Involvement of Two Types of TNF Receptor in TNF-α Induced Neutrophil Apoptosis

We previously demonstrated that tumor necrosis factor-α (TNF-α) induces rapid human neutrophil apoptosis. In this paper, we examined which of the TNF receptors, p55 kDa TNF receptor (55-R) or p75 kDa TNF receptor (75-R), or both are involved in this process using specific rabbit antisera. Antibodies to 55-R (anti55-R) or 75-R (anti75-R) alone did not induce neutrophil apoptosis. Further addition of cycloheximide and anti-rabbit immunoglobulin to anti55-R but not to anti75-R accelerated apoptosis, although anti75-R augmented the capacity of anti55-R to do so. These results suggest that 55-R is a prerequisite for TNF-α induced neutrophil apoptosis.     

Inhibition by Bacterial Lipopolysaccharide of Spontaneous and TNF-α-Induced Human Neutrophil Apoptosis In Vitro

In the previous paper (Takeda et al, Int. Immunol., 5, 691-694, 1993), we demonstrated that tumor necrosis factor-α (TNF-α) promptly accelerates apoptosis of human neutrophils in vitro. In order to determine the role of neutrophil apoptosis in defending against bacterial infection, we studied the effect of bacterial lipopolysaccharide (LPS) on this process. LPS inhibited spontaneous and TNF-α-induced human neutrophil apoptosis in vitro, as determined by 1) light and electron microscopy, 2) flow cytometry, and 3) agarose gel electrophoresis of DNA. Low concentrations of cycloheximide, a protein synthesis inhibitor, which alone did not affect neutrophil apoptosis, were able to reduce spontaneous apoptosis inhibition by LPS, suggesting the involvement of newly synthesized protein in this phenomenon.     

Nicotine blocks TNF-α-mediated neuroprotection to NMDA by an α-bungarotoxin-sensitive pathway

Excitotoxic neuronal death mediated by N-methyl-D -aspartate (NMDA) glutamate receptors can contribute to the extended brain damage that often accompanies trauma or disease. Both the inflammatory cytokine tumor necrosis factor-α (TNF-α) and nicotine have been identified as possible neuroprotective agents to NMDA assault. We find that TNF-α protection of a subpopulation of cultured cortical neurons to chronic NMDA-mediated excitotoxic death requires both the activation of the p55/TNFRI, but not p75/TNFRII, and the release of endogenous TNF-α. Nicotine protection to NMDA was mediated through an α-bungarotoxin-sensitive receptor. When coapplied, neuroprotection to NMDA by either TNF-α or nicotine was abolished but could be recovered with α-bungarotoxin. These results suggest that the cytokine TNF-α and α-bungarotoxin-sensitive nicotinic neurotransmitter receptors confer neuroprotection through potentially antagonistic pathways. © 1998 John Wiley & Sons, Inc. J Neurobiol 35: 29–36, 1998     

Transformation of mouse mammary epithelial cells with the Ha-ras but not with the neu oncogene results in a gene dosage-dependent increase in transforming growth factor-α production

An enhanced expression of transforming growth factor-α (TGFα) was demonstrated in two clones of NOG-8 mouse mammary epithelial cells, NOG-8 SR1 and NOG-8 SR2, that have been transformed by a v-Ha-ras oncogene. The amount of TGFα production in NOG-8 SR1 and NOG-8 SR2 cells was dependent on the level of p21^ras expression in these clones, which directly correlated with their cloning efficiency in soft agar. There was also a decrease in the number of epidermal growth factor (EGF) receptors on the NOG-8 SR1 and NOG-8 SR2 cells that is proportional to the amount of TGFα secreted. These effects were specific for ras because neu-transformed NOG-8 cells grew in soft agar at a comparable level to NOG-8 SR2 cells yet did not show any increase in TGFα production or change in EGF receptor expression.     

Paeonol from Paeonia suffruticosa prevents TNF-α-induced monocytic cell adhesion to rat aortic endothelial cells by suppression of VCAM-1 expression

Paeonol (2′-hydroxy-4′-methoxyacetophenone), the main active compound of the radix of Paeonia suffruticosa, has been reported to have a beneficial effect in prevention and treatment of cardiovascular diseases. Vascular cell adhesion molecule-1 (VCAM-1), plays a crucial role in case of early inflammatory responses, including atherosclerosis. In this study, we investigated the effect of paeonol on TNF-α-induced VCAM-1 expression in rat aortic endothelial cells (RAECs). The VCAM-1 expression in paeonol treated RAECs was measured. Paeonol inhibited TNF-α-induced VCAM-1 expression in a concentration-dependent manner. TNF-α induced p38 and extracellular signal-regulated kinase 1/2 (ERK1/2) activities that contributed to VCAM-1expression was obviously attenuated after pre-treating RAECs with paeonol. The decrease of VCAM-1 expression by paeonol pretreatment led to a reduction of monocytes adhesion to RAECs. Taken together, our results demonstrated that paeonol inhibited VCAM-1 expression by the attenuation of p38 and ERK1/2 signal transduction pathways. We concluded that paeonol had the potential therapeutic development for use in anti-inflammatory and vascular disorders.     

LYMPHOTOXIN-α (TNF-β) DURING SEPSIS

T cell release of lymphotoxin-α (LT-α, or TNF-β) is stimulated by pyrogenic exotoxins of Gram-positive bacteria and mitogens. In contrast to TNF-α, it is unknown whether LT-α plays any role in the pathogenesis of sepsis and, in particular, the pathogenesis of Gram-positive sepsis. Sera from patients with sepsis were examined for LT-α and compared with normal volunteers and pregnant women. LT-α was detected in 33% of sepsis sera (mean 608.4 pg/ml SE 306), 16% of normal sera (mean 167 pg/ml SE 87) and 23% of sera from pregnant women (mean 714 pg/ml SE 191). These differences were not significant and there were no differences within species sera when grouped by the type of causative organism, or disease severity. LT-α detected by immunoassay in serum was not bioactive, in contrast to that produced in cell culture. Recombinant soluble TNF receptors (rSTNFR) neutralized the bioactivity of recombinant LT-α at rSTNFR concentrations which did not interfere with immunoreactivity and which are known to prevailin vivo. Hence, LT-α is unlikely to have a critical role in the pathogenesis of sepsis. Much of the potential bioactivity of this lymphokine may be abrogated by TNFR in serum.     

Laminin 5 Promotes Activation and Apoptosis of the T Cells Expressing α3β1 Integrin

By introducing an α3 gene-containing plasmid into a human T cell line Jurkat, we prepared the T cells, which express a high level of the α3β1 integrin, to assess the role of laminin 5 in the skin immune system. The α3β1-expressing T cells adhered to laminin 5 and exhibited spreading. These adhered T cells showed a significant tyrosine phosphorylation of intracellular proteins including p59^fynupon T-cell receptor (TCR) stimulation. Six hours after cross-linking TCR, these cells on laminin 5 secreted a three times higher level of IL-2 than those on a BSA-coated plate. Twenty hours after the stimulation, 48% of the α3β1-expressing T cells on laminin 5 caused apoptosis. The protein level of cyclin D3 and E decreased, while that of p53 increased in these T cells. These data suggest that laminin 5 may play at least two regulatory roles for T cell functions: augmentation of IL-2 production by antigen-stimulated T cells and induction of apoptosis in these T cells.     

Nitric oxide activates the expression of IRAK-M via the release of TNF-α in human monocytes

The activation of interleukin receptor associated kinases (IRAK) is an important event in several inflammatory processes. However, exposing monocytes to a nitric oxide (NO) donor inhibits the activity of IRAK-1 and its molecular interaction with TNF receptor associated factor-6 (TRAF6). Despite the fact that NO is known to regulate many events in the immune and vascular system, the mechanism that underlies this inhibition remains unknown. We have recently demonstrated that IRAK-M inhibits the TLR/IRAK pathway during endotoxin tolerance and thus, we hypothesized that IRAK-M may be involved in the inhibition of IRAK-1 activity in the presence of NO. Hence, we have analyzed the expression of IRAK-M in human monocytes following exposure to a NO donor (GSNO) and we have observed that GSNO was capable of inducing IRAK-M mRNA and protein expression 8 and 20 h after stimulation, respectively. It is known that NO induces the expression of TNF-alpha in monocytes and we found that exposure to TNF-alpha induced IRAK-M mRNA expression in human monocytes within 2 h of stimulation. Furthermore, the expression of IRAK-M induced by GSNO was inhibited by the presence of a blocking antibody raised against TNF-alpha. Thus, our data indicate that stimulation of human monocytes with a NO donor results in a clear induction of IRAK-M and this is dependent on the release of TNF-alpha by this kind of cells.     

Enantioselective inhibition of TNF-α release by thalidomide and thalidomide-analogues

The question whether the immunomodulating activity of rac-thalidomide resides in either the (−)-(S)- or the (+)-(R)-enantiomer was addressed by synthesis and separation of pure enantiomers of thalidomide-analogues which carry a methyl-group at the asymmetric carbon atom and are thus prevented from racemization. The effect of the pure enantiomers of the thalidomide-analogues and also of the enantiomers of thalidomide on relapse of TNF-α was tested in vitro by using stimulated peripheral mononuclear blood cells. Both enantiomers of thalidomide inhibited the release of TNF-α equally well at low concentrations (5 and 12.5 μg/ml) but at higher concentrations (25 and 50 μg/ml) there was a weak but statistically significant selectivity towards the (−)-(S)-enantiomer. In the case of the configuration-stable thalidomide-analogues there was a very pronounced and statistically significant enantioselectivity towards the (S)-form even at lower concentrations (≥5 μg/ml). The (S)-enantiomers of the thalidomide-analogues differed in their inhibitory potency from (−)-(S)-thalidomide suggesting that the introduction of the methyl-group increases the TNF-α-inhibitory activity while the reduction of one of the carbonyl-functions in the glutarimide-moiety to a methylene-group decreases activity. The effect of these small molecular alterations on activity and the enantioselectivity towards the (S)-enantiomers may indicate that thalidomide and its analogues directly interact with one or several cellular target-proteins. © 1996 Wiley-Liss, Inc.     

Contrasting effects of interferon γ and interleukin 4 on responses of human vascular endothelial cells to tumour necrosis factor α

Tumour necrosis factor α (TNF-α) and interleukin 4 (IL-4) selectively synergise in inducing expression of the mononuclear cell adhesion receptor VCAM-1 (vascular cell adhesion molecule-1) on human umbilical vein endothelialcells (HUVEC), which results in increased adhesiveness of HUVEC for T lymphocytes. This process may be crucial for adherence of circulating lymphocytes prior to their passage from the blood into inflammatory tissues. IL-4 also amplifies production of interleukin 6 (IL-6) and monocyte chemotactic protein-(MCP-1) from TNF-α-activated HUVEC. In the present study we demonstrate that IL-4 enhances production of granulocyte-macrophage colon-stimulating factor (GM-CSF) from TNF-α-stimulated HUVEC. Moreover, using cultured adult saphenous vein and umbilical artery endothelial cells, we show identical effects of IL-4 on TNF-α-induced responses to those observed with endothelial cells of foetal origin. Additionaly, we report here that TNF-α and interferon γ (IFN-γ) synergise in the induction of both the lymphocyte adhesion receptor VCAM-1, and the TNF-α-inducible neutrophil adhesion receptor intercellular adhesion molecule-1, on all three endothelial cell types studied. In contrast, we found that GM-CSF secretion by endothelial cells treated with IFN-γ plus TNF-α was markedly decreased when compared to the response by TNF-α alone. These results suggest that the combined actions of several cytokines, acting sequentially or in concert, may exert differential effects on activation and accumulation of circulating lymphocytes at sites of inflammation.     

POTENTIATION OF TNF-α TOXICITY BY CONJUGATION WITH RICIN A-CHAIN

Hybrid toxins containing a cytokine moiety have been used effectively to selectively kill cells expressing the complementary cytokine receptor both in vivo and in vitro. To date all cytokines incorporated into hybrid toxins, e.g. interleukin 2 are biologically active as monomers, so attachment of a toxin group causes minimal interference with the cytokine structure. By contrast, the pro-inflammatory and anti-cancer cytokine tumour necrosis factor α (TNF-α) is biologically active as a homotrimer in which the grooves created between the hydrophobically associated monomers form the receptor binding region, so maintenance of this structure is crucial for activity. In this report the authors show that TNF-α can be modified by reaction with a crosslinking agent and by subsequent attachment of the toxin ricin A-chain without loss of TNF-α cytotoxic activity in the WEHI assay. Structural association of the hybrid toxin composed of TNF-α and ricin A-chain was confirmed by Western blot analysis. The hybrid toxin was toxic to HeLa cells (IC₅₀=4 pM) not sensitive to native TNF-α, and sensitive WEHI cells with substantially increased lethality (LD₅₀=0.01 fM). This increased TNF-α cytotoxic activity suggests that hybrid toxins containing TNF-α may have therapeutic applications in the treatment of cancer.     

TNF-α Suppresses Apelin Receptor Expression in Mouse Quadriceps Femoris-Derived Cells

Expression of the apelin receptor, APJ, in skeletal muscle (SM) is known to decrease with age, but the underlying mechanism remains unclear. Increased tumor necrosis factor (TNF)-α levels are observed in SM with age and are associated with muscle atrophy. To investigate the possible interconnection between TNF-α elevation and APJ reduction with aging, we investigated the effect of TNF-α on APJ expression in cells derived from the quadriceps femoris of C57BL/6J mice. Expression of Tnfa and Apj in the quadriceps femoris was compared between 4- (young) and 24-month-old (old) C57BL/6J mice (n = 10 each) using qPCR. Additionally, APJ-positive cells and TNF-α protein were analyzed by flow cytometry and Western blotting, respectively. Further, quadricep-derived cells were exposed to 0 (control) or 25 ng/mL TNF-α, and the effect on Apj expression was examined by qRT-PCR. Apj expression and the ratio of APJ-positive cells among quadricep cells were significantly lower in old compared to young mice. In contrast, levels of Tnfa mRNA and TNF-α protein were significantly elevated in old compared to young mice. Exposing young and old derived quadricep cells to TNF-α for 8 and 24 h caused Apj levels to significantly decrease. TNF-α suppresses APJ expression in muscle cells in vitro. The increase in TNF-α observed in SM with age may induce a decrease in APJ expression.     

Role of Curdlan Sulfate in the Binding of HIV-1 gp120 to CD4 Molecules and the Production of gp120-Mediated TNF-α

To clarify the mechanism by which curdlan sulfate (CRDS) inhibits human immunodeficiency virus (HIV)-1 infection, we examined its influence on the binding of gp120 to CD4 molecules on T cells and macrophages, as well as on the production of TNF-α by gp120-stimulated macrophages (which promotes HIV-1 replication). CRDS treatment of cells not only inhibited the binding of HIV-1 gp120 to CD4⁺ cells, but also inhibited TNF-α production induced by gp120. Inhibition of HIV-1 infection by CRDS may be related to these two actions.     

IL-10 overexpression differentially affects cartilage matrix gene expression in response to TNF-α in human articular chondrocytes in vitro

Cartilage-specific extracellular matrix synthesis is the prerequisite for chondrocyte survival and cartilage function, but is affected by the pro-inflammatory cytokine TNF-α in arthritis. The aim of the present study was to characterize whether the immunoregulatory cytokine IL-10 might modulate cartilage matrix and cytokine expression in response to TNF-α. Primary human articular chondrocytes were treated with either recombinant IL-10, TNF-α or a combination of both (at 10 ng/mL each) or transduced with an adenoviral vector overexpressing human IL-10 and subsequently stimulated with 10 ng/ml TNF-α for 6 or 24 h. The effects of IL-10 on the cartilage-specific matrix proteins collagen type II, aggrecan, matrix-metalloproteinases (MMP)-3, -13 and pro-inflammatory cytokines were evaluated by real-time RT-PCR and immunohistochemistry. Transduced chondrocytes overexpressed high levels of IL-10 which significantly up-regulated collagen type II expression. TNF-α suppressed collagen type II and aggrecan, but increased MMP and cytokine expression in chondrocytes compared to the non-stimulated controls. The TNF-α mediated down-regulation of aggrecan expression was significantly antagonized by IL-10 overexpression, whereas the suppression of collagen type II was barely affected. The MMP-13 and IL-1β expression by TNF-α was slightly reduced by IL-10. These results suggest that IL-10 overexpression modulates some catabolic features of TNF-α in chondrocytes.     

Production and recovery of recombinant protease inhibitor α1-antitrypsin

Human α₁-antitrypsin (AAT) was produced in the recombinant yeast Saccharomyces cerevisiae ATCC 20699 grown in batch and fed-batch culture. The final biomass concentration and antitrypsin concentration attained were 55 g·L⁻¹ and 1.23 g·L⁻¹, respectively, in the fed-batch. The maximum productivities of biomass and antitrypsin were 1.6 and > 0.04 g L⁻¹h⁻¹, respectively, or substantially greater than the highest productivity values reported in the past. For recovering the antitrypsin, the cell slurry was concentrated 4-fold (231 g·L⁻¹ biomass, 122 min of processing) by cross-flow microfiltration and the cells were disrupted by bead milling (3 passes of 3 min total retention time). The cell homogenate was treated with aluminum chloride or PBS (pH 7) to aid separation of the cell debris by flocculation and sedimentation. The clarified cell homogenate was subjected to ammonium sulfate fractionation to precipitate the recombinant antitrypsin. The AAT precipitated at 45–75% saturation of ammonium sulfate, depending on the age of the homogenate. The crude AAT in the homogenate degraded at room temperature (25°C), with a zero order deactivation rate of 1.815 × 10⁻³ ± 3.43 × 10⁻⁴ g AAT L⁻¹h⁻¹.