Transcription, translation and secretion of interleukin 1 and tumor necrosis factor: effects of tebufelone, a dual cyclooxygenase/5-lipoxygenase inhibitor.

We examined the effect of tebufelone, a dual cyclooxygenase (CO)/5-lipoxygenase (LO) inhibitor, on the synthesis, secretion and gene expression of interleukin (IL) 1 beta and tumor necrosis factor (TNF)-alpha by human peripheral blood mononuclear cells (PBMC). Basal concentrations of immunoreactive IL 1 beta and TNF-alpha after 18-24 h, in the absence or presence of tebufelone (less than or equal to 12.5 microM), were near the limit of detection (100 pg/ml). By contrast, preincubation (1 h) of cells, in amounts of tebufelone which decrease the formation of leukotriene (LT) B4, markedly enhanced (up to 500%) the synthesis of IL 1 beta and TNF-alpha following lipopolysaccharide (LPS), heat-killed Staphylococcus epidermidis or concanavalin A stimulation. Moreover, a disproportionate amount of the overall increase in IL 1 (alpha and beta) was secreted in contrast to the amount which remained cell associated, an effect unrelated to cell damage or leakage as tebufelone had no effect on either lactate dehydrogenase release by PBMC, or mitochondrial dehydrogenases of adherent monocytes as detected by enzymatic cleavage of the substrate 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide. There was no inverse correlation between the changes in prostaglandin (PG)E2 levels and TNF-alpha or IL 1 beta synthesis, and when PG formation was maximally inhibited by preincubating the cells in indomethacin, tebufelone, added 1 h before the stimulus, continued to enhance the synthesis of IL 1 beta although not that of TNF-alpha. The addition of the CO/5-LO inhibitor 2 h after LPS stimulation, however, did not interfere with IL 1 beta synthesis, suggesting that tebufelone interacts with an early event(s) in the activation of PBMC. For IL 1 beta and TNF-alpha, basal and stimulated (4 h post LPS) mRNA levels were not increased by tebufelone, despite a concomitant increase in the synthesis of IL 1 beta. In conclusion, we have demonstrated that tebufelone enhances IL 1 (alpha and beta) and TNF-alpha synthesis at concentrations which suppress leukotriene formation. These findings argue against a role of 5-LO products as necessary intermediates of IL 1 (alpha and beta) and TNF-alpha synthesis.     

Autoregulation of interleukin 1 production

Interleukin 1 (IL 1) alpha and beta are distinct cytokines with a common receptor on target cells. Both have been implicated in immunity, inflammation and connective tissue metabolism. Their production is stimulated by microbial products and also by other cytokines derived from accessory cells and lymphocytes. Following reports that IL 1 can stimulate its own production, we have tested the effects of recombinant IL 1 on the synthesis and release of IL 1 alpha and beta by human blood mononuclear cells (MNC). We confirmed that autoinduction occurs but report also the novel finding that this effect is very concentration dependent. At some concentrations within the range found in vivo, recombinant IL 1 not only failed to stimulate further IL 1 production but also inhibited the background level of synthesis in 20-h MNC cultures. The negative feedback appears unrelated to prostaglandin E2 and interferon-gamma levels and could not be reproduced by adding transforming growth factor beta. This previously unrecognized autoregulation may be relevant to clinical diseases associated with pathogenic over-production of IL 1.     

Dissociation between plasma and monocyte-associated cytokines during sepsis

We report our investigations of circulating interleukin (IL) 1 beta, IL 6 and tumor necrosis factor (TNF)-alpha, as well as cell-associated IL 1 alpha, IL 1 beta and TNF-alpha in plasma and monocytes of 21 patients with sepsis syndrome and 6 patients with non-septic shock. Longitudinal studies reveal that (a) the most frequent detectable plasma cytokines were TNF-alpha and IL 6, (b) the presence and the kinetics of circulating cytokines were independent of one other, (c) detectable levels of cytokines could be found for a long period of time, and (d) significantly higher levels of IL 6 were found for non-surviving patients. Because of the in vivo half-life of cytokines and of the existence of numerous specific high-affinity receptors, it is quite probable that detectable plasma cytokines represent the excess of produced mediators which have not been trapped by the target cells. TNF-alpha (410 +/- 65 pg/10(6) monocytes) and IL 1 beta (153 +/- 60 pg/10(6) monocytes) were frequently found associated to monocyte lysates (88% and 50%, respectively). Despite the fact that IL 1 alpha is the most abundant cytokine found associated to monocytes following in vitro activation, IL 1 alpha was rarely found in monocytes of intensive care unit patients (29%). No correlation was found to exist between the levels of plasma cytokines and cell-associated cytokines. Some patients had plasma TNF-alpha or IL 1 beta in the absence of the corresponding monocyte-associated cytokine. This observation suggests that cells other than monocytes can participate in the production of circulating cytokines. At the end of the longitudinal study (day 14 +/- 2), only 2/12 surviving patients still had plasma TNF-alpha, whereas 8/12 had monocyte-associated TNF-alpha. These results indicate that activation of monocytes still occurs in patients for whom no plasma cytokines can be detected. Thus, in addition to the measurement of plasma cytokine, measurement of cell-associated cytokine appears useful to assess cytokine production and monocyte activation in vivo.     

Proteolysis of the native murine IL 1 beta precursor is required to generate IL 1 beta bioactivity

We utilized antisera specific for murine IL 1 alpha and IL 1 beta proteins to characterize cell-associated IL 1 according to two distinct criteria, immunochemical detection of radiolabeled IL 1 polypeptides and inhibition of IL 1 activity in cell lysates. Evidence is presented that IL 1 alpha and IL 1 beta are each synthesized by LPS-stimulated but not by unstimulated murine macrophages and accumulate within the cell as intracellular precursors in sizes of 33 kDa and 37 kDa, respectively. As judged by the respective rates of synthesis, IL 1 alpha was about 4-fold more abundant than IL 1 beta. Most (greater than or equal to 95%) of the cell-associated IL 1 activity was inhibited in the presence of the antiserum to IL 1 alpha, suggesting that the precursor of IL 1 beta is mostly biologically inactive. Incubation of cell lysates with papain but not with trypsin or plasmin markedly stimulates an increase in the level of cell-associated IL 1 beta activity and leads to the cleavage of 15-16 kDa carboxyl-terminal fragments from the IL 1 beta precursor. Together, these data indicate that proteolysis of the IL 1 beta precursor is required to generate bioactive IL 1 beta molecules and provide a basis for further investigations of the specific role of proteinases in processing the IL 1 beta precursor to a bioactive form.     

Regulation of interleukin 1 generation in immune-activated fibroblasts

In the present study we have demonstrated that fibroblasts can generate the inflammatory cytokine interleukin 1 (IL 1) under conditions similar to those abundant in cellular immune responses. Thus, induction of IL 1 requires a sequential two-step protocol which consists of preactivation of mouse embryo fibroblasts (MEF) with crude preparations of T cell or macrophage-derived conditioned media (CM; 72 h), followed by a challenge with lipopolysaccharide (LPS; 24 h). Unstimulated fibroblasts or such cells activated by either CM or LPS produced only low levels of IL 1, while a synergism between both signals was observed for obtaining maximal IL 1-like activity in MEF. Each of a series of individual recombinant lymphokines and cytokines (IL 2, granulocyte/macrophage-colony-stimulating factor, tumor necrosis factor, IL 1 beta and interferons-alpha, beta and gamma) was shown to serve as an efficient priming signal for the induction of IL 1. IL 1-like activity in fibroblasts was detected in cell lysates or associated with the producing-cell membrane but not in culture fluids. Immune-stimulated fibroblasts, activated under such experimental conditions, were shown to actively transcribe mRNA of both IL 1 genes (alpha and beta). For the expression of IL 1-specific mRNA in fibroblasts a single stimulus, provided by either LPS or a lymphokine/cytokine, was sufficient; however, a more intense signal was observed when both stimuli were applied. The IL 1-like biological activity of fibroblast origin was significantly reduced by anti-IL 1 alpha antibodies. Thus, fibroblasts, when activated by immune and bacterial products, generate IL 1 which in turn possibly amplifies cellular immune responses or inflammatory processes in connective tissues.     

In vitro production of IL 1 beta, IL 1 alpha, TNF and IL2 in healthy subjects: distribution, effect of cyclooxygenase inhibition and evidence of independent gene regulation

Numerous studies have reported altered in vitro cytokine production in various diseases. In the present study we used specific immunoassays to quantitate production of interleukin 1 beta (IL 1 beta), IL 1 alpha, tumor necrosis factor (TNF) and IL 2 from human peripheral blood mononuclear cells (PBMC). The distribution of cell-associated and secreted cytokines was studied in PBMC of 21 individuals; in response to lipopolysaccharide (LPS) the proportion of cell-associated IL 1 beta ranged from 13% to 56%, for IL 1 alpha 29% to 98%, and for TNF 2% to 17%. In a larger cohort of 32 subjects, the total amount of immunoreactive cytokines produced in response to LPS or phytohemagglutinin was normally distributed within the study group. Mean production of IL 1 alpha in response to LPS was 10.1 ng/ml and exceeded production of IL 1 beta (5.6 ng/ml) and TNF (2.2 ng/ml). The distribution pattern was characterized by high intersubject variability extending over two orders of magnitude and the presence of high and low "producers". Production of IL 1 alpha and IL 1 beta correlated (R = 0.69). In contrast, production of IL 1 beta did not correlate with production of TNF or IL 2. Indomethacin present during stimulation of PBMC increased the amount of IL 1 beta produced and showed a high correlation (R = 0.83) compared to cultures without indomethacin. Thus, low production of IL 1 beta in certain subjects appears not to be due to inhibitable levels of cyclooxygenase products. In a retrospective study, PBMC from 12 subjects who had taken oral cyclooxygenase inhibitors during the preceding 7 days produced 43% more IL 1 beta than subjects who did not take these drugs (p less than 0.05). These studies demonstrate that the amount of cytokine synthesized by PBMC (a) is regulated independently for IL 1, TNF and IL 2; (b) correlates for IL 1 beta and IL 1 alpha; (c) is intrinsic for low and high "producers", and (d) production of IL 1 beta increases with the use of oral cyclooxygenase inhibitors.     

Induction of interleukin 6 by human and murine recombinant interleukin 1 in mice

Interleukin (IL) 6 is a pleistropic cytokine with activities, among others, on immune cells, hematopoietic precursor cells and hepatocytes. We have investigated the kinetics and amplitude of its in vivo induction in mice after injection of four different IL 1 species as well as murine (m) and human (h) tumor necrosis factor (TNF) and bacterial lipopolysaccharide (LPS) using a sensitive bioassay on 7TD1 cells to measure the IL 6 concentrations. Recombinant mIL 1 beta, administered as a single i.v. injection in mice, induced the appearance of IL 6 in the plasma with peak levels observed after 2 h. A dose-response correlation was found between serum IL 6 levels and injected IL 1 alpha concentrations at 3 and 8 h after the injection. We then compared the ability of h/mIL 1 alpha, h/mIL 1 beta, h/mTNF and LPS to induce IL 6 in mice. We found: (a) LPS is the most potent inducer of IL 6; (b) 3 h after injection, the four IL 1 preparations had induced IL 6 levels comparable with the IL 6 levels observed after TNF injection; (c) high doses of mIL 1, alpha or beta, but not hIL 1, resulted in a high IL 6 level persisting for over 8 h. We conclude that IL 1 is a potent inducer of IL 6 in vivo and that no major differences are observed between the four IL 1 preparations, as evaluated at 3 h after the injection. However, mIL 1 alpha and mIL 1 beta, in contrast to hIL 1 alpha and hIL 1 beta, induced a sustained IL 6 level over a longer time period. This pattern of prolonged IL 6 induction is even much more pronounced after mTNF injection, but not after hTNF injection.     

ICAM-1-independent lymphocyte transmigration across high endothelium: differential up-regulation by interferon gamma, tumor necrosis factor-alpha and interleukin 1 beta.

The adhesion of lymphocytes to cytokine-treated high endothelium was studied using cultured high endothelial cells (HEC). Pretreatment of the HEC layer with a variety of cytokines caused up-regulation of lymphocyte adhesion with the effects ordered interferon gamma (IFN-gamma) greater than tumor necrosis factor-alpha (TNF-alpha) greater than or equal to interleukin 1 beta (IL 1 beta). Increased lymphocyte adhesion was found to be independent of ICAM-1 as expression by HEC was not increased by cytokines and antibodies against ICAM-1 did not block adhesion. The peptide CS1 and anti-beta 1 integrin subunit antibodies, however, caused partial inhibition of lymphocyte adhesion thus indicating a role for fibronectin on HEC and alpha 4 beta 1 on lymphocytes. Study of the kinetics of lymphocyte adhesion showed that the effects of IFN-gamma and TNF-alpha were persistent and remained detectable 2.5 h after removal of the cytokines whereas the effects of IL 1 beta were transient and were not sustained beyond 1 h. All of the cytokines used caused transient increases in the number of surface-bound lymphocytes with IFN-gamma greater than TNF-alpha greater than or equal to IL 1 beta, however, the most dramatic effect was on the transmigration of lymphocytes across the HEC. Both IFN-gamma and TNF-alpha caused sustained increased transmigration with IFN-gamma having the greater effect. IL 1 beta had little effect on transmigration. This model demonstrates that the binding and transmigration of lymphocytes across HEC can be differentially regulated by the actions of individual cytokines. These results support the concept that locally produced cytokines regulate HEC function within the lymph node.     

Protection of neutropenic mice from lethal Candida albicans infection by recombinant interleukin 1

Natural and synthetic immunomodulators that increase nonspecific resistance to infection are also known to induce interleukin 1 (IL 1) production. Previous studies have demonstrated a protective effect of recombinant human IL 1 beta against death from infection caused by Pseudomonas aeruginosa. In the present study we investigated the effect of IL 1 beta or IL 1 alpha on the survival of neutropenic mice with a lethal Candida albicans infection. Mice with cyclophosphamide-induced neutropenia were injected with 3 X 10(5) C. albicans i.v. When 80 ng IL 1 beta was given as a single i.p. injection 24 h before the infection, survival compared to that in control animals was as follows: 100% vs. 97% at 24 h, 83% vs. 70% at 48 h and 70% vs. 23% at 72 h after the infection (p less than 0.01). The effect of IL 1 was also apparent when it was given 1/2 h before or 6 h after the infection. The results obtained with 80 ng IL 1 alpha given at 24 h before infection were similar to that obtained with IL 1 beta. The numbers of Candida cultured from the blood, liver, spleen, and kidney were not significantly different in IL 1 beta-treated and control animals. Passive transfer of serum obtained from mice pretreated with IL 1 to recipient mice did not provide protection against a subsequent lethal candidal infection. In conclusion, the present study demonstrates that IL 1 beta and IL 1 alpha prolong survival in neutropenic mice with a lethal C. albicans infection.     

Interleukin 1 and tumor necrosis factor-alpha additively increase the levels of granulocyte-macrophage and granulocyte colony-stimulating factor (CSF) mRNA in human fibroblasts

Recombinant interleukin (IL) 1 beta and tumor necrosis factor/cachectin (TNF-alpha) induce, usually within 2 h, a dose-dependent increase in the levels of granulocyte-macrophage colony-stimulating factor (GM-CSF) and G-CSF mRNA in cultured human fibroblasts. Maximal induction is reached at about 4-8 h and usually last for at least 48 h. IL 1 beta and TNF have additive effects on the levels of GM- and G-CSF mRNA, and on the secretion of G-CSF activity into the culture medium. IL 1 alpha has the same additive effect that IL 1 beta has with TNF, but no additive effect with IL 1 beta. In contrast, the high basic level of M-CSF (CSF-1) mRNA shows little or lower variations in response to IL 1, TNF-alpha or both IL 1 and TNF-alpha also induce, with similar kinetics, an increase in IL 1 beta but not mRNA level. In contrast to what is observed with macrophages and endothelial cells, E. coli lipopolysaccharide does not modify the fibroblast CSF mRNA level up to 48 h of culture.     

Induction of cell-associated interleukin 1 through stimulation of the adhesion-promoting proteins LFA-1 (CD11a/CD18) and CR3 (CD11b/CD18) of human monocytes.

Serum-free culture of human monocytes in the presence of monoclonal antibodies to the LFA-1 alpha chain (CD11a), CR3 alpha chain (CD11b) or beta chain (CD18) bound to Sepharose induced the dose-dependent production of cell-associated interleukin (IL) 1 activity and of IL 1 alpha and IL 1 beta antigens, but no release of extracellular IL 1 activity or antigen in the culture medium. Triggering of IL 1 production was also observed with insolubilized anti-CD11/CD18 F(ab')2 antibodies. Two cross-linked antibodies recognizing distinct epitopes on the CD11b molecule induced cell-associated IL 1. Soluble antibodies did not induce IL 1 production. The kinetics of induction of IL 1 by stimulation of adhesion-promoting proteins differed from those of IL 1 induction by adhesion to plastic. The lack of induction of IL 1 release by stimulation of the CD11/CD18 molecules resembled the intracellular accumulation of IL 1 induced by lipid A. Induction of IL 1 by adhesive processes may be a mechanism by which T cells trigger IL 1 production by monocytes during antigen presentation.     

Action of recombinant human interleukin 6, interleukin 1 beta and tumor necrosis factor alpha on the mRNA induction of acute-phase proteins

The rat hepatoma cell line Fao was used to study the role of three inflammatory mediators on the mRNA regulation of several acute-phase proteins. In the presence of 10(-6) M dexamethasone beta-fibrinogen mRNA levels increased 6-fold after addition of recombinant human IL 6 (rhIL 6). rhIL 1 beta or recombinant human tumor necrosis factor alpha (rhTNF alpha) had essentially no effect on beta-fibrinogen mRNA induction but led to a 20-fold increase in alpha 1-acid glycoprotein mRNA in the presence of dexamethasone. On the other hand, rhIL 6 was a much weaker stimulator of alpha 1-acid glycoprotein mRNA synthesis. All three mediators reduced albumin mRNA concentrations to about 30% of controls. Whereas the induction of beta-fibrinogen mRNA was potentiated by dexamethasone, the synthetic glucocorticoid analog was an absolute requirement for the stimulation of alpha 1-acid glycoprotein mRNA. The mRNA levels of the negative acute-phase protein albumin were induced 5-fold by dexamethasone alone. The beta-fibrinogen mRNA induction started immediately after addition of rhIL 6 and reached a maximum between 12 and 18 h. In contrast, the time-course for alpha 1-acid glycoprotein mRNA synthesis showed a lag phase of 8 h followed by an increase up to 20 h after rhIL 1 beta. rhTNF alpha led to an even more delayed increase in alpha 1-acid glycoprotein mRNA. Whereas in the case of beta-fibrinogen mRNA induction no synergistic effect was observed between various concentrations of the three mediators, the combination of rhIL 6/rhIL 1 beta as well as rhIL 6/rhTNF alpha or rhIL 1 beta/rhTNF alpha regulated synergistically alpha 1-acid glycoprotein and albumin mRNA. It is concluded that discrete acute-phase proteins are regulated differently by the inflammatory mediators IL 6, IL 1 beta and TNF alpha, indicating that the acute-phase response is more complex than previously assumed. The Fao cell line used in this study turned out to be an ideal model for acute-phase protein regulation, suitable for the discrimination between the inflammatory mediators IL 6 and IL 1/TNF alpha.     

Mechanism of in vitro antitumor effects of interleukin 1 (IL 1)

In vitro studies suggest that purified IL 1 beta derived from normal human peripheral blood monocytes and human myelomonocytic cell line THP-1 cell supernatants was capable of modest augmentation of NK activity of purified LGL and of promoting monocyte cytotoxicity for the human melanoma A375 target cells. In addition, purified IL 1 beta also has direct cytostatic and cytocidal effects for A375 cells. A375 melanoma cells were cloned to obtain a homogeneous population of IL 1 receptor-bearing target cells. Recombinant human IL 1 alpha inhibited the proliferation of these cells within 48-72 h in a dose-dependent manner. Similar doses of recombinant IL 1 alpha exhibited inhibitory effects on the ornithine decarboxylase (ODC) activity of A375 cells by 6-24 h. Putrescine, a nontoxic product of the ODC pathway, could prevent the cytostatic effect of recombinant IL 1 alpha on these tumor target cells. This observation indicates that inhibition of the ODC pathway is causally related to the antiproliferative effect of IL 1 on these tumor cells.     

Differential expression of interleukin 1 alpha by Thy-1+ and Thy-1- lung fibroblast subpopulations: enhancement of interleukin 1 alpha production by tumor necrosis factor-alpha

The purpose of this investigation was to determine whether subpopulations of murine lung fibroblasts produced interleukin 1 (IL 1). We previously identified two major populations of pulmonary fibroblasts based on the presence or absence of Thy-1. Thy-1+ and Thy-1- subsets synthesize fibronectin and type I and III collagen, but only the Thy-1- population displays class II major histocompatibility complex antigens after stimulation with interferon-gamma and presents antigen to T helper clones. Interestingly, in the current study we determined that only Thy-1- fibroblast lines and clones synthesized IL 1. Although constitutive production was low, tumor necrosis factor -alpha (TNF-alpha) stimulated 5-20-fold increases in IL 1 production in Thy-1- fibroblasts. The Thy-1+ fibroblasts did not produce IL 1 even after TNF-alpha treatment. Northern blot analysis of TNF-alpha treated cells revealed that in the Thy-1- subset increased mRNA levels for IL 1 alpha were detected, while IL 1 beta mRNA was not detected. Furthermore, IL 1 activity from TNF-alpha-treated Thy-1- fibroblast membranes and supernatants was completely neutralized by IL 1 alpha-specific antibodies. These observations support the hypothesis that the antigen-presenting Thy-1- subset is important for promoting the inflammation associated with pulmonary fibrosis. In addition, the existence of functional subsets of lung fibroblasts is further substantiated by differential expression of IL 1.     

A monoclonal antibody directed against the human intercellular adhesion molecule (ICAM-1) modulates the release of tumor necrosis factor-alpha, interferon-gamma and interleukin 1

ICAM-1 is a cell surface glycoprotein which is one of the ligands for the leukocyte function-associated antigen (LFA-1). It is involved in leukocyte adhesion to endothelial cells as well as in immune functions requiring cell-cell contact. The quantitative expression of ICAM-1 in various cell types can be either induced or enhanced by treatment with cytokines, such as interferon-gamma (IFN-gamma), tumor necrosis factor (TNF)-alpha or interleukin 1 (IL 1), a phenomenon which results in the augmentation of binding to LFA-1-positive cells. In contrast, treatment with anti-ICAM-1 antibodies blocks this binding. A monoclonal antibody (mAb), termed 7F7, which recognizes an epitope on ICAM-1, was used to investigate the role of ICAM-1 in cytokine production by T lymphocytes and monocytes. Production of TNF-alpha. IFN-gamma and IL1 was significantly inhibited (p less than 0.01) by the incubation of mAb 7F7 with phytohemagglutinin-activated blood mononuclear cells (MNC) or isolated E rosette-positive T lymphocytes. The maximal level of inhibition was reached with 1 microgram/ml of purified antibody. A similar inhibition was obtained using saturating concentrations of 400 microliters/ml of mAb 7F7 hybridoma supernatant corresponding to an inhibitory activity of 1 microgram of purified mAb. In contrast, granulocyte/macrophage-colony-stimulating factor release showed a heterogeneous response over five experiments with an increase found in three experiments and a decrease in two experiments. Addition of increasing concentrations of supernatant or purified mAb to unstimulated MNC or T lymphocyte cultures had no effect on cytokine release. The observed inhibition of the production of TNF-alpha. IFN-gamma and IL 1 by antibody-mediated blockade of the ICAM-1 structure probably represents a negative circuit that serves to tune the activation of leukocytes and to avoid an overproduction of cytokines.     

Cytopathic changes in rat microglial cells induced by pathogenic Acanthamoeba culbertsoni: morphology and cytokine release.

To determine whether pathogenic Acanthamoeba culbertsoni trophozoites and lysate can induce cytopathic changes in primary-culture microglial cells, morphological changes were observed by transmission electron microscopy (TEM). In addition, the secretion of two kinds of cytokines, tumor necrosis factor alpha (TNF-alpha) and interleukin-1beta (IL-1beta), from microglial cells was observed. Trophozoites of pathogenic A. culbertsoni made contact with microglial cells and produced digipodia. TEM revealed that microglial cells cocultured with amoebic trophozoites underwent a necrotic process, accompanied by lysis of the cell membrane. TEM of microglial cells cocultured with amoebic lysate showed that the membranes of the small cytoplasmic vacuoles as well as the cell membrane were lysed. The amounts of TNF-alpha secreted from microglial cells cocultured with A. culbertsoni trophozoites or lysate increased at 6 h of incubation. The amounts of IL-1beta secreted from microglial cells cocultured with A. culbertsoni trophozoites at 6 h of incubation was similar to those secreted from the control group, but the amounts decreased during cultivation with A. culbertsoni lysate. These results suggest that pathogenic A. culbertsoni induces the cytopathic effects in primary-culture rat microglial cells, with the effects characterized by necrosis of microglial cells and changes in levels of secretion of TNF-alpha and IL-1beta from microglial cells.     

Cytokine regulation of human monocyte interleukin-1 (IL-1) production in vitro. Enhancement of IL-1 production by interferon (IFN) gamma, tumour necrosis factor-alpha, IL-2 and IL-1, and inhibition by IFN-alpha.

IL-1 production (secreted and cell-associated) was measured in monocyte cultures stimulated by a variety of agents in vitro. Monocytes either adherent to conventional plastic culture plates in serum-free conditions, or in suspension in culture medium containing serum were stimulated to produce IL-1 during culture. In non-adherent, serum-free conditions, monocytes produced very low or undetectable amounts of IL-1 during 20 h of culture. Lipopolysaccharide (LPS) induced equivalent amounts of secreted and cell-associated IL-1, although at very low concentrations more cell-associated IL-1 was produced. IL-1 production in response to LPS could be augmented by crude lymphokine, IFN-gamma, or tumour necrosis factor (TNF) alpha. TNF-alpha preferentially augmented the production of cell-associated IL-1 in LPS-stimulated cultures. TNF-alpha induced a significant amount of IL-1 (mainly cell-associated) directly but could also induce IL-1 secretion when combined with IL-2 or IFN-gamma, or when in the presence of serum. IL-2 acted synergistically with low concentrations of IFN-gamma or IL-1 to induce significant levels of IL-1 production. IFN-alpha did not induce any IL-1 production, but was a potent inhibitor of IL-1 production induced by a variety of stimuli. These results suggest that IL-1 production may be enhanced or reduced by different cytokines at concentrations likely to be found in chronic inflammatory lesions.     

Direct and mononuclear cell mediated effects on interleukin 6 production by glioma cells in infection with herpes simplex virus type 1

To clarify the mechanism of interleukin (IL)-6 elevation in the cerebrospinal fluid of viral meningitis and/or encephalitis patients, we investigated how herpes simplex virus type 1 (HSV1)-infection enhances IL-6 production in human glioma cells (the U373MG and T98G cells). Although human glioma cells did not show enhanced IL-6 production by direct HSV1-infection, the cell-free supernatant from HSV1-stimulated mononuclear cells (MNC) culture and lipopolysaccharide, as a positive control, markedly elevated IL-6 production at both mRNA and polypeptide levels. Ultra violet-irradiated HSV1 induced the secretion of the IL-6 inducing factor(s) from MNC, whereas heat-inactivated HSV1 did not show this activity. This finding indicated that the adsorption of virus on the surface of MNC may be sufficient for induction of secretion. The supernatant from the culture of HSV1-stimulated MNC contained detectable amounts of IL-1beta, tumor necrosis factor (TNF) alpha, interferon (IFN) gamma and IL-6, and its IL-6-inducing activity was inhibited only by anti-IL-1beta antibodies. Moreover, recombinant IL-1beta markedly enhanced IL-6 production in glioma cells with a concomitant elevation of its mRNA level. Taken together, the results suggest that in HSV1-infection of the CNS, enhancement of IL-6 production in glial cells is mediated not by direct infection to glial cells but rather by IL-1beta released from HSV1-stimulated MNC. These findings may help elucidate the mechanisms underlying cerebro-parenchymal inflammatory progression and repair in herpes simplex encephalitis.