Induction of interleukin-1 in human monocytes by the superantigen staphylococcal enterotoxin A requires the participation of T cells

Nanogram quantities of the bacterial superantigen Staphylococcal Enterotoxin A (SEA) induced significant amounts of extracellular IL-1 alpha and IL-1 beta in human peripheral blood mononuclear cells. Induction of maximal IL-1 alpha and IL-1 beta levels by lipopolysaccharide (LPS) required microgram quantities. LPS induced detectable extracellular IL-1 content within 3-6 hr and maximal levels were detected already after 12 hr. Induction of IL-1 production by SEA showed a delayed release with peak values after 24-48 hr. IL-1 beta was the major species of IL-1 seen in both SEA- and LPS-stimulated culture supernatants. SEA was in general a relatively stronger inducer of extracellular IL-1 alpha than LPS. SEA-induced extracellular IL-1 production in human monocytes was entirely dependent on the presence of T cells, whereas addition of T cells to LPS-stimulated purified human monocytes only marginally enhanced the extracellular IL-1 production. The capacity to induce extracellular IL-1 production in monocytes in response to SEA was high in the CD4+ 45RO+ memory T cell subset, whereas CD4+ 45RA+ naive T cells and CD8+ T cells had lower IL-1-inducing capacity. The T cell help for IL-1 production could not be replaced by a panel of T cell-derived recombinant lymphokines added to SEA-stimulated monocytes, including IFN-gamma and TNF, indicating the participation of cell membrane-bound ligands or hitherto unidentified soluble mediators.     

Selection of novel analogs of thalidomide with enhanced tumor necrosis factor alpha inhibitory activity.

BACKGROUND: Tumor necrosis factor alpha (TNF alpha) is thought to mediate both protective and detrimental manifestations of the inflammatory response. Recently, thalidomide (alpha-N-phthalimidoglutarimide) was shown to partially inhibit monocyte TNF alpha production (by 50-70%) both in vivo and in vitro. More efficient inhibition of TNF alpha may, however, be necessary to rescue the host from more acute and extensive toxicities of TNF alpha-mediated inflammation. MATERIALS AND METHODS: Three structural analogues of thalidomide were selected for study based on increased activity against TNF alpha production. The parent drug and the analogs were tested in vitro in human peripheral blood mononuclear cell cultures for their effects on lipopolysaccharide (LPS) induced cytokine protein and mRNA production using ELISAs and Northern blot hybridization. The in vitro effects of the drugs were then confirmed in vivo in a mouse model of LPS induced lethality. RESULTS: The new compounds (two esters and one amide) showed increased inhibition of TNF alpha production by LPS-stimulated human monocytes, relative to the parent drug thalidomide. The analogs and the parent drug enhanced the production of interleukin 10 (IL-10), but had little effect on IL-6 and IL-1 beta protein and mRNA production. When tested in vivo, the amide analog protected 80% of LPS-treated mice against death from endotoxin induced shock. CONCLUSIONS: Analogs of thalidomide designed to better inhibit TNF alpha production in vitro have correspondingly greater efficacy in vivo. These finding may have therapeutic implication for the treatment of human diseases characterized by acute and extensive TNF alpha production such as tuberculous meningitis or toxic shock.     

Processing and secretion of tumor necrosis factor alpha in endotoxin-treated Mono Mac 6 cells are dependent on phorbol myristate acetate.

Lipopolysaccharide (LPS, endotoxin) is a potent stimulator of tumor necrosis factor alpha (TNF alpha) synthesis and secretion in mouse macrophage tumor cells (Golenbock, D. T., Hampton, R. Y., Qureshi, N., Takayama, K., and Raetz, C. H. R. (1991) J. Biol. Chem. 266, 19490-19498). In contrast, addition of LPS (10 ng/ml) to human monomyelocytic (Mono Mac 6) cells induces very little production of TNF alpha, as judged by immunoassay of the growth medium. When 30 ng/ml 4-beta-phorbol-12-myristate 13-acetate (PMA) is added together with LPS, large amounts of TNF alpha are secreted. PMA alone is inactive. Maximal TNF alpha levels in the medium are achieved at 1 ng/ml of LPS. Protein kinase C inhibitors, such as H7 (1-(5-isoquinolinylsulfonyl)-2-methylpiperazine), staurosporine, and sphingosine, reduce TNF alpha secretion stimulated by PMA. The effect of PMA has been investigated at each stage of TNF alpha biogenesis. Treatment of Mono Mac 6 cells with LPS alone results in rapid, transient, and full expression of TNF alpha mRNA. Concomitant addition of PMA does not increase TNF alpha mRNA synthesis any further, but it prolongs the half-life of TNF alpha mRNA about 3-fold. However, mRNA stabilization does not account for the striking effect of PMA on TNF alpha secretion. Analysis of TNF alpha synthesis and secretion by immunoprecipitation indicates that LPS alone is fully effective in stimulating the formation of the intracellular 26-kDa TNF alpha precursor. LPS alone is not sufficient to allow processing of the precursor and secretion of mature 17-kDa TNF alpha. The rate of TNF alpha secretion observed immediately after the addition of PMA to LPS-pretreated cells is similar to the maximum rate from LPS/PMA-treated cells, but without the lag observed in cells after being exposed to LPS and PMA simultaneously. In summary, PMA is required for the completion of TNF alpha precursor processing and secretion in LPS-treated human Mono Mac 6 cells, whereas murine RAW cells are able to complete the terminal steps of TNF alpha processing in the absence of PMA.     

Macrophages rapidly internalize their tumor necrosis factor receptors in response to bacterial lipopolysaccharide

The effect of bacterial lipopolysaccharide (LPS) on macrophage receptors for tumor necrosis factor/cachectin (TNF-R) was studied. At equilibrium, iodinated recombinant human TNF alpha (rTNF alpha) bound to 1100 +/- 200 sites/cell on macrophage-like RAW 264.7 cells with a Kd of 1.3 +/- 0.1 x 10(-9) M. Preexposure of RAW 264.7 cells to 10 ng/ml LPS for 1 h at 37 degrees C resulted in complete loss of cell surface TNF alpha binding sites. 50% loss ensued after 1 h with 0.6 ng/ml LPS, or after 15 min with 10 ng/ml LPS. Complete loss of TNF alpha binding sites occurred without change in numbers of complement receptor type 3. No decrease in TNF-R followed preexposure to LPS at 4 degrees C, nor could LPS displace 125I-rTNF alpha from its binding sites. Although TNF-R disappeared from the surface of intact macrophages following exposure to LPS, specific TNF alpha binding sites were unchanged in permeabilized macrophages, indicating that TNF-R were rapidly internalized. Conditioned media from LPS-treated RAW 264.7 cells induced 30% down-regulation of TNF-R on macrophages from LPS-hyporesponsive mice (C3H/HeJ), suggesting that a soluble macrophage product may be responsible for a minor portion of the LPS effect. Additional evidence against endogenous TNF alpha being the major cause of TNF-R internalization was the rapid onset of the effect of LPS on TNF-R compared to the reported onset of TNF alpha production, the relatively high concentrations of exogenous rTNF alpha required to mimic the effect of LPS, and the inability of TNF alpha-neutralizing antibody to block the effect of LPS. LPS-induced down-regulation of TNF-R was complete or nearly complete not only in RAW 264.7 cells, but also in primary macrophages of both human and murine origin, was less marked in human endothelial cells, and was absent in human granulocytes and melanoma cells and mouse L929 cells. Thus, in situ, macrophages and some other host cells may be resistant to the actions of TNF alpha produced during endotoxinemia, because such cells may internalize their TNF-R in response to LPS before TNF alpha is produced.     

Oligodeoxynucleotides enhance lipopolysaccharide-stimulated synthesis of tumor necrosis factor: dependence on phosphorothioate modification and reversal by heparin.

BACKGROUND: Specific inhibition of target proteins by antisense oligodeoxynucleotides is an extensively studied experimental approach. This technique is currently being tested in clinical trials applying phosphorothioate-modified oligonucleotides as therapeutic agents. These polyanionic molecules, however, may also exert non-antisense-mediated effects. MATERIALS AND METHODS: We examined the influence of oligonucleotides on lipopolysaccharide (LPS)-stimulated tumor necrosis factor alpha (TNF alpha) synthesis in freshly isolated human peripheral blood mononuclear cells. Oligonucleotides (18 mer) with different degrees of phosphorothioate modification were studied. RESULTS: The addition of phosphorothioate oligonucleotides (5 microM) caused amplification of TNF synthesis of up to 410% compared with the control with LPS alone. Without LPS stimulation, phosphorothioate oligonucleotides did not induce TNF production. We demonstrate that the enhancement of LPS-stimulated TNF production by phosphorothioate oligonucleotides does not rely on the intracellular presence of oligonucleotides and is not mediated by LPS contamination. Partially phosphorothioate-modified oligonucleotides and unmodified oligonucleotides did not increase TNF synthesis. High concentrations of the polyanion heparin reversed the oligonucleotide-induced enhancement of TNF synthesis. CONCLUSIONS: The data suggest that amplification of TNF synthesis may be caused by binding of the polyanionic phosphorothioate oligonucleotide to cationic sites on the cell surface. Such binding sites have been proposed for polyanionic glycoaminoglycans of the extracellular matrix, which have also been described to augment LPS-stimulated TNF synthesis. The present results are relevant to all in vitro studies attempting to influence protein synthesis in monocytes by using phosphorothioate oligonucleotides. The significance of our findings for in vivo applications of phosphorothioates in situations where there is a stimulus for TNF synthesis, such as in sepsis, should be elucidated.     

Exogenous heat shock protein HSP70 reduces response of human neuroblastoma cells to lipopolysaccharide

The effect of exogenous heat shock protein HSP70 and lipopolysaccharide (LPS) on the production of reactive oxygen species (ROS), TNFα secretion, and mRNA expression by human neuroblastoma SK-N-SH cells. It was shown that exogenous HSP70 protects neuroblastoma cells from the action of LPS. The protection mechanism of HSP70 includes a reduction in the production of ROS and TNFα and a decrease in the expression of TLR4 and IL-1β mRNA in SK-N-SH cells induced by LPS.     

In vitro and in vivo TNFalpha synthesis modulation by methylguanidine, an uremic catabolyte.

This study was performed in order to examine whether the uraemic toxin, methylguanidine (MG), can modulate tumor necrosis factor alpha (TNF alpha) release by activated macrophages. In this study we have evaluated the ability of MG to influence TNF alpha release in vitro, in Escherichia coli lypopolysaccharide- (LPS)-stimulated J774 cells preincubated overnight with MG, and in vivo in rats treated with MG before and after LPS challenge. Parallel experiments employing N(G)-nitro-L-arginine methyl esther (L-NAME) were also carried out for comparison. The effect of LPS (6 x 10(3) u/ml) on TNF alpha release by J774, following overnight incubation with MG or L-NAME (1 mM), was examined 3 hours after LPS challenge. LPS-stimulated J774 released 287.83+/-88 u/ml TNF alpha into the culture medium. MG (1 mM) significantly inhibited TNF alpha release by 73% (P<0.05). L-NAME (1 mM) significantly inhibited TNF alpha release too by 72.88% (P<0.05). The effect of MG and L-NAME have been also studied in vivo. Serum TNF alpha levels in LPS treated rats 2 h after LPS challenge were 88.33+/-31.7 u/ml as compared to the serum TNF alpha levels of control rats (undetectable). Treatment of rats with MG (30 mg/kg, i.p.) strongly and significantly reduced TNF alpha release (98.71% inhibition; with P<0.001); in the same experimental setting L-NAME (10 mg/kg, i.p.) also significantly reduced TNF alpha serum levels (76.47% inhibition; with P<0.01). These results could indicate that immune disfunction related to uremia may be related to the inhibitory capability of uremic catabolyte, MG, on TNF alpha synthesis and release.     

Differential induction of interleukin-6 production in monocytes, endothelial cells and smooth muscle cells.

Studying the production of IL-6 (interleukin-6) by monocytes, endothelial cells and smooth muscle cells we observed that cytokine inducers like IL-1, TNF alpha (tumor necrosis factor alpha), LPS (lipopolysaccharide), SAC (Staphylococcus Aureus Cowan 1) and PMA could be divided roughly into two categories. Bacterial products such as LPS or SAC have a potent IL-6 inducing effect on monocytes and minor or no effect on endothelial- and smooth muscle cells. The other category comprising IL-1, TNF alpha and PMA induces IL-6 production in endothelial- and smooth muscle cells. Only IL-1 induces IL-6 production in monocytes as well as in endothelial cells and smooth muscle cells. In addition to IL-6, also IL-1 and TNF alpha are produced by monocytes however with different kinetics. None of the stimuli had any inhibitory effect on IL-6 production with the exception of PMA. Whereas PMA induced IL-6 production in endothelial cells and it potentiated the induction of IL-6 by IL-1 in these cells, it inhibited LPS-stimulated IL-6 production in monocytes. In line with the effects of PMA, staurosporin induced IL-6 production in monocytes and it inhibited IL-1 driven IL-6 production by endothelial cells.     

Granulocyte-macrophage colony stimulating factor modulates the production of TNF alpha by differentiated U937 cells infected with Leishmania major

In this work, the production of tumor necrosis factor alpha (TNF alpha) during interaction of human phagocytes with the intracellular parasite Leishmania major was further investigated. The human monocytic cell line U937, differentiated with a combination of 1 alpha, 25 dihydroxyvitamin D3 (VD) and retinoic acid (RA), or with granulocyte macrophage colony stimulating factor (GM-CSF) was used. Differentiated U937 cells were infected with Leishmania major promastigotes, and TNF alpha was assayed in cell culture supernatants. It was found that the cytokine was produced only by U937 cells differentiated with VD/RA and further incubated with GM-CSF and LPS or interferon gamma (IFN gamma). L. major induced TNF alpha production only in the presence of GM-CSF. No direct relationship was found, however, between production of TNF alpha and resistance of differentiated U937 cells to infection with L. major.     

N-acetylcysteine and glutathione as inhibitors of tumor necrosis factor production.

TNF is a major mediator in the pathogenesis of endotoxic shock, and its inhibition has a protective effect in various animal models of sepsis or endotoxin (lipopolysaccharide, LPS) toxicity. LPS treatment also induces an oxidative damage mediated by increased production of reactive oxygen intermediates. N-Acetylcysteine (NAC) is an antioxidant and a precursor of the synthesis of glutathione (GSH) and was reported to protect against LPS toxicity and LPS-induced pulmonary edema. In this study we investigated the effect of NAC on TNF production and LPS lethality in mice. The results indicated that oral administration of NAC protects against LPS toxicity and inhibits the increase in serum TNF levels in LPS-treated mice. The inhibition was not confined to the released form of TNF, since NAC also inhibited LPS-induced spleen-associated TNF. On the other hand, the inhibitor of GSH synthesis, DL-buthionine-(SR)-sulfoximine (BSO), had the opposite effect of potentiating LPS-induced TNF production, and this was associated with a decrease in liver GSH levels. Repletion of liver GSH with NAC reversed this effect. NAC was also active in inhibiting TNF production and hepatotoxicity in mice treated with LPS in association with a sensitizing dose of Actinomycin D. These data indicate that GSH can be an endogenous modulator of TNF production in vivo. On the other hand, NAC pretreatment did not inhibit other effects of LPS, particularly induction of serum IL-6, spleen IL-1 alpha, and corticosterone, in the same experimental model, suggesting that the observed effect could be specific for TNF.     

Endotoxin and Staphylococcus aureus enterotoxin A induce different patterns of cytokines.

The effects of Staphylococcus aureus enterotoxin A (SEA) and lipopolysaccharide (LPS) in cytokine production were assessed at the single cell level in cells obtained from healthy blood donors. Cytokine production was studied with UV-microscopy of fixed and permeabilized cells stained with cytokine specific monoclonal antibodies. The cytokines evaluated included tumour necrosis factor (TNF)-alpha, interleukin (IL)-1 alpha, IL-1 beta, IL-6, IL-8, IL-10, IL-2, IL-4, interferon (IFN)-gamma and TNF-beta. LPS exhibited marked production of IL-1 alpha, IL-1 beta, TNF-alpha, IL-6 and IL-8. After LPS stimulation IL-1 alpha, IL-1 beta, TNF-alpha and IL-8 were the dominating products, all peaking at or before 4 hours after cell stimulation. In addition, IL-10 production was evident after 12 hours of cell stimulation. The T-lymphocyte-derived cytokines TNF-beta, IL-2, IFN-gamma and IL-4 were never detected in the cultures. All cytokine production, except IL-8, was downregulated at 96 hours. In contrast, peak production of IL-1 alpha, IL-1 beta and IL-8, which were the dominant products, occurred after 12 hours in the SEA-stimulated cultures. Further, a significant T-lymphocyte production of TNF-beta, TNF-alpha, IFN-gamma and IL-2 was found with peak production 12-48 hours after initiation. Only low amounts of IL-6 were evident. The two types of cytokine pattern and kinetics found may correspond to the different clinical conditions after invasive Gram-negative Escherichia coli vs Gram-positive Staphylococcus aureus infections in humans, with a much more rapid onset of disease after E. coli infections.(ABSTRACT TRUNCATED AT 250 WORDS)     

Nitric oxide enhances PGI(2)production by human pulmonary artery smooth muscle cells

To evaluate the effect of exogenous nitric oxide (NO) and endogenous NO on the production of prostacyclin (PGI(2)) by cultured human pulmonary artery smooth muscle cells (HPASMC) treated with lipopolysaccharide (LPS), interleukin-1(beta)(IL-1(beta)), tumor necrosis factor alpha (TNF(alpha)) or interferon gamma (IFN(gamma)), HPASMC were treated with LPS and cytokines together with or without sodium nitroprusside (SNP), NO donor, N(G)-monomethyl-L-arginine (L-NMMA), NO synthetase inhibitor, and methylene blue (MeB), an inhibitor of the soluble guanylate cyclase. After incubation for 24 h, the postculture media were collected for the assay of nitrite by chemiluminescence method and the assay of PGI(2)by radioimmunoassay. The incubation of HPASMC with various concentrations of LPS, IL-1(beta)or TNF(alpha)for 24 h caused a significant increase in nitrite release and PGI(2)production. However, IFN(gamma)slightly increased the release of nitrite and had little effect on PGI(2)production. Although the incubation of these cells for 24 h with SNP did not cause a significant increase in PGI(2)production, the incubation of HPASMC with SNP and 10 microg/ml LPS, or with SNP and 100 U/ml IL-1(beta)further increase PGI(2)production and this enhancement was closely related to the concentration of SNP. However, stimulatory effect of SNP on PGI(2)production was not found in TNF(alpha)- and IFN(gamma)- treated HPASMC. Addition of L-NMMA to a medium containing LPS or IL-1(beta)reduced nitrite release and attenuated the stimulatory effect of those agents on PGI(2)production. MeB significantly suppressed the production of PGI(2)by HPASMC treated with or without LPS or IL-1(beta). The addition of SNP partly reversed the inhibitory effect of MeB on PGI(2)production by HPASMC. These experimental results suggest that NO might stimulate PGI(2)production by HPASMC. Exogenous NO together with endogenous NO induced by LPS or cytokines from smooth muscle cells might synergetically enhance PGI(2)production by these cells, possibly in clinical disorders such as sepsis and acute respiratory distress syndrome.     

Several functions of immune cells in mice changed by oxidative stress caused by endotoxin

We have studied natural killer (NK) activity, lymphoproliferative response, the release of several cytokines (IL-2, TNF alpha and IL-1 beta) and the ROS production in peritoneal leukocytes obtained 0, 2, 4, 12 and 24 h after lipopolysaccharide (LPS) injection. Lethal septic shock (100 % mortality occurred at 30 h after LPS administration) was caused in female BALB/c mice by intraperitoneal injection of 100 mg/kg of E. coli LPS. Cytotoxicity and lymphoproliferation assay were preformed together with the measurement of IL-1 beta, IL-2 and TNF alpha production, and quantification of ROS. Natural killer activity, spontaneous lymphoproliferative response, IL-2, TNF alpha, IL-beta release and ROS production were increased after LPS injection. In conclusions, ROS and proinflammatory mediators produced by immune cells in response to LPS are involved in the oxidative stress of endotoxic shock. This oxidative state alters some functional characteristics of leukocytes (proliferation and NK activity).     

Dual role of tumor necrosis factor alpha in brain injury

Brain injury (ischemia, trauma) is among the leading cause of mortality and disability in the western world. It induces increased production of tumor necrosis factor (TNF alpha) by brain resident cells. There is conflicting evidence on the role of this response in the injured brain, showing its potential effect in both processes of repair and of damage. This review presents data from clinical and experimental studies on the stimulation of TNF alpha production in brain injury and on the deleterious consequence of this acute response. Its inhibition by pharmacologic agents, neutralizing antibodies or soluble receptors has protective effects. In contrast, there are reports (from in-vitro studies or knock-out mice) on the beneficial effects of TNF alpha. To reconcile these apparently conflicting reports, the exact timing and extent of TNF alpha activation must be taken into account, as well as the presence of other mediators such as reactive oxygen species. It is suggested that the appropriate context of mediators, at any given time after brain injury may well determine whether the effect of TNF alpha is protective or toxic.     

Activation of the production of the tumor-necrosis factor by the combined action of lipopolysaccharide and muramyl dipeptide in vitro and in vivo

The effect of bacterial lipopolysaccharide (LPS), muramyl dipeptide (MDP) and their combination on the production of tumour necrosis factor by spleen cells in vitro and on tumour regression in vivo has been studied. TNF activity was detected in spleen cell supernatants and serum of mice treated with drugs, using L929 cells as targets. The combination of LPS and MDP was more effective in TNF production than each of the drugs used alone in vitro and in vivo. The injection of LPS and MDP to A/Sn mice with subcutaneous nodes of sarcoma SA-I resulted in total tumour necrosis. The treatment of mice with these drugs in water solutions was more effective, however, more toxic than the administration of LPS-treated splenocytes in MDP solution.     

Synergistic interactions of interleukin 1, interferon-beta, and tumor necrosis factor in terminally differentiating a mouse myeloid leukemic cell line (M1). Evidence that interferon-beta is an autocrine differentiating factor

The effect was investigated of combinations of cytokines known to be cytostatic for some tumor cells, namely interleukin 1 alpha (IL-1 alpha), interferon-beta (IFN-beta), and tumor necrosis factor (TNF), on the growth and differentiation of the mouse myeloid leukemic cell line, M1, cells. IL-1 alpha, IFN-beta, and TNF by themselves are antiproliferative for M1 cells. Treatment of cells with a mixture of any two of the three cytokines resulted in at least additive growth inhibition. None of these cytokines by themselves induced differentiation of M1 cells as assessed by increased expression of Fc receptors (FcR), stimulation of phagocytic activity and by morphologic criteria. However, as little as 1 U/ml IL-1 alpha in conjunction with IFN-beta or TNF increased FcR expression, phagocytic activity and morphologic changes in addition to inhibiting the growth of M1 cells. The combination of IFN-beta and TNF did not induce differentiation, although the growth of the cells was markedly inhibited. Both TNF and lipopolysaccharide (LPS) induced the in vitro production of IFN activity by M1 cells. Furthermore, the induction of differentiation of M1 cells by a combination of IL-1 alpha with either IFN-beta, TNF, or LPS was inhibited by antibody against mouse IFN-beta. Therefore, it appears that IFN-beta provides one of the two required signals for differentiation of M1 cells by these combinations of stimulants, the other being IL-1. Furthermore, the cytostatic effect of TNF by itself on M1 cells was also partly blocked by anti-IFN-beta antibody, suggesting that IFN-beta is also involved in the growth inhibitory effect of TNF for M1 cells. In contrast, the cytostatic effect of IL-1 on M1 cells was not blocked by anti-IFN-beta antibody. In conclusion, both the cytostatic and differentiative effect of TNF appear to be mediated by IFN-beta. Thus, the combination of IL-1 and IFN-beta or inducers of IFN-beta resulted in terminal differentiation of M1 cells. Northern blot analysis using cDNAs for murine IFN-beta1 or human IFN-beta2 showed an increased expression of mRNA for IFN-beta1 but not for IFN-beta2 by stimulation with TNF or LPS, strongly suggesting that IFN-beta 1 rather than IFN-beta 2 is responsible for TNF or LPS effects.     

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.