Interferonγ and tumour necrosis factorα induce a synergistic antiproliferative response in human Ewing's sarcoma cells in vitro

Three human cell lines derived from Ewing's sarcoma (RM-82, VH-64, and WE-68) were investigated to establish the influence of recombinant human interferon (rhIFN) and tumour necrosis factor (rhTNF) on cell proliferation and survival and to characterize IFN and TNF receptor expression. Incorporation of [³H]thymidine into cells was inhibited by rhIFN after 24 h of incubation. Half-maximal inhibition was observed with 10–80 U/ml rhIFN. A maximal effect (50%–70% inhibition of cell proliferation) was achieved by treatment of cells with 250 U/ml rhIFN. The influence of rhTNF on proliferation was found to differ among cell lines and varied with the concentration and the duration of exposure of cells to this cytokine. In WE-68 and VH-64 cells [³H]thymidine incorporation was not affected by rhTNF up to 2000 U/ml after 96 h of incubation, where-as in RM-82 cells the incorporation was inhibited by 35% after 48 h of incubation with 100 U/ml rhTNF. However, all cell lines showed a synergistic antiproliferative response to the combination of rhIFN and rhTNF after 24 h of incubation. The human recombinant cytokines interleukin(IL)-1, IL-1, IL-2, IL-3, IL-4, IL-6 and granulocyte/macrophagecolony-stimulating factor, tested alone and in combination with rhIFN and rhTNF, had no influence on cell proliferation. Binding studies in the cell lines with¹²⁵I-rhIFN revealed a dissociation constant (K _d ) of 160–306 pM and approximately 8000–13500 receptors/cell. Binding experiments with¹²⁵I-rhTNF indicated 430–1250 receptors/cell withK _d ranging from 13 pM to 162 pM. These data indicate that, among various cytokines, only IFN and TNF are capable of potently reducing Ewing's sarcoma cell growth in vitro. Our data suggest that IFN alone or in combination with TNF may be useful in the design of novel strategies in Ewing's sarcoma therapy.     

High release of tumor necrosis factor α, interferon γ and interleukin-6 by adherent lymphokine-activated killer cells phenotypically derived from T cells

Summary Adherent lymphokine-activated killer cells (A-LAK) are highly potent cytotoxic cells, which are shown to be derived not only from natural killer (NK)/K cells but phenotypically also from T cells. The generation and phenotypical and functional characterisation of these T-cell-derived A-LAK are described. In contrast to non-adherent cells (NA-LAK) and unseparated LAK (UN-LAK), these mostly CD3⁺CD56⁺ CD8⁺ cells display a high degree of expansion following initial interleukin-2 (rIL-2) activation and further culturing in autologous conditioned medium. A comparison of cytotoxic activities of cultured cells reveals a significantly higher oncolytic ability of A-LAK cells against both K562 and Daudi cells than that of cultured controls of NA-LAK and UN-LAK. In addition, A-LAK are characterised by a marked endogenous cytokine release of interferon, tumour necrosis factor and IL-6 as well as by their shedding of p55 IL-2 receptor after exposure to IL-2. The results demonstrate A-LAK to be the lymphocyte subpopulation with the most cytotoxic activity and endogenous cytokine release after exposure to IL-2. The improvement of techniques for long-term cultures may be of interest for future therapeutic approaches.Abbreviations used A-LAK adherent, lymphokine-activated killer cells - NK natural killer - CTCM complete tissue-culture medium - IL-2 interleukin-2 - IFN interferony - MNC peripheral blood mononuclear cells - TNF tumour necrosis factor This work was supported by the grant 01GA8802 of the Bundesministerium für Forschung und Technologie (BMFT)     

Induction of glycogenolysis in cultured Ewing's sarcoma cells by dopamine and β-adrenergic agonists

Summary This study describes hormonal regulation of glycogen metabolism in Ewing's sarcoma cells. ³H-Glycogen synthesized in cultured Ewing's sarcoma WE-68 cells from ³H-glucose was hydrolyzed in a concentration-dependent manner by various catecholamines. The order of potency for the glycogenolytic effects of catecholamines was isoproterenol dopamine > norepinephrine > epinephrine. The concentrations giving half-maximal effectiveness (EC₅₀) were about 2×10^-8 M, 3×10^-8 M, 8×10^-8 M, and 5×10^-7 M for isoproterenol, dopamine, norepinephrine, and epinephrine, respectively. Higher concentrations of each of the catecholamines were necessary to elicit EC₅₀ stimulation of cyclic AMP production in Ewing's sarcoma cells. Glycogenolysis induced by dopamine was blocked by chlorpromazine, a dopamine D₁-receptor antagonist, but not by haloperidol, a dopamine D₂-receptor antagonist. The glycogenolytic action of norepinephrine was markedly reduced by propranolol, a -adrenoreceptor antagonist, and was not affected by yohimbine, an -adrenoreceptor antagonist. In addition, chlorpromazine also antagonized the glycogenolytic response to norepinephrine. Dibutyryl cyclic AMP, 3-isobutyl-1-methylxanthine, and the diterpene forskolin were also found to induce ³H-glycogen hydrolysis. Our data indicate that cate-cholamines exert their glycogenolytic effects in Ewing's sarcoma cells by stimulation of cyclic AMP formation via -adrenergic recepetors and dopamine D₁-receptors.     

Interleukin 1beta and tumour necrosis factor alpha secreting cells are increased in the peripheral blood of patients with primary Sjögren's syndrome

Methods: PBMC spontaneously secreting tumour necrosis factor α (TNFα), interleukin 1ß (IL1ß), and interleukin 6 (IL6) were assessed by enzyme linked immunospot (ELISPOT) analysis in a cohort of 31 patients with pSS fulfilling the modified European classification criteria. Nineteen healthy volunteers served as controls. ELISPOT results were correlated with glandular and extraglandular manifestations and autoantibody titres—that is, rheumatoid factor (RF) isotypes, anti-Ro/SS-A, anti-La/SS-B as determined by an enzyme linked immunosorbent assay (ELISA) technique. Results: The number of TNFα and IL1ß secreting cells was significantly higher in patients with pSS than in controls. No differences were detected in the number of IL6 secreting PBMC. Patients with recurrent parotid swelling (RPS) had a significantly increased number of IL1ß secreting PBMC. Moreover, the number of IL1ß secreting PBMC correlated with the disease duration (r_s=0.479; p<0.01) and with the concentration of IgM RF (r_s=0.63; p<0.01) and IgG RF (r_s=0.42; p<0.05). Other autoantibodies did not correlate with cytokine secreting PBMC. Conclusion: The increased systemic secretion of IL1ß and TNFα in patients with pSS points to a pathogenic impact of these cytokines in this autoimmune disease. In particular the correlation of IL1ß secreting PBMC with RPS and RF production indicates that IL1ß is a crucial regulator in the development of local and systemic disease manifestations.     

The roles of TNF-α and the soluble TNF receptor I on sleep architecture in OSA

Objective  Patients with obstructive sleep apnea (OSA) have been described to have increased levels of inflammatory cytokines (particularly TNF-α) and have severely disturbed sleep architecture. Serum inflammatory markers, even in normal individuals, have been associated with abnormal sleep architecture. Not much is known about the role the TNF receptor plays in the inflammation of OSA nor if it is associated with changes in sleep architecture or arousals during the night. We hypothesized that the TNF receptor might play an important role in the inflammation as well as sleep architecture changes in patients with OSA. Design  Thirty-six patients with diagnosed (AHI > 15) but untreated OSA were enrolled in this study. Baseline polysomnograms as well as TNF-α and soluble TNF receptor I (sTNF-RI) serum levels were obtained on all patients. We evaluated the association between serum levels of TNF-α and sTNF-RI with various polysomongraphic characteristics, including sleep stages and EEG arousals. Results  sTNF-RI levels were significantly correlated with snore arousals (r value 0.449, p value 0.009), spontaneous movement arousals (r value 0.378, p value 0.025), and periodic limb movement arousals (r value 0.460, p value 0.008). No statistically significant correlations were observed with TNF-α to any polysomnographic variables. To control for statistical significance with multiple comparisons, a MANOVA was performed with TNF-α and sTNF-RI as dependent variables and sleep architecture measures and arousals as independent variables. The model for sTNF-RI was statistically significant (F value 2.604, p value 0.03), whereas the model for TNF-α was not, suggesting sleep quality significantly affects sTNF-RI. Hierarchal linear regression analysis demonstrated that sTNF-RI was independently associated with spontaneous movement arousal index scores after controlling for age, body mass index, and sleep apnea severity. Conclusions  These findings suggest that sTNF-RI is associated with arousals during sleep, but not with other measures in patients with OSA.     

Binding of antibodies raised against tumour necrosis factor alpha (TNFα) to blood vessels and macrophages in inflamed synovial tissue

Summary The distribution of tumour necrosis factor (TNF) in rheumatoid synovium has been investigated. Ten rheumatoid synovia were compared with seven normal synovia and a range of other tissues, using one polyclonal and six monoclonal antibodies. A common staining pattern was obtained with five reagents. Absence of staining of tissue with the other reagents may relate to binding to different epitopes. Excluding cross-reactivity with smooth muscle seen with two reagents, results with the first five reagents were as follows. Normal tissues showed either no staining or faint staining of venular endothelium. In addition skin keratinocytes and colonic mucus showed staining. Rheumatoid synovium showed staining of venules and weaker staining of mononuclear cells, both as individual cells in the deep tissue and more uniformly in areas of the lining layer. The majority of cells that stained for TNF double-stained for CD68 (macrophages). These represented less than 10% of CD68-positive cells. Isolated cells staining for TNF stained for CD3 (T-cells), forming less than 1% of the total T-cells. The presence of staining of venular endothelium suggests that the cytokine may be synthesised by endothelial cells or may be taken up after production by macrophages.     

Interleukin-27 enhances the production of tumour necrosis factor-α and interferon-γ by bone marrow T lymphocytes in aplastic anaemia

Aplastic anaemia (AA) is considered as an immune-mediated bone marrow failure syndrome. The mechanism is involved with a variety of immune molecules including interferon-γ (IFN-γ), tumour necrosis factor-α (TNF-α) and interleukins (ILs). IL-27 is a novel member of the IL-12 family, which mediates T cell response and enhances the production of IFN-γ. However, little is known about the role of IL-27 in the development of AA. This study investigated the role of IL-27 and its receptor IL-27R in the pathogenesis of AA. Both the mRNA expression of IL-27/IL-27R subunits in the bone marrow mononuclear cells (BMMNCs) and the levels of IL-27 in the marrow plasma in AA were found to be higher than in controls. Increased IL-27 correlated with the disease severity of AA. Subsequently, we stimulated marrow T lymphocytes with recombinant human (rh)IL-27 and found that rhIL-27 enhanced the production of TNF-α and IFN-γ in both CD4(+) and CD8(+) T lymphocytes from AA patients. We also detected increased TNF-α and IFN-γ in the supernatants of BMMNCs from AA patients after IL-27 stimulation. In conclusion, our data suggest that elevated IL-27 and IL-27-induced TNF-α and IFN-γ overproduction might be involved in the pathogenesis of AA.     

Influence of IFNα-2b and BCG on the release of TNF and IL-1 by Kupffer cells in rats with hepatoma

!NTRODUCTIONKuPffer eells are residential macroPhages in the1 iver,which Play a eritieal role in the maintenaneeof normal liver function and in immunal surveilaneeof hePatoeellular earcinoma(HCC)and othereaneers〔l].The bio一ogieal immune modulants havebeen used for treating Patients with HCC and othereaneers[2〕.In our previous studies,the eombineduse of biological immune modulants showed bettereffeets.The normal rats and hePatoma rats indueedby DEN(Diethylnitrosamine)were treated …     

IL-1β and TNF-α produced by peripheral blood mononuclear cells before and during interferon therapy in patients with chronic hepatitis C

We investigated the spontaneous and phytohemagglutinin-stimulated production of interleukin-1 (IL-1) and tumor necrosis factor- (TNF-) by peripheral blood mononuclear cells in patients with chronic hepatitis C during treatment with interferon- (IFN-). Spontaneous productions of these were significantly higher in patients with chronic hepatitis C than in healthy subjects. For patients prescribed interferon, stimulated production of TNF- was significantly higher in complete responders than in partial responders, but the differences were small between the other cytokine levels and outcome of IFN treatment. Spontaneous production of these cytokines was higher in patients with genotype III with complete response than in genotype III patients with a partial response, but this was not the case in patients with genotype II. There was a negative correlation between these cytokines and histological activity index. Spontaneous production of cytokines was decreased only in complete responders after the administration of interferon. These data suggest that the elevated production of cytokines in patients with chronic hepatitis C may be due to host response to the virus, and monitoring cytokines along with alanine aminotransferase and hepatitis C virus RNA during treatment may provide more precise information of the effectiveness of therapy.     

Interleukin-4 (IL-4) induces down-modulation and shedding of the p55 tumour necrosis factor receptor and inhibits TNF α's effect on rheumatoid synovial fibroblasts

Recombinant human interleukin-4 (rhIL-4) and rhIL-1 each produced a rapid down-modulation of tumour necrosis factor receptor (TNFR) on rheumatoid synovial fibroblasts (RSF) in vitro. This was associated with a staurosporine-resistant increase in p55 soluble TNFR levels, in culture media, suggesting that downmodulation was due to enhanced receptor shedding via a protein kinase C-independent mechanism. Pretreatment with rhIL-4 reduced the subsequent tumour necrosis factor (TNF) stimulation of prostaglandin E (PGE) and matrix metalloproteinase-3 (MMP-3) production by RSF. Thus, the potential anti-synovial monokine properties of rhIL-4 are not confined to inhibiting monokine production but also include the ability to interfere with their action on cells that constitute a substantial proportion of the rheumatoid synovium.     

Different distributions of interstitial cells of Cajal and platelet-derived growth factor receptor-α positive cells in colonic smooth muscle cell/interstitial cell of Cajal/plateletderived growth factor receptor-α positive cell syncytium in mice

AIM To investigate the distribution and function of interstitialcells of Cajal(ICCs) and platelet-derived growth factor receptor-α positive(PDGFRα+) cells in the proximal and distal colon.METHODS The comparison of colonic transit in the proximal and distal ends was performed by colonic migrating motor complexes(CMMCs). The tension of the colonic smooth muscle was examined by smooth muscle spontaneous contractile experiments with both ends of the smooth muscle strip tied with a silk thread. Intracellular recordings were used to assess electrical field stimulation(EFS)-induced inhibitory junction potentials(IJP) on the colonic smooth muscle. Western blot analysis was used to examine the expression levels of ICCs and PDGFRα in the colonic smooth muscle.RESULTS Treatment with NG-nitro-L-arginine methyl ester hydrochloride(L-NAME) significantly increased the CMMC frequency and spontaneous contractions, especially in the proximal colon, while treatment with MRS2500 increased only distal CMMC activity and smooth muscle contractions. Both CMMCs and spontaneous contractions were markedly inhibited by NPPB, especially in the proximal colon. Accordingly, CyPPA sharply inhibited the distal contraction of both CMMCs and spontaneous contractions. Additionally, the amplitude of stimulationinduced nitric oxide(NO)/ICC-dependent slow IJPs(sIJPs) by intracellular recordings from the smooth muscles in the proximal colon was larger than that in the distal colon, while the amplitude of electric field stimulationinduced purinergic/PDGFRα-dependent fast IJPs(fIJPs) in the distal colon was larger than that in the proximal colon. Consistently, protein expression levels of c-Kit and anoctamin-1(ANO1) in the proximal colon were much higher, while protein expression levels of PDGFRα and small conductance calcium-activated potassium channel 3(SK3) in the distal colon were much higher.CONCLUSION The ICCs are mainly distributed in the proximal colon and there are more PDGFRα+ cells are in the distal colon, which generates a pressure gradient between the two ends of the colon to propel the feces to the anus.