of both Cy (380 mg/kg) and 5-fluorouracil (250 mg/kg). In contrast, post-treatment of mice with single or multiple doses of rhIL-1 alpha was not chemoprotective. Pretreatment of mice with rhIL-1 alpha increased the acute LD90 of Cy from 380 mg/kg to greater than 500 mg/kg in normal mice, an LD90 dose-modifying effect of at least 1.25, was accompanied by a more rapid recovery from neutropenia and a less severe reduction in the number of bone marrow single lineage monocyte, myeloid, or myelomonocytic colonies. Some of the mice (10 to 50%) that were successfully protected by pretreatment with rhIL-1 alpha died after day 50. These mice consistently presented with extensive pulmonary inflammation and fibrosis at death. Mice bearing murine renal cancer (Renca) were also protected from the acute toxic effects of Cy (450 mg/kg) by pretreatment with rhIL-1 alpha. Renca-bearing mice pretreated with rhIL-1 alpha and either sublethal (300 mg/kg) or lethal (450 mg/kg) doses of Cy exhibited enhanced survival times over those of untreated Renca-bearing mice. Interestingly, the cause of death in Renca-bearing mice that ultimately failed treatment with rhIL-1 alpha plus 300 mg/kg Cy was recurrent tumor, whereas most mice treated with rhIL-1 alpha plus 450 mg/kg Cy had no detectable tumor, although several died from late pulmonary inflammation and fibrosis. Thus, the dose escalation of Cy in rhIL-1 alpha-pretreated mice results in greater antitumor effects of Cy. However, the dose escalation of some cytotoxic agents allowed by the use of myelostimulatory agents can result in late fatal complications not detected in acute toxicity testing.     

Pig MAP/ITIH4 and haptoglobin are interleukin-6-dependent acute-phase plasma proteins in porcine primary cultured hepatocytes.

The acute-phase expression of pig MAP (major acute-phase protein)/ITIH4 (inter-alpha-trypsin inhibitor heavy chain 4) and haptoglobin were analysed in primary cultures of isolated pig hepatocytes in response to recombinant human (rh) cytokines: tumor necrosis factor-alpha (TNF-alpha), interleukin-1 (IL-1), interleukin-6 (IL-6), as well as to bacterial lipopolysaccharide (LPS). Analysis of pig MAP/ITIH4 and haptoglobin mRNAs was carried out by RT-PCR amplification. Secreted proteins from the cytokine-treated hepatocytes were quantified by immunochemical techniques. Time-course and dose-response experiments show that pig MAP/ITIH4 and haptoglobin belong to the type II acute-phase proteins, as they are specifically induced by rhIL-6 and not by rhTNF-alpha or rhIL-1. Stimulation of cultured pig hepatocytes with rhIL-6 for 48 h at doses of 1000 U.mL-1 showed a fourfold to fivefold increase in pig MAP/ITIH4 concentration in the medium, while the concentration of haptoglobin only increased twofold. A similar increase in the concentration of pig MAP/ITIH4 was also observed in media of LPS-treated hepatocytes with the simultaneous generation of IL-6 by the Kupffer cells present in the cultures. Albumin secretion decreased after stimulation with doses of 100 or 1000 U.mL-1 rhTNF-alpha, rhIL-1 or rhIL-6. Therefore, it can be concluded that pig MAP/ITIH4 behaves as a major acute-phase protein produced by porcine hepatocytes under the effect of inflammatory cytokines.     

The effects of infection of thermal injury by Pseudomonas aeruginosa PAO1 on the murine cytokine response.

Pseudomonas aeruginosa infection, one of the major complications of burn wounds, may lead to sepsis and death. Using the Multi-Probe Template/RNase protection assay, we have compared the expression of different cytokine genes within the skin and livers of thermally injured mice infected with P. aeruginosa PAO1. Thermal injury alone enhanced or up-regulated certain cytokines, including macrophage colony-stimulating factor (M-CSF), interleukin 1 (IL-1)RI, IL-1 beta, macrophage inflammatory protein (MIP)-1 beta and MIP-2; while PAO1 challenge alone up-regulated tumour necrosis factor alpha (TNF-alpha) and transforming growth factor beta (TGF-beta) expression. The combination of thermal injury plus PAO1 infection enhanced the expression of several pro-inflammatory and haematopoietic cytokines [stem cell factor (SCF), leukocyte inhibitory factor (LIF), IL-6 and TNF-alpha]; induced the expression of granulocyte-macrophage colony-stimulating factor (GM-CSF) and G-CSF by 5 h and the expression of additional cytokines, including TGF-beta, TNF-beta, lymphotoxin beta (LT-beta), interferon gamma (IFN-gamma), and IFN-beta by 40 h post-burn/infection. While the most intense cytokine expression occurred in the skin, the majority of cytokines tested were also expressed in the liver by 40 h post-burn/infection. These results suggest that in P. aeruginosa infection of burn wounds: (1) up-regulation of the expression of different cytokines, locally and within the livers of burned mice, is an indication of P. aeruginosa -induced sepsis; and (2) IL-6 and G-CSF play an important role in the host response mechanism.     

Modulation of hematologic and immunologic effects of high dose chemotherapy by interleukin-2 in a murine tumor model.

To determine if intensive chemotherapy consisting of cyclophosphamide (C), etoposide (E), and cisplatin (P) (CEP) may be usefully combined with recombinant human interleukin-2 (rhIL-2), we examined a murine tumor model designed to approximate a common clinical situation: macroscopic, drug-resistant cancer. Using C57BL/6 mice with extensive tumor burden 10 days after intravenous B16 melanoma cell injection, we observed (1) C, E, and P synergize to enhance survival but do not cure mice at the highest tolerable dose (C = 200 mg/kg, E = 60 mg/kg, and P = 3 mg/kg); (2) rhIL-2 at 3 x 10(5) U (subcutaneously) daily for 4 days administered 10-18 days after B16 injection significantly improves survival; (3) CEP plus rhIL-2 is more effective than CEP alone only when rhIL-2 is administered before CEP; (4) CEP suppresses IL-2-induced lymphokine-activated killer cell activity in the spleen; and (5) rhIL-2 protects mice incompletely from the immunologic and hematologic suppression of CEP. Our results suggest that intensive chemotherapy combined with rhIL-2 may be beneficial. The success of any such combination may be schedule dependent.     

Gut-derived norepinephrine plays a critical role in producing hepatocellular dysfunction during early sepsis

Although plasma norepinephrine (NE) increases and hepatocellular function is depressed during early sepsis, it is unknown whether gut is a significant source of NE and, if so, whether gut-derived NE helps produce hepatocellular dysfunction. We subjected rats to sepsis by cecal ligation and puncture (CLP), and 2 h later (i.e., early sepsis) portal and systemic blood samples were collected and plasma levels of NE were assayed. Other rats were enterectomized before CLP. Hepatocellular function was assessed with an in vivo indocyanine green (ICG) clearance technique, systemic levels of tumor necrosis factor (TNF)-alpha, interleukin (IL)-1beta, and IL-6 were determined, and the effect of NE on hepatic ICG clearance capacity was assessed in an isolated, perfused liver preparation. Portal levels of NE were significantly higher than systemic levels at 2 h after CLP. Prior enterectomy reduced NE levels in septic animals. Thus gut appears to be the major source of NE release during sepsis. Enterectomy before sepsis also attenuated hepatocellular dysfunction and downregulated TNF-alpha, IL-1beta, and IL-6. Perfusion of the isolated livers with 20 nM NE (similar to that observed in sepsis) significantly reduced ICG clearance capacity. These results suggest that gut-derived NE plays a significant role in hepatocellular dysfunction and upregulating inflammatory cytokines. Modulation of NE release and/or hepatic responsiveness to NE should provide a novel approach for maintaining hepatocellular function in sepsis.     

Involvement of the liver, but not of IL-6, in IL-1-induced desensitization to the lethal effects of tumor necrosis factor.

C57BL/cnb mice were found to be protected against a lethal combination of recombinant murine (m) TNF and GalN by pretreatment with several cytokines. At certain doses, rmTNF and human (h) TNF protected completely. The clearest protection was induced by rIL-1: all four rIL-1 species (both m and h, as well as alpha and beta) protected when given 12 h before the challenge. LPS and rmIFN-gamma protected weakly, whereas rmIL-6 and rhIL-6 did not protect at all. Also adrenocorticotropic hormone, dexamethasone, or dexamethasone in combination with rhIL-6 could not protect. A single IL-1 injection also completely protected mice against a lethal dose of mTNF in the absence of GalN sensitization. The desensitization by IL-1 cannot be explained by a faster clearance of the challenge TNF. In addition, we demonstrate that the IL-1-induced desensitization was only observed when a functioning liver was present, that IL-1-pretreated animals did not show decreased numbers of hepatocyte TNF receptors, and that the amount of TNF-induced IL-6 was not reduced.     

Induction of interleukin 2 receptor (TAC) by tumor necrosis factor in YT cells

The effect of human recombinant tumor necrosis factor alpha (TNF-alpha) and interleukin 1 beta (IL-1 beta) on interleukin 2 receptor (IL-2R) expression on YT cells was examined. IL-2R expression was assessed by flow cytometric analysis with a monoclonal antibody to IL-2R (anti-TAC). TNF-alpha, like IL-1 beta, induced increased levels of IL-2R expression on YT cells with similar kinetics of induction. Maximum induction occurred at 20 to 30 hr. On a molar basis. TNF was less active than IL-1 beta. RNA isolated from TNF-alpha- or IL-1 beta-treated YT cells contained increased levels of IL-2R-specific mRNA as indicated by slot blot analysis by using an IL-2R-specific mRNA probe. Kinetic and IL-1 beta mRNA expression studies indicated that the TNF effect was a direct one. Because IL-2R expression is known to be associated with lymphocyte activation, the present results suggest that TNF-alpha may play a role in the regulation of immune responses.     

Interleukin 10 mitigates the development of the zymosan-induced multiple organ dysfunction syndrome in mice.

We investigated the effect of interleukin 10 on the development of zymosan-induced multiple organ dysfunction syndrome (MODS) and on plasma concentrations and production capacity of tumour necrosis factor (TNF)-alpha by peritoneal cells. Groups of C57BL/6 mice received a single intraperitoneal injection with zymosan, a cell wall component of Saccharomyces cerevisiae, at day 0. Daily doses of human recombinant interleukin 10 (IL-10: 10 or 50 microg/kg) were given intraperitoneally either starting directly before administration of zymosan (day 0), or 5 or 8 days after administration of zymosan. The animals were monitored for survival, condition, body weight and temperature. On day 12 all surviving animals were killed to obtain plasma, organs and peritoneal cells. Plasma concentrations of TNF-alpha and lipopolysaccharide-stimulated production of TNF-alpha by peritoneal cells were measured; organ weights were registered as an indicator for organ damage. IL-10 improves survival and clinical condition and also reduces organ damage, but only at the highest dose used and only when started simultaneously with the administration of zymosan. Circulating TNF-alpha concentrations 12 days after zymosan are not affected by any of the IL-10 schedules used. However, lipopolysaccharide-stimulated production of TNF-alpha by peritoneal cells is increased, in a dose- and time-dependent fashion. The anti-inflammatory cytokine IL-10 is able to attenuate the development of MODS in this model, but only when given simultaneously with zymosan, and in high dosages.     

Interleukin-1 deficiency in combination with macrophage depletion increases susceptibility to Pseudomonas aeruginosa bacteremia

We evaluated the role of IL-1 during Pseudomonas aeruginosa bacteremia by intravenously injecting P. aeruginosa strain D4 into IL-1-deficient and WT mice. The two strains showed equivalent mortality rates. However, when the mice were pretreated with cyclophosphamide, bacteremia-induced mortality was significantly greater in the IL-1-deficient mice than in the WT mice ( P < 0.01). We then investigated the role of neutrophils and macrophages in protecting IL-1-deficient mice from bacteremia by administering anti-Gr-1 antibody or liposomes containing dichloromethylene diphosphonate, respectively. After P. aeruginosa inoculation survival was significantly lower in the macrophage-depleted IL-1-deficient mice than in the WT mice. In contrast, neutrophil depletion did not have this effect. Compared to the macrophage-depleted WT mice, the macrophage-depleted IL-1-deficient bacteremic mice had higher bacterial counts in various organs 48 and 72 hr post-infection. They also had lower TNF-α, IL-6, and INF-γ concentrations in their livers during the early phase of sepsis. Thus, IL-1 deficiency becomes disadvantageous during P. aeruginosa bacteremia when it is accompanied by immunosuppression, particularly when macrophage functions are seriously impaired.     

Modulation of TNF release by choline requires alpha7 subunit nicotinic acetylcholine receptor-mediated signaling

The alpha7 subunit-containing nicotinic acetylcholine receptor (alpha7nAChR) is an essential component in the vagus nerve-based cholinergic anti-inflammatory pathway that regulates the levels of TNF, high mobility group box 1 (HMGB1), and other cytokines during inflammation. Choline is an essential nutrient, a cell membrane constituent, a precursor in the biosynthesis of acetylcholine, and a selective natural alpha7nAChR agonist. Here, we studied the anti-inflammatory potential of choline in murine endotoxemia and sepsis, and the role of the alpha7nAChR in mediating the suppressive effect of choline on TNF release. Choline (0.1-50 mM) dose-dependently suppressed TNF release from endotoxin-activated RAW macrophage-like cells, and this effect was associated with significant inhibition of NF-kappaB activation. Choline (50 mg/kg, intraperitoneally [i.p.]) treatment prior to endotoxin administration in mice significantly reduced systemic TNF levels. In contrast to its TNF suppressive effect in wild type mice, choline (50 mg/kg, i.p.) failed to inhibit systemic TNF levels in alpha7nAChR knockout mice during endotoxemia. Choline also failed to suppress TNF release from endotoxin-activated peritoneal macrophages isolated from alpha7nAChR knockout mice. Choline treatment prior to endotoxin resulted in a significantly improved survival rate as compared with saline-treated endotoxemic controls. Choline also suppressed HMGB1 release in vitro and in vivo, and choline treatment initiated 24 h after cecal ligation and puncture (CLP)-induced polymicrobial sepsis significantly improved survival in mice. In addition, choline suppressed TNF release from endotoxin-activated human whole blood and macrophages. Collectively, these data characterize the anti-inflammatory efficacy of choline and demonstrate that the modulation of TNF release by choline requires alpha7nAChR-mediated signaling.     

Subcutaneous administration of recombinant glycosylated interleukin 6 in patients with cancer: pharmacokinetics, pharmacodynamics and immunomodulatory effects

This is the first report of the serum profile of a glycosylated recombinant form of human IL-6 (rhIL-6) administered subcutaneously (1-10 microg/kg/day) in a phase I/II trial as a thrombopoietic agent in patients with advanced cancer. The pharmacodynamic effects of IL-6 were also examined. Detailed pharmacokinetic measurements were made in four patients. Peak concentrations at 5-8 h and a median t0.5 of ca. 5 h were similar to those previously reported for non-glycosylated IL-6. However, higher peak concentrations and apparent differences in effective dose levels to those previously reported with the non-glycosylated form were seen. Indications of an apparent attenuation in circulating IL-6 concentrations with continuing injections were seen in eight of 10 patients examined but anti-IL-6 antibody generation was seen in only two patients. Soluble interleukin 6 receptor concentrations generally decreased. No major changes in T cell subsets were seen but expression of CD25 and CD54 by T lymphocytes significantly increased, accompanied by marked increases in soluble CD25 (sIL-2R) and CD54 (sICAM-1). No consistent change in B cells, monocytes or NK cells were seen. No evidence for induction of TNF-alpha was found. This study demonstrates similar biological effects of glycosylated rhIL-6 to those reported for the non-glycosylated form but illustrates several apparent differences which are discussed further.     

Interleukin-1 beta (IL-1beta) induces tumor necrosis factor alpha (TNF-alpha) expression on mouse myeloid multipotent cell line 32D cl3 and inhibits their proliferation

Interleukin-1 alpha (IL-1alpha) and beta (IL-1beta) are well known factors that stimulate hematopoiesis, nevertheless there are reports that show that they can also inhibit this activity. While both IL-1alpha and IL-1beta induce the expression of hematopoietic cytokines, such as growth factors and their receptors on myeloid cells, helping thus to regulate hematopoiesis, it is not known if their inhibitory activity is also mediated through the induction of other specific cytokines. In this work we show that recombinant human IL-1beta (rhIL-1beta) inhibits the proliferation of a mouse IL-3-dependent myeloid multipotent cell line (32D cl3), without inducing its differentiation. We show that rhIL-1beta induces in 32D cl3 cells the expression of the tumor necrosis factor alpha (TNF-alpha) gene, a well known growth inhibitor, and that the rhIL-1beta growth inhibition property on 32D cl3 cells is partially due to this secreted TNF-alpha, hinting thus that the inhibition of hematopoiesis by IL-1 is mediated through other induced cytokines.     

Interleukin-1 alpha (IL-1 alpha) and tumor necrosis factor alpha (TNF alpha) regulate insulin-like growth factor binding protein-1 (IGFBP-1) levels and mRNA abundance in vivo and in vitro.

TNF alpha and IL-1 alpha are thought to contribute to impaired anabolism in a variety of clinical states, including sepsis, cancer cachexia and the AIDS wasting syndrome. We asked whether cytokines exert direct effects on hepatic production of IGFBP-1, an important modulator of IGF bioavailability. C57BL/6 mice were treated with 100 micrograms/kg of recombinant IL-1 alpha or TNF alpha by intraperitoneal injection. Western ligand blotting and immunoprecipitation with specific antisera revealed that serum levels of IGFBP-1 (but not IGFBP-2, -3, -4, -5 or -6) are increased approximately 4 fold 2 h after treatment and then decline. Northern blotting confirms that hepatic IGFBP-1 mRNA abundance also is increased acutely in both IL-1 alpha- and TNF alpha-treated animals. Similar results obtained in adrenalectomized mice indicate that adrenal activation is not required for this effect. Cell culture studies show that cytokines exert direct effects on the production of IGFBP-1 by HepG2 hepatoma cells, increasing IGFBP-1 levels in conditioned medium and the abundance of IGFBP-1 mRNA approximately 3-fold. In contrast, transient transfection studies with IGFBP-1 promoter/luciferase reporter gene constructs show that IGFBP-1 promoter activity is reduced after 18 hr cytokine treatment. We conclude that IL-1 alpha and TNF alpha increase circulating levels of IGFBP-1, reflecting direct effects on hepatic IGFBP-1 mRNA abundance. Stimulation of hepatic IGFBP-1 production may contribute to alterations in IGF bioactivity and impaired anabolism in clinical conditions where cytokine production is high. Additional studies are required to identify specific mechanisms mediating effects of cytokines on hepatic production of IGFBP-1.     

Role of alpha(2)-macroglobulin in fever and cytokine responses induced by lipopolysaccharide in mice

Alpha(2)-macroglobulin (alpha(2)M) is not only a proteinase inhibitor in mammals, but it is also a specific cytokine carrier that binds pro- and anti-inflammatory cytokines implicated in fever, including interleukin (IL)-1beta, IL-6, and tumor necrosis factor-alpha (TNF-alpha). To define the role of alpha(2)M in regulation of febrile and cytokine responses, wild-type mice and mice deficient in alpha(2)M (alpha(2)M -/-) were injected with lipopolysaccharide (LPS). Changes in body temperature as well as plasma levels of IL-1beta, IL-6, and TNF-alpha and hepatic TNF-alpha mRNA level during fever in alpha(2)M -/- mice were compared with those in wild-type control mice. The alpha(2)M -/- mice developed a short-term markedly attenuated (ANOVA, P < 0.05) fever in response to LPS (2.5 mg/kg ip) compared with the wild-type mice. At 1.5 h after injection of LPS, the plasma concentration of TNF-alpha, but not IL-1beta or IL-6, was significantly lower (by 58%) in the alpha(2)M -/- mice compared with their wild-type controls (ANOVA, P < 0.05). There was no difference in hepatic TNF-alpha mRNA levels between alpha(2)M -/- and wild-type mice 1.5 h after injection of LPS. These data support the hypotheses that 1) alpha(2)M is important for the normal development of LPS-induced fever and 2) a putative mechanism of alpha(2)M involvement in fever is through the inhibition of TNF-alpha clearance. These findings indicate a novel physiological role for alpha(2)M.     

Suppressive activity of a macrolide antibiotic, roxithromycin, on pro-inflammatory cytokine production in vitro and in vivo

This study was designed to examine the influence of a macrolide antibiotic, roxithromycin (RXM), on the production of pro-inflammatory cytokines, interleukin (IL)-1beta and tumor necrosis factor (TNF)-alpha. In the first experiments, we examined the effect of RXM on in vitro cytokine production from lipopolysaccharide (LPS)-stimulated human peripheral blood monocytes. The monocytes were cultured in the presence of various doses of the agent. After 24 h, the culture supernatants were obtained and assayed for IL-1beta and TNF-alpha contents by enzyme-linked immunosorbent assay. RXM suppressed the in vitro production of IL-1beta and TNF-alpha in response to LPS stimulation. This was dose dependent and first noted at a concentration of as little as 0.05 microg/ml, which is much lower than therapeutic blood levels. In the second part of the experiments, we examined the influence of RXM on the appearance of IL-1beta and TNF-alpha in mouse lung extract induced by LPS inhalation. RXM was administered orally into BALB/c mice at a single dose of 2.5 mg/kg once a day for 5-12 weeks. These mice were then instilled with LPS into the trachea and examined for the presence of cytokines in aqueous lung extracts. Pretreatment of mice with RXM for 5 weeks did not influence of the appearance of both IL-1beta and TNF-alpha in aqueous lung extracts. However, pretreatment for more than 7 weeks dramatically suppressed the cytokine appearance in the extracts.     

Ciliary neurotrophic factor inhibits brain and peripheral tumor necrosis factor production and, when coadministered with its soluble receptor, protects mice from lipopolysaccharide toxicity.

BACKGROUND: The receptor of ciliary neurotrophic factor (CNTF) contains the signal transduction protein gp130, which is also a component of the receptors of cytokines such as interleukin (IL)-6, leukemia-inhibitory factor (LIF), IL-11, and oncostatin M. This suggests that these cytokines might share common signaling pathways. We previously reported that CNTF augments the levels of corticosterone (CS) and of IL-6 induced by IL-1 and induces the production of the acute-phase protein serum amyloid A (SAA). Since the elevation of serum CS is an important feedback mechanism to limit the synthesis of proinflammatory cytokines, particularly tumor necrosis factor (TNF), we have investigated the effect of CNTF on both TNF production and lipopolysaccharide (LPS) toxicity. MATERIALS AND METHODS: To induce serum TNF levels, LPS was administered to mice at 30 mg/kg i.p. and CNTF was administered as a single dose of 10 micrograms/mouse i.v., either alone or in combination with its soluble receptor sCNTFR alpha at 20 micrograms/mouse. Serum TNF levels were the measured by cytotoxicity on L929 cells. In order to measure the effects of CNTF on LPS-induced TNF production in the brain, mice were injected intracerebroventricularly (i.c.v.) with 2.5 micrograms/kg LPS. Mouse spleen cells cultured for 4 hr with 1 microgram LPS/ml, with or without 10 micrograms CNTF/ml, were also analyzed for TNF production. RESULTS: CNTF, administered either alone or in combination with its soluble receptor, inhibited the induction of serum TNF levels by LPS. This inhibition was also observed in the brain when CNTF and LPS were administered centrally. In vitro, CNTF only marginally affected TNF production by LPS-stimulated mouse splenocytes, but it acted synergistically with dexamethasone (DEX) in inhibiting TNF production. Most importantly, CNTF administered together with sCNTFR alpha protected mice against LPS-induced mortality. CONCLUSIONS: These data suggest that CNTF might act as a protective cytokine against TNF-mediated pathologies both in the brain and in the periphery.