Granulocyte-macrophage colony stimulating factor induces both HLA-DR expression and cytokine production by human monocytes

The effect of recombinant human granulocyte-macrophage colony stimulating factor (GM-CSF) on the expression of HLA-DR, and the production of the cytokines interleukin-1 (IL-1) and tumor necrosis factor alpha (TNF alpha) by human peripheral blood monocyte-enriched populations was investigated. GM-CSF was shown to induce both the expression of HLA-DR and the cytokines IL-1 and TNF alpha in a dose-dependent manner. In contrast, interferon-gamma (IFN-gamma), which induced major histocompatibility complex (MHC) class II expression, did not induce IL-1 or TNF alpha production. However, IFN-gamma enhanced the cell surface expression of HLA-DR and the production of IL-1 and TNF alpha on monocyte-enriched cells stimulated by GM-CSF. By itself, GM-CSF did not induce surface class II expression on the human monocytic tumour cell line THP-1, whereas it synergized with IFN-gamma to induce surface expression. These cells responded to GM-CSF by producing IL-1 and TNF alpha; Northern blotting showed that mRNA levels of IL-1 and TNF alpha were transiently induced, similar to other cytokines. Our results indicate that GM-CSF is a major macrophage activating factor that is capable of inducing both the expression of HLA-DR and the cytokines involved in T-cell activation by macrophages; therefore, GM-CSF may be of importance in potentiating antigen presenting function.     

Melanoma differentiation-associated gene-7/IL-24 gene enhances NF-kappa B activation and suppresses apoptosis induced by TNF

Melanoma differentiation-associated gene-7 (mda-7), also referred to as IL-24, is a novel growth regulatory cytokine that has been shown to regulate the immune system by inducing the expression of inflammatory cytokines, such as TNF, IL-1, and IL-6. Whether the induction of these cytokines by MDA-7 is mediated through activation of NF-kappaB or whether it regulates cytokine signaling is not known. In the present report we investigated the effect of MDA-7 on NF-kappaB activation and on TNF-induced NF-kappaB activation and apoptosis in human embryonic kidney 293 cells. Stable or transient transfection with mda-7 into 293 cells failed to activate NF-kappaB. However, TNF-induced NF-kappaB activation was significantly enhanced in mda-7-transfected cells, as indicated by DNA binding, p65 translocation, and NF-kappaB-dependent reporter gene expression. Mda-7 transfection also potentiated NF-kappaB reporter activation induced by TNF receptor-associated death domain and TNF receptor-associated factor-2. Cytoplasmic MDA-7 with deleted signal sequence was as effective as full-length MDA-7 in potentiating TNF-induced NF-kappaB reporter activity. Secretion of MDA-7 was not required for the potentiation of TNF-induced NF-kappaB activation. TNF-induced expression of the NF-kappaB-regulated gene products cyclin D1 and cyclooxygenase-2, were significantly up-regulated by stable expression of MDA-7. Furthermore, MDA-7 expression abolished TNF-induced apoptosis, and suppression of NF-kappaB by IkappaBalpha kinase inhibitors enhanced apoptosis. Overall, our results indicate that stable or transient MDA-7 expression alone does not substantially activate NF-kappaB, but potentiates TNF-induced NF-kappaB activation and NF-kappaB-regulated gene expression. Potentiation of NF-kappaB survival signaling by MDA-7 inhibits TNF-mediated apoptosis.     

Signaling of ATP receptors in glia-neuron interaction and pain

ATP causes the activation of p38 or ERK1/2, mitogen activated protein kinases (MAPKs) resulting in the release of tumor necrosis factor-alpha (TNF) and Interleukin-6 (IL-6) from microglia. We examined the effect of TNF and IL-6 on the protection from PC12 cell death by serum deprivation. When PC12 cells were incubated with serum-free medium for 32 hr, their viability decreased to 30 %. IL-6 alone slightly protected the death of PC12 cells, whereas TNF alone did not show any protective effect. In the meanwhile, when PC12 cells were pretreated with TNF for 6 hr and then incubated with IL-6 under the condition of serum-free, the viability of PC12 cells dramatically increased. TNF induced an increase of IL-6 receptor (IL-6R) expression in PC12 cells at 4-6 hr. These data suggested that 6 hr pretreatment with TNF increased IL-6R expression in PC12 cells, leading to an enhancement of IL-6-induced neuroprotective action.To elucidate the role of p38 in pathological pain, we investigated whether p38 is activated in the spinal cord of the neuropathic pain model. In the rats displaying a marked allodynia, the level of phospho-p38 was increased in the microglia of injury side in the dorsal horn. Intraspinal administration of p38 inhibitor suppressed the allodynia. These results demonstrate that neuropathic pain hypersensitivity depends upon the activation of p38 signaling pathway in microglia in the dorsal horn following peripheral nerve injury.     

Tumour-necrosis-factor-mediated cytotoxicity is correlated with phospholipase-A2 activity, but not with arachidonic acid release per se.

L929, a murine fibrosarcoma cell line highly sensitive to the anti-proliferative and cytotoxic action of tumour necrosis factor (TNF), was used as a target cell in our studies. We [Suffys et al. (1987) Biochem. Biophys. Res. Commun. 149, 735-743], as well as others, have previously provided evidence that a phospholipase (PL), most probably a PL-A2-type enzyme, is likely to be involved in TNF-mediated cell killing. We now further document this conclusion and provide suggestive evidence that the enzyme activity specifically involved in TNF cytotoxicity differs from activities associated with the eventual cell death process itself or with non-toxic serum treatment. We also show that the 5,8,11,14-icosatetraenoic acid (arachidonic acid, delta 4 Ach) released by PL, and possibly metabolized, is unlikely to be a key mediator of the TNF-mediated cytotoxicity. These conclusions are based on the following experimental findings. 1. TNF treatment of cells, prelabelled for 24 h with [3H] delta 4Ach or [14C] delta 3Ach (delta 3Ach identical to 5,8,11-icosatrienoic acid) resulted in an early, time-dependent and concentration-dependent release of radioactivity in the supernatant preceding actual cell death. The extent of this response was moderate, albeit reproducible and significant. Analysis of the total lipid fraction from cells plus supernatant revealed that only release of arachidonic acid from phospholipids, but not its metabolization was induced by TNF. However, the release of less unsaturated fatty acids, such as linoleic acid (Lin) or palmitic acid (Pam), was not affected during the first hours after TNF addition. 2. An L929 subclone, selected for resistance to TNF toxicity, was found to be defective in TNF-induced delta 4Ach libration. 3. Interleukin-1 (IL1) was not cytotoxic for L929 and did not induce release of delta 4Ach. 4. Release of delta 4Ach was not restricted to TNF; the addition of serum to the cells also induced release of fatty acids into the medium. In this case, however, there was no specificity, as all fatty acids tested, including Lin and Pam, were released. 5. Inhibition of PL-A2 activity by appropriate drugs markedly diminished TNF-induced delta 4Ach release and resulted also in a strong decrease in TNF-induced cytotoxicity. 6. Other drugs, including serine protease inhibitors, which strongly inhibit TNF-induced cytotoxicity, also decreased the TNF-induced delta 4Ach release, whereas LiCl potentiated both TNF-mediated effects. 7. Protection of cells against TNF toxicity by means of various inhibitors was not counteracted by addition of exogenous fatty acids, including delta 4Ach.(ABSTRACT TRUNCATED AT 400 WORDS)     

Febrile and acute hyperthermia enhance TNF-induced necrosis of murine L929 fibrosarcoma cells via caspase-regulated production of reactive oxygen intermediates

Previous studies have demonstrated the essential role of TNF-induced reactive oxygen intermediates (ROI) in the necrosis of L929 cells. We investigated the molecular basis for the interaction of hyperthermia and TNF in these cells. Hyperthermia, both febrile (40.0-40.5 degrees C) and acute (41.5-41.8 degrees C), strongly potentiated TNF killing, and sensistization was significantly quenched by the antioxidant, BHA. The broad-spectrum caspase inhibitor, Z-VAD, has been shown to markedly increase the TNF sensitivity of L929 cells at 37 degrees C; we observed that hyperthermia would also enhance the sensitivity of L929 cells to TNF + Z- VAD and that BHA could significantly quench the response, as well. The basis for hyperthermic potentiation was unlikely thermally-increased sensitivity to ROI, as treatment with hydrogen peroxide for 24 h killed L929 cells essentially equivalently, whether incubated continuously at 37 degrees C or at 40.0-40.5 degrees C, or for 2 h at 41.5-41.8 degrees C. However, febrile and acute hyperthermia markedly increased TNF-induced production of ROI, with or without Z-VAD. Hyperthermia dramatically accelerated the onset of this production, as well as the onset of necrotic death, as determined by oxidation of dihydro-rhodamine and propidium iodide staining, respectively, both of which were significantly quenchable with BHA. We conclude that hyperthermia potentiates TNF-mediated killing in this cell model primarily by increasing the afferent, and not the efferent, phase of TNF-induced necrosis.     

Granulocyte macrophage-colony stimulating factor and interleukin-3 increase expression of type II tumour necrosis factor receptor, increasing susceptibility to tumour necrosis factor-induced apoptosis. Control of leukaemia cell life/death switching

Tumour necrosis factor (TNF) induces apoptosis in a range of cell types via its two receptors, TNFR1 and TNFR2. Here, we demonstrate that proliferation and TNFR2 expression was increased in human leukaemic TF-1 cells by granulocyte macrophage-colony stimulating factor (GM-CSF) and interleukin-3 (IL-3), with TNFR1 expression unaffected. Consequently, they switch from a proliferative to a TNF-induced apoptotic phenotype. Raised TNFR2 expression and susceptibility to TNF-induced apoptosis was not a general effect of proliferation as IL-1beta and IFN-gamma both proliferated TF-1 cells with no effect on TNFR expression or apoptosis. Although raised TNFR2 expression correlated with the apoptotic phenotype, stimulation of apoptosis in GM-CSF-pretreated cells was mediated by TNFR1, with stimulation of TNFR2 alone insufficient to initiate cell death. However, TNFR2 did play a role in apoptotic and proliferative responses as they were blocked by the presence of an antagonistic TNFR2 antibody. Additionally, coincubation with cycloheximide blocked the mitotic effects of GM-CSF or IL-3, allowing only the apoptotic responses of TNF to persist. TNF life/death was also observed in K562, but not MOLT-4 and HL-60 human leukaemic cell types. These findings show a cooperative role of TNFR2 in the TNF life/death switching phenomenon.     

Human papillomavirus type 16 E6-enhanced susceptibility of L929 cells to tumor necrosis factor alpha correlates with increased accumulation of reactive oxygen species.

Human papillomavirus type 16 (HPV-16) E6 has been shown to prevent or enhance apoptosis depending on the stimulus and cell type. Here we present evidence that HPV-16 E6 sensitized murine fibrosarcoma L929 cells to tumor necrosis factor alpha (TNF)-induced cytolysis. The E6-enhanced cytolysis correlated with a precedent increase in reactive oxygen species (ROS) level and antioxidant treatment could completely block the E6-dependent sensitization. These findings represent the first demonstration of a link between a viral oncogene-sensitized cytolysis and ROS. Previous studies have shown conflicting results regarding whether TNF-induced cytolysis of L929 cells is through necrosis or apoptosis. Here we report that, although L929 cells underwent DNA fragmentation after exposure to TNF, they retained the morphology of intact nuclei while gaining permeability to propidium iodide, features characteristic of necrosis rather than apoptosis. We confirmed that the broad spectrum caspase inhibitor benzyloxycarbonyl-Val-Ala-Asp(OMe)-fluoromethylketone markedly increased the susceptibility of L929 cells to TNF, and further demonstrated that E6 enhanced this susceptibility, which again correlated with increased ROS accumulation. We showed that the expression of E6 in L929 cells did not alter the stability of p53, and the cells retained a p53 response to actinomycin D. Furthermore, two E6 mutants defective for p53 degradation in other systems exhibited differential effects on TNF sensitization. These results suggest that the enhancement of TNF-induced L929 cytolysis by E6 is independent of p53 degradation. We also found that TNF-induced activation of NF-kappaB did not account for the enhanced TNF susceptibility by E6.     

A role for potassium in TNF-induced apoptosis and gene-induction in human and rodent tumour cell lines

Rat/mouse T cell hybridoma-derived PC60 R55/R75 cells were used as a model to study the role of intracellular potassium in TNF-induced apoptosis and gene induction. A reduction of intracellular potassium with nigericin or valinomycin (ionophores), or ouabain (Na(+)/K(+)-ATPase inhibitor) sensitized PC60 R55/R75 cells to TNF-induced apoptosis. TNF-induced GM-CSF release in PC60 R55/R75 cells was enhanced by nigericin or ouabain. Similar results were obtained with human cervix carcinoma cells HeLaH21 exposed to TNF. These results suggest a role for intracellular potassium in TNF-induced apoptosis and gene induction.     

IL-10 synergistically enhances GM-CSF-induced CCR1 expression in myelomonocytic cells

CC chemokine receptor 1 (CCR1) has been implicated in inflammation. The present study examined the signaling mechanisms that mediate GM-CSF/IL-10-induced synergistic CCR1 protein expression in monocytic U937 cells. GM-CSF alone markedly increased both the mRNA and protein expression of CCR1. IL-10 augmented GM-CSF-induced CCR1 protein expression with no effect on mRNA expression. PD098059 and U0126 (two MEK inhibitors), and LY294002 (a PI3K inhibitor) inhibited GM-CSF/IL-10-induced CCR1 gene and protein expression. PD098059, U0126, and LY294002 also attenuated chemotaxis of GM-CSF/IL-10-primed U937 cells in response to MIP-1alpha. Immunoblotting studies show that GM-CSF alone induced ERK2 phosphorylation; whereas, IL-10 alone induced p70(S6k) phosphorylation in U937 cells. Neither cytokine when used alone induced PKB/Akt phosphorylation. Combined GM-CSF/IL-10 treatment of U937 cells induced phosphorylation of ERK2, p70(S6k), and PKB/Akt. PD098059 and U0126 completely abrogated ERK2 phosphorylation; whereas, LY294002 completely blocked PKB/Akt and p70(S6k) phosphorylation. Our findings indicate that IL-10 may potentiate GM-CSF-induced CCR1 protein expression in U937 cells via activation of PKB/Akt and p70(S6k).     

IL-17 reduces TNF-induced Rantes and VCAM-1 expression

Functional diversity of the memory T-cell-derived cytokine IL-17 was explored at the receptor level. IL-17 inhibited TNF-induced chemokine Rantes expression in human synovial fibroblasts and mouse lung fibroblasts. This inhibitory activity of IL-17 (IC50=0.2 ng/ml) was 6-fold more potent than its stimulatory activity on TNF-alpha-induced IL-6 secretion (ED50=1.2 ng/ml), measured in the same cells. IL-17 also inhibited the TNF-mediated expression of adhesion molecule VCAM-1, and the NF-kappaB binding to the VCAM-1 promoter-specific site, along with the inhibitor of NF-kappaB, IkappaB-beta. Neutralization of the human IL-17 receptor (IL-17R) by M202 antibody competitively reverses the IL-17-induced IL-6 upregulation. However, M202 only partially affected the inhibitions by IL-17. Yet, IL-17R was essential for the Rantes inhibition, as assessed in lung-derived fibroblasts from IL-17R gene deficient mice. Therefore, inhibitory and stimulatory functions of IL-17 involve receptor IL-17R but show distinct dose-responses and in turn different sensitivities to an IL-17R antagonizing antibody.     

TNFalpha-induced glutathione depletion lies downstream of cPLA(2) in L929 cells.

Both glutathione (GSH) depletion and arachidonic acid (AA) generation have been shown to regulate sphingomyelin (SM) hydrolysis and are known components in tumor necrosis factor alpha (TNFalpha)-induced cell death. In addition, both have hypothesized direct roles in activation of N-sphingomyelinase (SMase); however, it is not known whether these are independent pathways of N-SMase regulation or linked components of a single ordered pathway. This study was aimed at differentiating these possibilities using L929 cells. Depletion of GSH with L-buthionin-(S,R)-sulfoximine (BSO) induced 50% hydrolysis of SM at 12 h. In addition, TNF induced a depletion of GSH, and exogenous addition of GSH blocked TNF-induced SM hydrolysis as well as TNF-induced cell death. Together, these results establish GSH upstream of SM hydrolysis and ceramide generation in L929 cells. We next analyzed the L929 variant, C12, which lacks both cytosolic phospholipase A(2) (cPLA(2)) mRNA and protein, in order to determine the relationship of cPLA(2) and GSH. TNF did not induce a significant drop in GSH levels in the C12 line. On the other hand, AA alone was capable of inducing a 60% depletion of GSH in C12 cells, suggesting that these cells remain responsive to AA distal to the site of cPLA(2). Furthermore, depleting GSH with BSO failed to effect AA release, but caused a drop in SM levels, showing that the defect in these cells was upstream of the GSH drop and SMase activation. When cPLA(2) was restored to the C12 line by expression of the cDNA, the resulting CPL4 cells regained sensitivity to TNF. Treatment of the CPL4 cells with TNF resulted in GSH levels dropping to levels near those of the wild-type L929 cells. These results demonstrate that GSH depletion following TNF treatment in L929 cells is dependent on intact cPLA(2) activity, and suggest a pathway in which activation of cPLA(2) is required for the oxidation and reduction of GSH levels followed by activation of SMases.     

Distinct roles of basal steady-state and induced H-ferritin in tumor necrosis factor-induced death in L929 cells

Tumor necrosis factor (TNF) alpha is a cytokine capable of inducing caspase-dependent (apoptotic) cell death in some cells and caspase-independent (necrosis-like) cell death in others. Here, using a mutagenesis screen for genes critical in TNF-induced death in L929 cells, we have found that H-ferritin deficiency is responsible for TNF resistance in a mutant line and that, upon treatment with TNF, this line fails to elevate levels of labile iron pool (LIP), critical for TNF-induced reactive oxygen species (ROS) production and ROS-dependent cell death. Since we found that TNF-induced LIP in L929 cells is primarily furnished by intracellular storage iron, the lesser induction of LIP in H-ferritin-deficient cells results from a reduction of intracellular iron storage caused by less H-ferritin. Different from some other cell lines, the H-ferritin gene in L929 cells is not TNF inducible; however, when H-ferritin is expressed in L929 cells under a TNF-inducible system, the TNF-induced LIP and subsequent ROS production and cell death were all prevented. Thus, LIP is a common denominator of ferritin both in the enhancement of cell death by basal steady-state H-ferritin and in protection against cell death by induced H-ferritin, thereby acting as a key determinant of TNF-induced cell death.     

Acidic sphingomyelinase induced by electrophiles promotes proinflammatory cytokine production in human bladder carcinoma ECV-304 cells

Electrophiles in environmental pollutants or cigarette smoke are high risk factors for various diseases caused by cell injuries such as apoptosis and inflammation. Here we show that electrophilic compounds such as diethyl malate (DEM), methyl mercury and cigarette smoke extracts significantly enhanced the expression of acidic sphingomyelinase (ASMase). ASMase activity and the amount of ceramide of DEM-treated cells were approximately 6 times and 4 times higher than these of non-treated cells, respectively. Moreover, we found that DEM pretreatment enhanced the production of IL-6 induced by TNF-α. Knockdown of ASMase attenuated the enhancement of TNF-α-dependent IL-6 production. On the other hand, enhancement of TNF-α-induced IL-6 production was observed in ASMase-overexpressing cells without DEM. Fractionation of the lipid raft revealed that the TNF receptor 1 (TNFR1) was migrated into the lipid raft in DEM-treated cells or ASMase-overexpressing cells. The TNF-α-induced IL-6 expression required the clustering of TNFR1 since IL-6 expression were decreased by the destruction of the lipid raft with filipin. These results demonstrated a new role for ASMase in the acceleration of the production of TNF-induced IL-6 as a pro-inflammatory cytokine and indicated that electrophiles could potentiate inflammation response by up-regulating of ASMase expression following formation of lipid rafts.     

Activation of two distinct anti-proliferative pathways, apoptosis and p38 MAP kinase-dependent cell cycle arrest, by tumor necrosis factor in human melanoma cell line A375

The proliferation of human melanoma cell line A375-6 cells is inhibited by several cytokines, including interleukin-1 (IL-1). A375-R8 cells, a subclone of A375-6, are resistant to IL-1-induced growth inhibition. The proliferation of both cell lines is inhibitable by tumor necrosis factor (TNF). In this study, we characterized the mechanisms of TNF-induced growth inhibition. TNF-induced growth inhibition in both cell lines was partially suppressed by a selective p38 mitogen-activated protein kinase (MAPK) inhibitor (SB203580), whereas a combination of SB203580 and Z-VAD-fmk, an inhibitor for a wide range of caspases, completely blocked TNF-induced growth inhibition, indicating that TNF-induced growth inhibition is mediated by both p38 MAPK and caspases. However, Z-VAD-fmk alone suppressed TNF-induced growth inhibition in A375-R8, but not A375-6, cells, suggesting that there may exist a TNF-induced anti-apoptotic mechanism in A375-6 cells which is lost or mutated in A375-R8 cells. Evidence in support of this notion includes (1) TNF-induced apoptosis only in A375-R8, but not A375-6 cells; (2) cycloheximide enabled TNF to induce apoptosis even in A375-6 cells; and (3) somatic hybrid cells between A375-6 and A375-R8 cells are resistant to TNF-induced apoptosis. Since TNF-induced NF-kappa B activation, cell cycle arrest, RB dephosphorylation, and E2F downregulation are indistinguishable in both cell lines, none of these factors is likely to be involved in the TNF-induced anti-apoptotic mechanism in A375-6 cells. Our results indicate that TNF activates two distinct anti-proliferative pathways including p38 MAPK-dependent cell cycle arrest and caspase-mediated apoptosis, as well as an anti-apoptotic mechanism in melanoma cells.     

Interdependence of the radioprotective effects of human recombinant interleukin 1 alpha, tumor necrosis factor alpha, granulocyte colony-stimulating factor, and murine recombinant granulocyte-macrophage colony-stimulating factor

Interleukin 1 alpha (IL-1 alpha), tumor necrosis factor alpha (TNF alpha), granulocyte-colony-stimulating factor (G-CSF), and granulocyte-macrophage colony-stimulating factor (GM-CSF) are molecularly distinct cytokines acting on separate receptors. The release of these cytokines can be concomitantly induced by the same signal and from the same cellular source, suggesting that they may cooperate. Administered alone, human recombinant (hr)IL-1 alpha and hrTNF alpha protect lethally irradiated mice from death, whereas murine recombinant GM-CSF and hrG-CSF do not confer similar protection. On a dose basis, IL-1 alpha is a more efficient radioprotector than TNF alpha. At optimal doses, IL-1 alpha is a more radioprotective cytokine than TNF alpha in C57BL/6 and B6D2F1 mice and less effective than TNF alpha in C3H/HeN mice, suggesting that the relative effectiveness of TNF alpha and IL-1 alpha depends on the genetic makeup of the host. Administration of the two cytokines in combination results in additive radioprotection in all three strains. This suggests that the two cytokines act through different radioprotective pathways and argues against their apparent redundancy. Suboptimal, nonradioprotective doses of IL-1 alpha also synergize with GM-CSF or G-CSF to confer optimal radioprotection, suggesting that such an interaction may be necessary for radioprotection of hemopoietic progenitor cells.     

Tumor necrosis factor-mediated cytotoxicity involves ADP-ribosylation

The mechanism of TNF-mediated cytotoxicity was studied in several cell lines, including L929 murine fibroblasts. TNF caused a time- and dose-dependent increase of ADP-ribosylation in L929 target cells parallel to cell death. During the course of TNF-mediated cytotoxicity in the presence of actinomycin D, an increase in ADP-ribosylation became apparent between 4 and 6 h after exposure to TNF. Intracellular NAD+ and ATP levels decreased parallel to but not preceding cell death. Two inhibitors of ADP-ribosylation, namely 3-aminobenzamide and nicotinamide, prevented TNF-mediated cytotoxicity. Another target, the human cervical carcinoma cell line ME-180, showed an increase in ADP-ribosylation when treated with TNF, and the cytotoxic action of TNF on this target cell was inhibited by these two inhibitors. In the absence of actinomycin D, treatment of L929 cells with TNF also increased ADP-ribosylation, and the cytotoxic action of TNF was inhibited by nicotinamide. These results indicate that ADP-ribosylation may be involved in the TNF-mediated cytotoxic reaction.