Human TNF-alpha Research Articles

2-Aminopurine selectively inhibits splicing of tumor necrosis factor alpha mRNA.

2-Aminopurine (2-AP) inhibits specific kinases that phosphorylate the alpha subunit of eukaryotic translation initiation factor 2. One of these, PKR, is also involved in signal transduction. We show here that 2-AP selectively inhibits expression of tumor necrosis factor alpha (TNF-alpha) mRNA in primary human lymphoid cells. 2-AP does not inhibit transcription of the human TNF-alpha gene, nor does it affect mRNA stability. Instead, the flow of short-lived precursor transcripts into mature TNF-alpha mRNA is blocked. When 2-AP is present during induction, unspliced TNF-alpha precursor transcripts accumulate at the expense of mRNA. Using RNase protection analysis with genomic probes for different exon-intron junctions, we show that 2-AP blocks splicing of TNF-alpha mRNA. Neither the TNF-beta nor the interleukin-1 beta gene shows such regulation. 2-AP also inhibits splicing of precursor RNA transcribed from an exogenous human TNF-alpha gene. Sequences within this gene thus confer sensitivity to 2-AP. Yet, control is not exerted at a specific splice site. Our results reveal the involvement of a 2-AP-sensitive component, expressed in functional form before induction, in the splicing of TNF-alpha mRNA.

Tumor Necrosis Factor (TNF)-α Inhibits Insulin Signaling through Stimulation of the p55 TNF Receptor and Activation of Sphingomyelinase

Tumor necrosis factor (TNF)-alpha plays a central role in the state of insulin resistance associated with obesity. It has previously been shown that one important mechanism by which TNF-alpha interferes with insulin signaling is through the serine phosphorylation of insulin receptor substrate-1 (IRS-1), which can then function as an inhibitor of the tyrosine kinase activity of the insulin receptor (IR). However, the receptors and the signaling pathway used by TNF-alpha that mediate the inhibition of IR activity are unknown. We show here that human TNF-alpha, which binds only to the murine p55 TNF receptor (TNFR), is as effective at inhibiting insulin-dependent tyrosine phosphorylation of IR and IRS-1 in adipocytes and myeloid 32D cells as murine TNF-alpha, which binds to both p55 TNFR and p75 TNFR. Likewise, antibodies that are specific agonists for p55 TNFR or p75 TNFR demonstrate that stimulation of p55 TNFR is sufficient to inhibit insulin signaling, though a small effect can also be seen with antibodies to p75 TNFR. Exogenous sphingomyelinase and ceramides, known to be formed by activation of p55 TNFR, inhibit IR and IRS-1 tyrosine phosphorylation and convert IRS-1 into an inhibitor of IR tyrosine kinase in vitro. Myeloid 32D cells expressing IR and IRS-1 are sensitive to this inhibition, but cells expressing IR and IRS-2 are resistant, pointing to an important difference in the biological function between IRS-1 and IRS-2. These data strongly suggest that TNF-alpha inhibits insulin signaling via stimulation of p55 TNFR and sphingomyelinase activity, which results in the production of an inhibitory form of IRS-1.

Monocyte Tumor Necrosis Factor Receptor Levels as a Predictor of Risk in Human Sepsis

To assess peripheral blood monocyte tumor necrosis factor receptor (TNFR) levels and plasma soluble tumor necrosis factor receptor (sTNFR) concentrations in critically ill patients with sepsis syndrome. Prospective, descriptive cohort study with no interventions. Surgical intensive care unit of a tertiary-care hospital associated with a university medical school. Twenty-one patients with a documented source of infection who met currently accepted criteria for sepsis syndrome/septic shock. Plasma sTNFR p55 and p75 values were quantified by enzyme-linked immunosorbent assay, and monocyte TNFR levels were assessed by fluorescence flow cytometry after the monocytes were stained with biotinylated human recombinant TNF-alpha and streptavidin-phycoerythrin. Compared with healthy controls, plasma sTNFR p55 and p75 values were significantly higher (P <.01) in both surviving and nonsurviving patients with sepsis; in nonsurviving patients with sepsis, however, only sTNFR p55 values were significantly (P < .05) higher than in surviving patients with sepsis. By contrast, monocytes from the nonsurviving patients with sepsis manifested a significant (P < .01) and sustained (up to 4 days) decrease in cell surface TNFR values compared with either the normal controls or the surviving patients with sepsis. Assessment of monocyte surface TNFR values may provide a rapid prognostic indicator for patients with sepsis who are at increased risk of death.

Development of an enzyme immunoassay for the measurement of human tumour necrosis factor-alpha (hTNF-alpha) using bispecific antibodies to hTNF-alpha and horseradish peroxidase.

The cytokine tumour necrosis factor-alpha (TNF-alpha) is involved in several pathological processes, and human recombinant TNF-alpha (hrTNF-alpha) is available for testing in preclinical and clinical research. The purpose of this work was the creation of tetradomas producing bispecific anti-hTNF-alpha/anti-HRP (horseradish peroxidase) antibodies and the development of a rapid and sensitive solid-phase enzyme immunoassay. Monoclonal antibodies obtained against hrTNF-alpha could recognize both natural and recombinant hTNF-alpha. The four chosen hybridomas produced IgG1 with an affinity constant of the order of 10(-9) M. Three of them recognized different epitopes. The clone selected for fusion with the hybridoma producing anti-HRP antibodies secreted antibodies against portion 30-50 of the hTNF-alpha N-terminal amino acid residues as found by Western-blot analysis with mutant and chimaeric proteins. The tetradoma producing bispecific anti-hTNF-alpha/anti-HRP antibodies was identified using a fluorescence-activated cell sorter. Bispecific antibodies were isolated by hydroxyapatite chromatography. A sandwich ELISA was developed: one of the monoclonal anti-TNF-alpha antibodies was absorbed to the solid phase as the catcher and was detected by a bispecific anti-hTNF-alpha/anti-HRP antibody. The detection limit of the assay was 1 ng/ml. With such ELISA, the level of hTNF-alpha could be conveniently estimated in different samples containing either natural or recombinant hTNF-alpha in an experimental environment or in clinical trials.

Acute stimulation of the hypothalamic-pituitary-adrenal axis by IL-1 beta, TNF alpha and IL-6: a dose response study.

We investigated the effects of i.v. and intracerebroventricular (i.c.v) administration of increasing doses of recombinant human IL-1 beta, TNF alpha and IL-6 on plasma corticosterone (B) levels in rats. Rats were equipped with a jugular cannula for repeated blood sampling anda subgroup of rats also received an i.c.v implanted cannula. I.v. administration of IL-1 beta, TNF alpha or IL-6 and i.c.v administration of IL-1 beta and IL-6 induced a significant dose-dependent increase in plasma B levels, whereas i.c.v injection of TNF alpha in doses up to 1000 ng/rat was not effective. I.v. pretreatment of rats with anti-CRH antiserum had no significant overall effect on the plasma B response to i.v. administered IL-1 beta (500 and 3000 ng/rat), whereas the plasma B response to i.v. TNF alpha or IL-6 administration (3000 ng/rat) were significantly reduced. I.v. pretreatment of the animals with recombinant human IL-1 receptor antagonist (IL-1ra) significantly blocked the plasma B response to i.v. treatment with IL-1 beta, whereas the TNF alpha- and IL-6-induced increases in plasma B levels were not affected. Our data show that 1) i.v. administration of IL-beta, TNF alpha or IL-6 and i.c.v administration of IL-1 beta or IL-6 dose-dependently stimulate the HPA axis; 2) when given i.v. or i.c.v, IL-1 beta is more powerful than TNF alpha and IL-6 in activating the HPA axis; 3) endogenous CRH is involved in the activation of the HPA axis by acute i.v. administration of TNF alpha and IL-6. It is most likely that in case of i.v. treatment with IL-1 beta a CRH-independent mechanism is involved. This study provides no arguments for the involvement of endogenous IL-1 in TNF alpha- or IL-6-induced activation of the HPA axis.

Distribution of TNF alpha and its reactive vascular adhesion molecules in fibrovascular membranes of proliferative diabetic retinopathy.

This study investigated the presence of the cytokine tumour necrosis factor alpha (TNF alpha) and the vascular adhesion glycoproteins ICAM-1, VCAM-1, E-selectin, P-selectin, and PECAM within fibrovascular membranes of eyes with proliferative diabetic retinopathy (PDR). The presence of these molecules was determined by immunohistochemical staining using monoclonal antibodies and the APAAP technique. Staining for TNF alpha was observed on the retinal vascular endothelium of five of 12 specimens, on infiltrating cells within all membranes, and on the extracellular matrix of nine specimens. This staining wa abolished by absorption of the monoclonal antibody with human recombinant TNF alpha. Likewise, ICAM-1 staining was given by infiltrating cells and extracellular matrix of nine membranes and by the endothelium of three of the specimens. VCAM-1, E-selectin, and P-selectin staining was observed on the vascular endothelium of 5/12, 4/12, and 3/12 epiretinal membranes respectively. PECAM was expressed by the endothelium of 4/12 specimens, by infiltrating cells of 8/12 membranes, and also by the extracellular matrix of two of the specimens. The widespread distribution of TNF alpha and the nature of the adhesion molecules expressed by vascular endothelial cells in PDR membranes suggest that local activation of TNF alpha and enhanced expression of vascular cell adhesion molecules may play an important role in the development of the proliferative phase of diabetic retinopathy.

A novel Escherichia coli lipid A mutant that produces an antiinflammatory lipopolysaccharide.

A unique screen was used to identify mutations in Escherichia coli lipid A biosynthesis that result in a decreased ability to stimulate E-selectin expression by human endothelial cells. A mutation was identified in the msbB gene of E. coli that resulted in lipopolysaccharide (LPS) that lacks the myristoyl fatty acid moiety of the lipid A. Unlike all previously reported lipid A mutants, the msbB mutant was not conditionally lethal for growth. Viable cells or purified LPS from an msbB mutant had a 1000-10,000-fold reduction in the ability to stimulate E-selectin production by human endothelial cells and TNF alpha production by adherent monocytes. The cloned msbB gene was able to functionally complement the msbB mutant, restoring both the LPS to its native composition and the ability of the strain to stimulate immune cells. Nonmyristoylated LPS acted as an antagonist for E-selectin expression when mixed with LPS obtained from the parental strain. These studies demonstrate a significant role for the myristate component of LPS in immune cell activation and antagonism. In addition, the msbB mutant allowed us to directly examine the crucial role that the lipid A structure plays when viable bacteria are presented to host defense cells.

Induction of alpha-helix in the beta-sheet protein tumor necrosis factor-alpha: acid-induced denaturation.

Acid-induced unfolding of proteins often results in an intermediate structure, called the molten globule structure or "A" state, which retains at least partial secondary structure but lacks a rigid tertiary structure. Acid-induced unfolding has been studied extensively for alpha-helical proteins, while few studies have been done on proteins containing only beta-strands. Tumor necrosis factor-alpha (TNF-alpha) is a trimer in which the individual subunits consist of antiparallel beta-sheet, organized into a jellyroll beta-sandwich. We have found previously [Narhi et al. (1996) Biochemistry 35, 11447-11453] that thermal denaturation of TNF-alpha results in an aggregate which contains a substantial amount of alpha-helix and that the addition of trifluoroethanol induces alpha-helix in both murine and human TNF-alpha. Here we show that acid also can induce alpha-helix in these proteins. At acidic pH (below 4), both human and murine TNF-alpha convert to a monomeric form, as determined by sedimentation and diffusion constants obtained from sedimentation velocity experiments. The sedimentation coefficient indicated that this monomer was only slightly expanded relative to the native state. Near-UV circular dichroic (CD) analysis showed a loss of tertiary structure. These structural features coincide with the notion that the acid-induced structure of TNF-alpha is a molten globule. What is unique in this protein is that TNF-alpha acquires alpha-helical structure, which is not present in the native structure as determined by both CD and Fourier transform infrared spectroscopy. Even more surprising is that TNF-alpha at pH 3.3 undergoes a very gradual noncooperative change in secondary structure upon heating, which results in an increase in alpha-helical content. At pH 2.2 in the absence of salt, TNF-alpha shows considerable alpha-helix, although heating does not change the spectrum. At pH 2.2, physiological salt decreases the amount of alpha-helix at ambient temperature, and upon heating, we see the noncooperative increase in alpha-helix as observed at pH 3.3 with low salt. The addition of salt at low pH induces reassociation but to a range of oligomers rather than a unique trimer structure. This acid-induced formation of an alpha-helical monomer of TNF-alpha may be related to its known interaction with lipid bilayers.

Activation of nuclera factor-kappa B and control of the expression of cell adhesion molecules in human prostate cancer cells

Cell adhesion molecules (CAMs) have been considered to play key roles in cancer metastasis through binding of cancer cells to the endothelial cells or matrix. We have found that constitutive expression of E-selectin, ICAM-1 (intercellular adhesion molecule-1) and VCAM-1 (vascular cell adhesion molecule-1) on the cell surface of prostate cancer cell lines, PC-3 LNCaP, and DU145. In an attempt to explain the highly metastatic potential of these cells, we investigated expression of CAMs. Since these expressed CAMs are known to be regulated by a transcription factor NF kappa B. We have examined if NF kappa B is activated in these prostateccell lines. Expression of E-selectin, ICAM-1. Sialyl-Lewis X, LFA-1 (lymphocyte function-associated antigen-1), integrin alpha 4, and integrin beta 1 were examined by indirect immunofluorescent staining and Western blotting using specific monoclonal antibodies. We then examined whether pro-inflammatory cytokines known to stimulate NF kappa B, IL-1 and TNF, could drive the nuclear translocation of NF kappa B Ten ng/ml of recombinant human IL-1 beta or TNF alpha was added to the cell culture and the CAMs level, as well as nuclear translocation of NF kappa B, was examined. The staining levels of ICAM-1, VCAM-1, and integrin beta 1 were highly elevated in PC-3 and DU145. In surprising, NF kappa B was retained in the cytoplasm (as an inactive form) and not detectable in the nucleus (active form) as demonstrated by immunofluorescence. Although time-dependent expression of these molecules were explored, the level of ICAM-1 and VCAM-1 on the cell surface were unchanged. However, nuclear translocation of NF kappa B could be induced. These observations indicate that while the signaling pathway to NF kappa B is intact, it is not directed to induction of its target CAM genes. It is possible that other transcription factors might be involved in these cells and that they might to be constitutively stimulated during the carcinogenesis of prostate cancer.

The possible role of TNF-? and IL-2 in inducing tumor-associated metabolic alterations

This study was conducted to investigate the role of tumor necrosis factor-alpha (TNF-alpha) and interleukin-2 (IL-2) in inducing cancer cachexia, and the results were compared with those obtained from our previous study on Fisher 344 rats with methylcholanthrene-induced sarcoma. Three groups of male Fisher 344 rats received one of the following regimens: 4 x 10(4) IU of human recombinant TNF-alpha per rat per day subcutaneously (sc) for 5 consecutive days (n = 5), 3.5 x 10(5) U human recombinant IL-2 per rat per day sc for 14 consecutive days (n = 5), or normal saline (n = 5). The activities of both phosphoenolpyruvate carboxykinase (PEPCK) and malic enzyme (ME) were increased slightly in the IL-2 group. Furthermore, LPL activity was significantly increased in the adipose tissue of the TNF group and in the cardiac muscle of the IL-2 group, but not in that of the TNF group. These results show that there is a significant difference between the metabolic alterations seen in the tumor-bearing state and those induced by either TNF-alpha or IL-2 alone. Thus, it is unlikely that IL-2 or TNF-alpha is the sole mediator of cancer cachexia in this tumor and rat model.

Morphological effects of tumor necrosis factor-alpha on the blood vessels in rat experimental brain tumors.

The morphological changes in the vascular endothelium caused by the administration of tumor necrosis factor-alpha (TNF-alpha) were studied in an experimental model of rat brain tumors. Wistar rats bearing implanted C6 glioma received human natural-type TNF-alpha (1.7 x 10(5) U/m2) through the carotid artery and were sacrificed 3 or 24 hours later. The endothelial cells of the tumor blood vessels, demonstrated by the immunoreaction to factor VIII-related antigen, were enlarged after TNF-alpha administration. Morphometry demonstrated that the nuclei of these endothelial cells were also increased in size. The endothelial cells in the brain remote to the tumor were not affected. An in vitro binding study demonstrated that TNF-alpha binding sites were distributed in the vascular endothelial cells within the tumor but not in the brain remote to the tumor. The selective effect of TNF-alpha on the tumor blood vessels in experimental brain tumors may be related to the selective distribution of the TNF-alpha binding site.

Protective Effect of Celosian, an Acidic Polysaccharide, on Chemically and Immunologically Induced Liver Injuries.

Hepatoprotective effect of celosian, an acidic polysaccharide isolated from the water extract of the seed of Celosia argentea, was investigated using chemical and immunological liver injury models. Celosian inhibited the elevation of serum enzyme (GPT, GOT, LDH) and bilirubin levels on carbon tetrachloride (CC1(4))-induced liver injuries in rat. In addition, the hepatoprotective effect of celosian was also observed in this model of liver injury by histopathological findings. Moreover, celosian suppressed rises in GPT or mortality on fulminant hepatitis induced by D-galactosamine/lipopolysaccharide (D-Ga1N/LPS) or Propionibacterium acnes/LPS in mice. These findings suggested that celosian is an active component in protection against chemical and immunological hepatitis and the activity was found to be a dose dependent. Celosian showed a concentration dependent inhibitory effect on lipid peroxide (LPO) generation in vitro. Though celosian did not reduce the release of tumor necrosis factor-alpha (TNF-alpha), it protected against recombinant human TNF-alpha (rhTNF-alpha)-induced liver injury in D-galactosamine sensitized mice.

Intracerebroventricular injection of tumor necrosis factor-alpha induces thermal hyperalgesia in rats.

To investigate the role of tumor necrosis factor-alpha (TNF-alpha) in the brain in nociception, we injected recombinant human TNF-alpha (rhTNF-alpha; 1 pg-10 ng/rat) into the lateral cerebroventricle (LVC) in rats and observed the changes in paw withdrawal latency to radiant heat by using the plantar test for 90 min after injection. LCV injections of TNF-alpha at doses of 10 pg, 100 pg and 1 ng reduced paw withdrawal latency, showing a maximal response at a dose of 10 pg which peaked 60 min after injection. TNF-alpha at doses of 1 pg and 10 ng had no effect on nociception during the test period. The TNF-alpha (10 pg)-induced reduction in paw withdrawal latency was blocked by simultaneous injection of diclofenac (1 ng), a cyclooxygenase inhibitor, or interleukin-1 receptor antagonist (IL-1 ra, 10 ng). LCV injection of neither diclofenac (1 ng) nor IL-1 ra (10 ng) had any effect on nociception by itself. The results suggest that TNF-alpha in the brain induces thermal hyperalgesia and that the brain TNF-alpha-induced hyperalgesia is mediated by the central action of interleukin-1 and activation of the cyclooxygenase pathway of the arachidonate.

Tumor necrosis factor-alpha inhibits lipid and lipoprotein transport by Caco-2 cells

Cytokines, important mediators of inflammation, have been shown to cause disturbances in circulating and hepatic lipid metabolism. Although the intestine plays a major role in dietary fat transport and largely contributes to plasma lipoproteins, the effects of cytokines on intestinal lipid handling remain unknown. In the present study, the modulation of lipid, apoprotein, and lipoprotein synthesis and secretion by tumor necrosis factor-alpha (TNF-alpha) was investigated in Caco-2 cells. Highly differentiated and polarized cells (20 days in culture) were incubated for 20 h with recombinant human TNF-alpha (100-500 ng/ml). No cytotoxic effect of TNF-alpha cells was observed, as indicated by the determinations of Caco-2 cell viability and monolayer transepithelial resistance. Moreover, no differences in cell maturation (sucrase activity) or cell proliferation ([3H]thymidine incorporation and cell cycle analysis) were detected between treated and control cultures. Significant inhibition of lipid secretion by TNF-alpha was observed, with the greatest reduction at 500 ng/ml. TNF-alpha significantly decreased Caco-2 cell secretion of phospholipids (22%), triglycerides (30%), and cholesteryl ester (37%). It also significantly diminished the export of newly synthesized low-density lipoproteins (LDL; 20%) and high-density lipoproteins (HDL; 13%), with a lesser effect on very low-density lipoproteins (VLDL; 3%). The lipid composition of these lipoproteins was minimally affected. De novo synthesis of apo A-I, apo B-100, and apo B-48 was also markedly reduced by TNF-alpha. Sphingomyelinase activity was not increased and cell content of sphingomyelin was not altered, suggesting that inhibitory effects on lipid and apoprotein of TNF-alpha were not mediated by the ceramide pathway. Our results indicate that TNF-alpha may play a role in modulating intestinal lipid metabolism, thus affecting circulating lipoproteins.

Changes in endogenous cytokines, adhesion molecules and platelets during cytokine-induced tumour necrosis

The aim of this study was to investigate mechanisms of anti-tumour activity and necrosis induced by combinations of tumour necrosis factor alpha (TNF-alpha) and interferon gamma (IFN-gamma). In a breast cancer xenograft model, locally injected recombinant human TNF-alpha arrested growth of established tumours in the absence of overt necrosis. Macroscopic necrosis occurred when rat IFN-gamma, which had no anti-tumour activity as a single agent, was given systemically. Treatment with TNF-alpha and IFN-gamma caused focal engorgement of tumour capillaries with erythrocytes, intravascular recruitment of polymorphonuclear cells and platelet adherence to the tumour vascular endothelium 4 h after the combined treatment. This was followed by destruction of tumour vascular endothelium and both necrosis and apoptosis of tumour cells. Concomitant with these changes, semiquantitative reverse transcriptase-polymerase chain reaction (RT-PCR) analysis revealed the increase of stromal (murine) mRNA levels for TNF-alpha, TNF receptor 55 kDa, TNF receptor 75 kDa, intracellular adhesion molecule 1, vascular cell adhesion molecule 1, P-selectin and interleukin 6 (IL-6). Thus, the effect of the combined TNF-alpha and IFN-gamma therapy involved the selective destruction of the tumour vasculature, death of tumour cells and increased expression of a series of stromal cytokines, cytokine receptors and adhesion molecules, which could be implicated in the observed events.

Low Temperatures and Hypertonicity Do Not Block Cytokine-induced Stimulation of the Sphingomyelin Pathway but Inhibit Nuclear Factor-κB Activation

In order to better understand the significance of tumor necrosis factor-alpha (TNF-alpha) and interleukin-1 beta (IL-1 beta)-receptor internalization in the sphingomyelin pathway signal transduction, we investigated receptor signaling under conditions in which receptor internalization is blocked. We demonstrate that human recombinant TNF-alpha and IL-1 beta both induced sphingomyelin and phosphatidylcholine hydrolysis at either 4, 14, or 37 degrees C in human skin fibroblasts and U937 monocytic cells. Cytokine-induced sphingomyelin degradation also occurred when endocytosis was inhibited by incubating the cells in hypertonic medium. While internalization was not required for the production of ceramide, activation of the transcription factor NF-kappa B was strongly reduced when cells were stimulated with TNF at low temperature or in hypertonic medium. Under these conditions, activation of NF-kappa B by the cell-permeant C2- ceramide (N-acetylsphingosine), by exogenous sphingomyelinase or by phorbol myristate acetate was also inhibited. These results suggest that low temperature and hypertonicity, two inhibitors of receptor internalization: (i) do not affect the TNF-alpha- or IL-1 beta-induced sphingomyelin hydrolysis, but (ii) do inhibit a step distal to ceramide of the intracellular signaling pathway leading to NF-kappa B activation.

Stimulation of prostaglandin (PG) F2 alpha and PGE2 release by tumour necrosis factor-alpha and interleukin-1 alpha in cultured human luteal phase endometrial cells.

Tumour necrosis factor-alpha (TNF-alpha) and interleukin-1 alpha (IL-1 alpha) are important mediators of cell signalling in the uterus. Prostaglandins (PG) have been implicated in the increase of endometrial vascular permeability which occurs during the implantation process. This study evaluates the effect of these two pleiotropic cytokines on PGF2 alpha and PGE2 release from human luteal phase endometrial glandular epithelial cells (GEC) and stromal cells (STC) in culture. Basal PGF and PGE release did not differ significantly from each other or among cell types, and declined significantly with increasing number of days in culture. On day 3, basal PG release had decreased to half of that on day 1 of culture. However, both cell types were still able to respond to the addition of exogenous arachidonic acid (5 microM) on day 3 of culture, with PG release by GEC being elevated 7- to 10-fold and by STC moderately, but still significantly, on day 4. The permissive effect of arachidonic acid on the stimulation of PG release may indicate the down-regulation of phospholipase A2 with continued time in culture. However, the addition of arachidonic acid (5 microM) on day 0 of culture, while able to cause significantly increased PG release from GEC, had no effect on STC. In contrast, the addition of a combination of arachidonic acid (5 microM), and either recombinant human TNF-alpha (10 micrograms rhTNF-alpha/l) or 10 micrograms rhIL-1 alpha/l, had a synergistic action and caused the significantly increased release of PGF and PGE from both cell types, compared with that achieved with either arachidonic acid or the cytokine alone (although GEC responded more than STC). During the first 24 h after the addition of rhTNF-alpha or rhIL-1 alpha, both cytokines stimulated PG release from both cell types in a dose- and time-dependent fashion. Neither cycloheximide (10 microM) nor actinomycin D (10 microM) affected basal PG release, but both blocked cytokine-induced PG release from both cell types. These results suggest that there is a differential control of human endometrial cell PG biosynthesis, and that PG release may be regulated through gene activation.

Hemoglobin interference with an enzyme-linked immunosorbent assay for the detection of tumor necrosis factor-alpha

The purpose of this study was to evaluate the effect of hemoglobin (Hb) on an enzyme-linked immunosorbent assay (ELISA) for the detection of tumor necrosis factor-alpha (TNF-alpha). Detection of TNF-alpha was performed by using the commercially available ELISA systems Factor-Test ttm hTNF-alpha and Predicta tm Tumor Necrosis Factor-alpha Kit (Genzyme, Cambridge, MA) in buffered samples containing 0.00 pg ml −1 or 200 pg ml −1 of recombinant human TNF-alpha, spiked with human Hb. The results suggest that Hb interferes with the detection of TNF-alpha by ELISA. A more pronounced effect was observed with the Factor-Test tm hTNF-alpha system. The observed effects were directly proportional to the concentration of Hb ranging from 1 to 20 mg ml −1. It appeared that in freshly spiked samples, Hb cross-reacted with monoclonal anti-TNF-alpha antibodies. When such an interaction occurred, Hb underwent a chemical reaction with hydrogen peroxide to yield a potent oxidant, capable of oxidizing the assay's substrates o-phenylenediamine dihydrochloride or 3,3',5,5'-tetramethylbenzidine dihydrochloride hydrate. The fact that Hb might interact with other proteins and possesses catalytic, peroxidase-like activity, suggests the possibility that this molecule may mimic the action of horseradish peroxidase in peroxidase-based ELISA systems and produce false positive results. It was also found that incubation of the TNF samples with Hb outside the ELISA system may have caused its instability. Non-denaturing size-exclusion chromatography revealed that incubation of recombinant human TNF-alpha with Hb caused its partial dissociation and consequent formation of immunologically less reactive TNF monomers possibly producing false negative results.

Exposure of tumor necrosis factor-alpha to luminal membrane of bovine brain capillary endothelial cells cocultured with astrocytes induces a delayed increase of permeability and cytoplasmic stress fiber formation of actin.

Tumor necrosis factor-alpha (TNF-alpha), a proinflammatory cytokine, has long been known to be involved in the pathogenesis of central nervous system infections and of certain neurodegenerative diseases. However, the possible role of the blood-brain barrier (BBB), the active interface between the blood circulation and brain tissue, remained unknown during these pathological conditions. In our in vitro reconstructed BBB model, 1-hr exposure of recombinant human TNF-alpha (in concentrations of 50, 250, and 500 U/ml, respectively) to the luminal membrane of bovine brain capillary endothelial cells (BBCEC) did not change significantly the transendothelial flux of either sucrose (m.w. 342 Da), or inulin (m.w. 5 kDa) up to 4 hr (early phase), except for a slight decrease (P < 0.05) in sucrose permeation at 2-4 hr with the highest dose of TNF-alpha. On the other hand, at 16 hr after the 1-hr challenge with TNF-alpha (delayed phase) at all 3 concentrations, significant increase was induced in the permeability of BBCEC monolayers for both markers. These changes of permeability were accompanied by a selective reorganization of F-actin filaments into stress fibers, while the intracellular distribution of vimentin remained similar to the control. These results suggest that BBCEC can respond directly to TNF-alpha by a delayed increase of permeability and reorganization of actin filaments.

TNF-R55-Specific Form of Human Tumor Necrosis Factor-α Induces Collagenase Gene Expression By Human Skin Fibroblasts

Tumor necrosis factor-alpha (TNF-alpha) is a potent inhibitor of connective tissue formation. The cellular effects of TNF-alpha are mediated by two distinct cell-surface receptors, TNF-R55 and TNF-R75, both present on various types of cells, including fibroblasts. In this study we wanted to elucidate the role of TNF-R55 as a mediator of the connective tissue effects of TNF-alpha by using a mutant, TNF-R55-specific form of human TNF-alpha. This mutant TNF-alpha markedly induced collagenase and stromelysin-1 gene expression in dermal fibroblasts, the maximal activation (up to 42-fold) being 65%-89% of that noted with wild-type human TNF-alpha. In addition, TNF-R55-specific TNF-alpha suppressed type I collagen mRNA levels as potently as wild-type TNF-alpha (by 60%). The enhancement of collagenase gene expression by TNF-R55-specific TNF-alpha was augmented by simultaneous treatment of normal and scleroderma skin fibroblasts with interferon-gamma, indicating specific enhancement of TNF-R55 signaling pathway by interferon-gamma. These results show that stimulation of the TNF-R55 signaling pathway is sufficient for the inhibitory effects of TNF-alpha on extracellular matrix formation by dermal fibroblasts. It is conceivable that due to reduced systemic toxicity, TNF-R55-specific forms of human TNF-alpha may prove to be feasible in the therapy of fibrotic disorders.