Induction of interleukin-2 receptor by tumor necrosis factor α on cultured ovarian tumor-associated lymphocytes

Summary We have recently reported that autologous tumor-specific cytotoxic T lymphocyte (CTL) lines and clones can be developed from lymphocytes infiltrating ovarian malignant ascites (TAL). In this study, we investigated the biological effects of tumor necrosis factor (TNF) in the induction, expansion, long-term proliferation and lytic function of CD8⁺ TAL. TNF up-regulated the IL-2 receptor (IL-2R) chain (Tac antigen) on the surface of CD3⁺ CD8⁺ CD4^– TAL, enhanced the proliferation of autologous tumor-specific CTL, and potentiated their lytic function in long-term cultures. Furthermore, in the induction and expansion phase of CD8⁺ TAL, the presence of TNF was associated with a selective increase in CD8⁺ IL-2R⁺ (Tac⁺) cells, and subsequent decrease in CD4⁺ IL-2R⁺ (Tac⁺) cells. These results suggest that the observed facilitation of the outgrowth of CD8⁺ cells in TAL cultures may be due, at least in part, to the up-regulation of IL-2R, and indicate the usefulness of TNF in the analysis of signalling in autologous tumor-reactive CTL.     

Protective effect of α-lipoic acid against antimycin A cytotoxicity in MC3T3-E1 osteoblastic cells

Oxidative stress represents a major cause of cellular damage and death in the process of osteoporosis. Antimycin A (AMA) has been shown to stimulate mitochondrial superoxide anions and reactive oxygen species (ROS). α-Lipoic acid (α-LA) is a naturally occurring essential coenzyme in mitochondrial multienzyme complexes and acts as a key player in mitochondrial energy production. However, whether α-LA affects the cytotoxicity of AMA in osteoblastic cells is unknown. In this study, we investigated the protective effects of α-LA against AMA-induced cytotoxicity using the MC3T3-E1 osteoblast-like cell line. Our results indicated that α-LA treatment attenuated AMA-induced cytotoxicity and LDH release in a dose-dependent manner. Notably, a significant recovery effect of α-LA on mineralization inhibited by AMA was found. Our results also demonstrated that treatment with 50 μM AMA leads to a reduction of mitochondrial membrane potential (MMP) and the complex IV dysfunction, which was inhibited by pretreatment with α-LA in a dose-dependent manner. In addition, treatment with α-LA significantly reduced the generation of ROS and mitochondrial superoxide production induced by AMA. In addition, our result suggests that PI3K/Akt and CREB pathways are related to the protective effect of α-LA. Importantly, Hoechst 33258 staining results indicated that pretreatment with α-LA prevented AMA-induced apoptosis. Mechanistically, we found that α-LA prevents MC3T3-E1 cells from apoptosis through attenuating cytochrome C release and reducing the level of cleaved caspase-3.     

Enhancement of epidermal growth factor receptor expression on glioma cells by recombinant tumor necrosis factor α

Summary Recombinant tumor necrosis factor (rTNF; optimal dose 1000 U/ml) significantly increased the density of epidermal growth factor receptor (EGF-R) in three of four glioma cell lines in culture as determined by binding analysis of anti-EGF-R monoclonal antibody (mAb) 425. Since enhancement of EGF-R expression by rTNF- was inhibited when cells were treated with the protein synthesis inhibitor cycloheximide, the effects of rTNF may be protein-synthesis-dependent. The dose of rTNF that was optimal for up-regulation of EGF-R on glioma cells did not inhibit the growth of these cells.¹²⁵I-labeled mAb 425 lysed glioma cells in culture following its internalization into the cells. After glioma cells had been treated with rTNF, the growth-inhibitory effects of the mAb were significantly enhanced, probably a reflection of the increase in EGF-R density on the tumor cell surfaces. The rTNF effects were specific to the EGF-R and did not affect unrelated glioma-associated antigens. In our previous clinical trials,¹²⁵I-labeled mAb 425 showed immunotherapeutic effects in glioma patients. The present study provides the basis for considerations of combined immunotherapy of glioma patients with¹²⁵I-labeled mAb 425 and rTNF.     

Synthesis of prostaglandin E2 and prostaglandin F2α by toadfish red blood cells

Saline washed red blood cells of the toadfish convert [1-¹⁴C] arachidonic acid to products that cochromatograph with prostaglandin E₂ and prostaglandin F_2α. This synthesis is inhibited by indomethacin (10 μg/ml). Conversion of arachidonic acid to prostaglandin E₂ was confirmed by mass spectrometry. When saline washed toadfish red blood cells were incubated with a mixture of [1-¹⁴C]-arachidonic acid and [5,6,8,9,11,12,14,15,-³H]-arachidonic acid, comparison of the isotope ratios of the radioactive products indicated that prostaglandin F_2α was produced by reduction of prostaglandin E₂. The capacity of toadfish red blood cells to reduce prostaglandin E₂ to prostaglandin F_2α was confirmed by incubation of the cells with [1-¹⁴C] prostaglandin E₂.     

Inhibition of prostaglandin I2 (PGI2) formation by LDL-cholesterol or LDL-peroxides?

A hypothesis of Gryglewski et al. explains the correlation between increased level of LDL and development of atherosclerosis by inhibition of PGI₂ synthesis by increased peroxide content of LDL. The aim of the present paper was to examine this hypothesis. The major results are: 1) Preparation of LDL in the presence of .02 % butylated hydroxytoluene did not reduce the lipid peroxide content of LDL from men and women and not change the inhibition or stimulation of the in vitro biosynthesis of PGI₂ by LDL isolated from blood of men or women, respectively. 2) In the LDL and HDL, respectively, of healthy men we found nearly the same lipid peroxide levels (nmole malondialdehyde (MDA)/mg lipoprotein-cholesterol) as in the lipoproteins of male patients with hyperlipidemia type IIa or IV, but the peroxide concentration is three times higher in HDL as in LDL. 3) LDL isolated from healthy men inhibited in dose dependent fashion the generation of PGI₂ from PGH₂ by aortic microsomes whereas LDL from premopausal women stimulated PGI₂ formation even calculated as LDL lipid peroxides (in nM MDA/ml). The results call into question the hypothesis that diminished PGI₂ formation by atherosclerotic vessels is related to inhibition of PGI₂ synthetase by lipid peroxides present in LDL in the lesions. A new working hypothesis is presented that also the fatty acid pattern and the lipid class composition in the LDL are important for their influence on the PGI₂ formation.     

Interleukin-4 plus tumor necrosis factor α augments the antigenicity of melanoma cells

Immune cytokines are important regulators of the immune response to neoplastic cells. We previously reported that interleukin 4 (IL-4) and either tumor necrosis factor α (TNF) or interferon γ (IFN) synergistically inhibit melanoma cell growth and induce cell differentiation. In the present study we used various combinations of IL-4, IFN and TNF to enhance the antigenicity of melanoma cells. IL-4 plus TNF significantly increased the ability of melanoma cells to stimulate cytotoxic T cells (CTL) and act as targets of these CTL; IL-4 plus IFN was somewhat less effective, while TNF plus IFN was not as effective. IL-4 plus TNF also increased the expression of HLA class I and HLA-DR antigens on melanoma cells. The CTL lines examined in this study were CD3⁺CD4⁺ and oligoclonal. These preclinical results suggest that the immune response to melanoma whole-cell vaccines might be enhanced by pretreating vaccine cells with IL-4 plus TNF.     

Inhibitory effects of tamoxifen and tumor necrosis factor α on human glioblastoma cells

We reported previously that tumor necrosis factor α (TNFα) inhibited proliferation and invasiveness of human malignant glial cells. Because tamoxifen, an estrogen antagonist, has also been shown to inhibit growth of such cells, we hypothesized that a combination of tamoxifen and TNFα might be more effective than either reagent alone. TNFα (1–100 ng/ml) or tamoxifen (80 ng/ml-2 μg/ml) alone inhibited proliferation of a human glioblastoma cell line (WITG3) in a dose-dependent fashion; in combination, tamoxifen and TNFα yielded additive growth inhibition. Apoptotic cells characterized by nuclear fragmentation were detectable after 48 h of TNFα or tamoxifen exposure and were significantly increased by combination treatment. In non-neoplastic human astroglia and fibroblasts, proliferation was unaffected by tamoxifen, and enhanced by TNFα as previously reported. Staurosporine (2–50 nM), which has been reported to augment the effects of TNFα, was less effective than tamoxifen against WITG3 and, in addition, was markedly inhibitory to non-neoplastic glial cells. Binding studies yielded no evidence of WITG3 estrogen or progesterone receptors, nor of tamoxifen effects on TNFα receptors. Data suggest that TNFα and tamoxifen in combination display growth-regulatory properties, which (a) are more inhibitory to human glioblastoma cells than either agent alone, (b) do not affect non-neoplastic glia, (c) do not require either estrogen/ progesterone receptors or alteration of external TNFα receptors, and (d) may involve apoptosis.     

Interleukin-1 binding and prostaglandin E2 synthesis by amnion cells in culture: regulation by tumor necrosis factor-α, transforming growth factor-β, and interleukin-1 receptor antagonist

Proinflammatory cytokines may promote preterm labor in the setting of intrauterine infection. Tumor necrosis factor (TNF) and interleukin-1 (IL-1) synergistically stimulate the production of prostaglandin E₂ (PGE₂) by amnion cells. Transforming growth factor-β (TGF-β) inhibits the cytokine-stimulated PGE₂ production. In the present study, we investigated the binding of IL-1β on human amnion cells in culture. Untreated amnion cells possessed 540±60 IL-1 receptors per cell, with a dissociation constant of 1.4±0.4 nM. Cells treated with TGF-β1 (10 ng/ml) had 570±110 receptors per cell. TNF-α (50 ng/ml) increased the number of IL-1 receptors to 2930±590. TGF-β1 inhibited the receptor upregulation by TNF-α. Cells treated with TGF-β1 and TNF-α expressed 1140±590 receptors per cell. The binding affinity was not changed by the cytokines. IL-1 receptor antagonist (IL-1ra) inhibited the stimulation of amnion cell PGE₂ production by IL-1β, but not by TNF-α. Amnion cells secreted large amounts of IL-1ra (1.1±0.3 ng/10⁵ cells). Treatment of the cells with TGF-β1 or TNF-α did not affect the release of IL-1ra. We conclude that IL-1 receptor expression is an important step in the regulation of the effects of cytokines on amnion cell PGE₂ production.     

Induction of abortion by intra- and extra-amniotic prostaglandin F2α administration

Legal abortion was induced by intrauterine administration of prostaglandin F_2α in 115 patients during the 11th – 20th week of pregnancy. An intra-amniotic method was used in 61 of the cases, an extra-amniotic one in 54 cases. The average total dose administered was 35.1 mg (range 5 – 65 mg) in the intra-amniotic group, and 6358 μg (range 1500 – 14000 μg) in the extra-amniotic group. Abortion rate was 92 % in the intra-amniotic material and 72 % in the extra-amniotic material, and side-effects, mainly gastrointestinal irritation, were noted in 74 % of the intra-amniotic cases and 54 % of the extra-amniotic ones. If total doses of 4750 μg or more were administered in the extra-amniotic cases, abortion rate went up to 80 %, but the frequency of side-effects simultaneously increased to 64 %. No serious complications occurred. Intrauterine prostaglandin induction is well suited therapeutic abortions in the second trimester, and the intra-amniotic technique is more practicable than the extra-amniotic one. The latter is applicable in cases where the puncture of the amniotic cavity is difficult to achieve, e.g. in cases of fetus mortuus and hydatiform mole.     

Stimulatory effect of β-alanyl-L-histidinato zinc on cell proliferation is dependent on protein synthesis in osteoblastic MC3T3-E1 cells

The effect of -alanyl-L-histidinato zinc (AHZ) on bone metabolism was investigated in osteoblastic MC3T3-El cells. Cells were cultured for 3 days at 37°C in a CO₂ incubator in plastic dishes containing -modified minimum essential medium supplemented with 10% fetal bovine serum. After the cultures, the medium was exchanged for that containing 0.1% bovine serum albumin plus various concentrations of AHZ or other reagents, and the cells were cultured further for appropriate periods of time. The presence of AHZ (10^–7–10^–5M) stimulated the proliferation of cells. AHZ (10^–6 and 10^–5M) increased deoxyribonucleic acid (DNA) content in the cells with 48hr-culture. This increase was completely blocked by the presence of cycloheximide (10^–6M) or hydroxyurea (10^–3M). Also, the presence of cycloheximide (10^–6M) completely inhibited the AHZ (10^–5M)-induced increase in the proliferation of cells. Meanwhile, parathyroid hormone (10^–7M), estrogen (10^–9M) and insulin (10^–M) significantly increased cellular DNA content. However, these hormonal effects clearly lowered in comparison with that of AHZ (10^–5M). Dibutyryl cyclic AMP (10^–4M) and zinc sulfate (10^–5M) did not cause a significant increase in cellular DNA content. The present results support the view that AHZ has a direct specific proliferative effect on osteoblastic cellsin vitro and that this effect is dependent on protein synthesis.     

Endogenous interferon α/β produced by Kupffer cells inhibits interleukin-1, tumor necrosis factor α production and interleukin-2-induced activation of nonparenchymal liver cells

Summary We have previously reported liver-specific interferon (IFN) / production by murine Kupffer cells that was not observed with other tissue macrophages incubated in the absence of stimulators such as IFN or lipopolysaccharide (LPS). Consequently, while interleukin-2 (IL-2) alone induced pronounced lymphokine-activated killer (LAK) activity from splenocytes, combination of anti-IFN/ antibody with IL-2 was required to generate significant LAK activity from nonparenchymal liver cells. This endogenous IFN/ production by Kupffer cells was not induced by LPS because (a) addition of polymyxin B did not abolish the positive effects of anti-IFN/ antibody on nonparenchymal liver cells, and (b) similar results were obtained when comparing the responses of LPS-responsive C3HeB/FeJ and LPS-hyporesponsive C3H/HeJ mice. The possibility of hepatotropic infection was also ruled out in that anti-IFN/ antibody enhanced hepatic but not splenic LAK cell induction in vitro in both conventional and germfree C3H/HeN mice. IFN/ played an autoregulatory role by down-regulating the production of IL-1 and tumor necrosis factor by Kupffer cells. However, the augmenting effect of anti-IFN/ antibody on LAK induction from non-parenchymal liver cells was not mediated through an increase in the level of either IL-1 or TNF, as specific antisera against either cytokine did not abrogate this positive effect. Finally, flow-cytometry analysis showed that IFN/ significantly diminished the expression of IL-2 receptor chain, indicating an inhibition of LAK cell generation at a relatively early stage of induction.This work is supported by NIH grant RO1-28 835 and by Medical Research Funds from the Veterans Administration     

Macropinocytosis of extracellular glutathione ameliorates tumor necrosis factor α release in activated macrophages

A number of inflammatory lung diseases have abnormally low glutathione (GSH) levels in the airway fluids. Lung macrophages are common mediators of inflammation, make up the majority of cells that are found in the airway epithelial lining fluid (ELF), and are commonly elevated in many lung diseases. Several animal models with altered ELF GSH levels are associated with similar alterations in the intracellular GSH levels of bronchoalveolar lavage (BAL) cells. The possible mechanisms and outcomes for this association between ELF GSH levels and intracellular BAL cell GSH are unknown. To investigate these issues, macrophages were grown in media supplemented with 500 μM GSH. GSH supplementation resulted in a 2-3 fold increase in macrophage intracellular GSH levels. The increase in macrophage intracellular GSH levels was associated with a significant reduction in NF-κB nuclear translocation and tumor necrosis factor α (TNFα) release upon LPS stimulation. Furthermore, co-treatment of macrophages with GSH and inhibitors of GSH breakdown or synthesis did not block GSH accumulation. In contrast, treatment with cytochalasin D, an inhibitor of actin dependent endocytosis, and amiloride, an inhibitor of macropinocytosis blocked, at least in part, GSH uptake. Furthermore, using two cigarette smoke exposure paradigms that result in two different GSH levels in the ELF and thus in the BAL cells resulted in modulation of cytokine release when stimulated with LPS ex vivo. These data suggest that macrophages are able to utilize extracellular GSH which can then modulate inflammatory signaling in response to proinflammatory stimuli. This data also suggests the lung can modulate inflammatory responses triggered by proinflammatory stimuli by altering ELF GSH levels and may help explain the dysregulated inflammation associated with lung diseases that have low ELF GSH levels.     

Inhibitory effect of tumor necrosis factor α on gluconeogenesis in perfused rat liver

Tumor necrosis factor α (TNFα) is a cytokine involved in many metabolic responses in both normal and pathological states. Considering that the effects of TNFα on hepatic gluconeogenesis are inconclusive, we investigated the influence of this cytokine in gluconeogenesis from various glucose precursors. TNFα (10 μg/kg) was intravenously injected in rats; 6 h later, gluconeogenesis from alanine, lactate, glutamine, glycerol, and several related metabolic parameters were evaluated in situ perfused liver. TNFα reduced the hepatic glucose production (p < 0.001), increased the pyruvate production (p < 0.01), and had no effect on the lactate and urea production from alanine. TNFα also reduced the glucose production (p < 0.01), but had no effect on the pyruvate production from lactate. In addition, TNFα did not alter the hepatic glucose production from glutamine nor from glycerol. It can be concluded that the TNFα inhibited hepatic gluconeogenesis from alanine and lactate, which enter in gluconeogenic pathway before the pyruvate carboxylase step, but not from glutamine and glycerol, which enter in this pathway after the pyruvate carboxylase step, suggesting an important role of this metabolic step in the changes mediated by TNFα.     

Potentiation of antitumor effects of tumor necrosis factor α and interferon γ by macrophage-colony-stimulating factor in a MmB16 melanoma model in mice

The efficacy of systemic infusion of recombinant human macrophage-colony-stimulating factor (M-CSF) in combination with local treatment with human recombinant tumor necrosis factor (TNF) and mouse recombinant interferon (IFN) was studied in vivo on a subclone of B16 melanoma (MmB16) in mice. Short-term intravenous administration of M-CSF at a dose of 10⁶ units daily had no antitumor effect in vivo. Similarly, local treatment of tumor with TNF (5 g daily) did not produce any therapeutic effect. However, simultaneous administration of the same dose of TNF with IFN (1000 units daily) resulted in a synergistic effects manifested by the retardation of tumor growth. Addition of systemic infusion of M-CSF to the local therapy with TNF and IFN induced further augmentation of antitumor efficacy and delayed progression of MmB16 melanoma. The strengthened antitumor effect of combination therapy including M-CSF, TNF and IFN was most probably due to the increased release of monocytes from the bone marrow, their recruitment into the site of tumor growth and subsequent local stimulation of their antitumor activity.