Human TNF-alpha Research Articles

A novel anti-inflammatory drug, SDZ ASM 981, for the treatment of skin diseases: in vitro pharmacology.

SDZ ASM 981, a novel ascomycin macrolactam derivative, has high anti-inflammatory activity in animal models of allergic contact dermatitis and shows clinical efficacy in atopic dermatitis, allergic contact dermatitis and psoriasis, after topical application. Here we report on the in vitro activities of this promising new drug. SDZ ASM 981 inhibits the proliferation of human T cells after antigen-specific or non-specific stimulation. It downregulates the production of Th1 [interleukin (IL)-2, interferon-gamma] and Th2 (IL-4, IL-10) type cytokines after antigen-specific stimulation of a human T-helper cell clone isolated from the skin of an atopic dermatitis patient. SDZ ASM 981 inhibits the phorbol myristate acetate/phytohaemagglutinin-stimulated transcription of a reporter gene coupled to the human IL-2 promoter in the human T-cell line Jurkat and the IgE/antigen-mediated transcription of a reporter gene coupled to the human tumour necrosis factor (TNF)-alpha promoter in the murine mast-cell line CPII. It does not, however, affect the human TNF-alpha promoter controlled transcription of a reporter gene in a murine dendritic cell line (DC18 RGA) after stimulation via the FcgammaRIII receptor. SDZ ASM 981 also prevents the release of preformed pro-inflammatory mediators from mast cells, as shown in the murine cell line CPII after stimulation with IgE/antigen. In summary, these results demonstrate that SDZ ASM 981 is a specific inhibitor of the production of pro-inflammatory cytokines from T cells and mast cells in vitro.

Effects of hTNFalpha expression in T cells on haematopoiesis in transgenic mice.

A transgenic line of mice carrying one copy of the hTNFalpha gene under the control of its own promoter and the CD2 locus control region has been analysed for the effects of TNFalpha on haematopoiesis. A low level constitutive expression of hTNFalpha in lymphoid tissue was observed. Human TNFalpha binds to and activates the murine p55 receptor, but not the p75 receptor. This implies that the observed effects of hTNFalpha in mice were mediated only through the p55 receptor. Various lymphoid tissues were depleted of lymphocytes, especially thymus, spleen and peripheral blood. Effects on thymus development were detected already at 3 wk of age, more general effects on haematopoiesis were evident by 5 wk: a drop in total blood leukocytes, mainly due to a 67% decline in lymphocytes. At 16 wk the mice had developed anaemia, whereas platelets, neutrophils and monocytes had increased. The fall in lymphocytes was due to lowered levels of T cells as well as B cells. The cause of the shortened lifespan of the transgenic mice was probably not the haematological effects of hTNFalpha directly. Absence of trophic factors supplied by the normal T cell population remains possible.

A novel lipopolysaccharide-induced transcription factor regulating tumor necrosis factor alpha gene expression: molecular cloning, sequencing, characterization, and chromosomal assignment.

Lipopolysaccharide (LPS) is a potent stimulator of monocytes and macrophages, causing secretion of tumor necrosis factor alpha (TNF-alpha) and other inflammatory mediators. Given the deleterious effects to the host of TNF-alpha, it has been postulated that TNF-alpha gene expression must be tightly regulated. The nature of the nuclear factor(s) that control TNF-alpha gene transcription in humans remains obscure, although NF-kappaB has been suggested. Our previous studies pertaining to macrophage response to LPS identified a novel DNA-binding domain located from -550 to -487 in the human TNF-alpha promoter that contains transcriptional activity, but lacks any known NF-kappaB-binding sites. We have used this DNA fragment to isolate and purify a 60-kDa protein binding to this fragment and obtained its amino-terminal sequence, which was used to design degenerate probes to screen a cDNA library from THP-1 cells. A novel cDNA clone (1.8 kb) was isolated and fully sequenced. Characterization of this cDNA clone revealed that its induction was dependent on LPS activation of THP-1 cells; hence, the name LPS-induced TNF-alpha factor (LITAF). Inhibition of LITAF mRNA expression in THP-1 cells resulted in a reduction of TNF-alpha transcripts. In addition, high level of expression of LITAF mRNA was observed predominantly in the placenta, peripheral blood leukocytes, lymph nodes, and the spleen. Finally, chromosomal localization using fluorescence in situ hybridization revealed that LITAF mapped to chromosome 16p12-16p13.3. Together, these findings suggest that LITAF plays an important role in the activation of the human TNF-alpha gene and proposes a new mechanism to control TNF-alpha gene expression.

Gonococcal infection of human fallopian tube mucosa in organ culture: relationship of mucosal tissue TNF-alpha concentration to sloughing of ciliated cells.

An experimental model consisting of gonococcal infection of human fallopian tube mucosa in organ culture has proven useful in studying the molecular pathogenesis of acute gonococcal salpingitis and postsalpingitis sequelae. Gonococcal infection of human fallopian tube mucosa in organ culture results in the sloughing of ciliated epithelial cells from the mucosa. This damage to the mucosa can be quantified on fallopian tube pieces by an assay of the percent of the periphery that has ciliary activity (PPCA) remaining at specific time points after infection. Although assay of the PPCA has been quite valuable, it is labor-intensive, somewhat subjective, and requires that the observers have training and experience. A more practical assay for genital mucosal damage is desirable for further investigations that employ the fallopian tube experimental model. Gonococcal infection of fallopian tube mucosa in organ culture also results in the production of easily quantified tumor necrosis factor-alpha (TNF-alpha) by the mucosa. Furthermore, treatment of the organ cultures with recombinant human TNF-alpha (rHuTNFalpha) alone also causes sloughing of ciliated cells from the mucosa. These findings strongly suggest that TNF-alpha is a mediator of the mucosal damage that attends gonococcal infection. To determine: (1) whether the PPCA values and the TNF-alpha concentrations in fallopian tube mucosal tissues correlate closely enough to allow prediction of the PPCA from a measurement of the mucosal tissue TNF-alpha concentration; and (2) whether the correlation of the TNF-alpha mucosal tissue concentration with the sloughing of ciliated cells (measured by the PPCA) supports the hypothesis that induction of TNF-alpha by gonococcal infection, with resultant sloughing of ciliated cells, is likely to be a major pathogenic mechanism of gonococcal salpingitis and might mediate postsalpingitis infertility and ectopic pregnancy. A metaanalysis was performed on studies from three research groups (two laboratories in the United States and one in the United Kingdom, using identical techniques for quantifying the PPCA, TNF-alpha, or both. There was a close and statistically significant correlation between the TNF-alpha mucosal tissue concentration and the proportion of ciliated cells lost from the mucosa as measured by the PPCA (r = 0.95, p < 0.001). Therefore, as the mucosal tissue concentration of endogenous TNF-alpha increased, the loss of ciliated cells from the epithelium increased proportionately. During gonococcal infection of human fallopian tube mucosa in organ culture, the mucosal tissue concentration of TNF-alpha can be used to predict the PPCA, and therefore, the extent of mucosal damage. This finding should facilitate studies of the molecular pathogenesis of infectious diseases involving human genital mucosa. Further, the close correlation of mucosal TNF-alpha concentration with genital mucosal damage, evaluated by the PPCA, supports the hypothesis that induction of the proinflammatory cytokine, TNF-alpha, by gonococcal infection, with resultant inflammation and sloughing of ciliated cells, is an important pathogenic mechanism of gonococcal salpingitis and may mediate postsalpingitis infertility and ectopic pregnancy as well.

Physiological effects and adjuvanticity of recombinant brushtail possum TNF-alpha.

The present paper describes the physiological properties of recombinant possum TNF-alpha and an adjuvant effect on antibody responses to the model protein antigen, keyhole limpet haemocyanin (KLH). For these studies recombinant possum TNF-alpha was produced in the yeast Pichia pastoris. The recombinant cytokine was secreted into the culture medium and purified by gel filtration. Possum TNF-alpha produced in this expression system was N-glycosylated and bioactive in two different assays. In a murine fibroblast L929 cytotoxicity assay, the possum TNF-alpha had lower specific activity compared to human TNF-alpha, while in a possum-specific assay, possum TNF-alpha enhanced the proliferation of PHA-stimulated possum thymocytes and was more active than human TNF-alpha. The physiological effect of the recombinant possum TNF-alpha was investigated in groups of possums administered doses of 6, 30 or 150 micrograms of cytokine. For each dose, TNF-alpha caused profound effects on the numbers of circulating leucocytes characterized by a three-to-four-fold increase in neutrophil numbers at 6-24 h after injection and an initial sharp decrease in lymphocyte numbers. The efficacy of TNF-alpha as an immunological adjuvant was determined in possums administered KLH (125 micrograms) in an aqueous or Al(OH)3-based formulation with or without added recombinant TNF-alpha (150 micrograms). Serum antibody responses to KLH were monitored by ELISA. The TNF-alpha stimulated two-fold and four-fold increases in antibody levels in aqueous and Al(OH)3-based vaccine formulations, respectively. The strongest antibody responses were observed in the group of possums that received KLH formulated in Al(OH)3 with addition of TNF-alpha.

Western blot analysis of bile or intestinal fluid from patients with septic shock or systemic inflammatory response syndrome, using antibodies to TNF‐α, IL‐1α and IL‐1β

Septic shock or systemic inflammatory response syndrome (SIRS) often develops in patients following burns, traumatic injury, surgery or biliary obstruction. Although the inflammatory cytokines TNF-alpha and IL-1 have been strongly implicated in the development of these syndromes, treatment of patients by the systemic administration of inhibitors of TNF-alpha or IL-1 has shown limited effectiveness. Recent reports suggest that septic shock may be perpetuated by inflammatory cytokines secreted by the liver in response to bacterial translocation resulting from cytokine-induced gastrointestinal damage. The present study sought to demonstrate the presence of high levels of inflammatory cytokines in the bile or small intestine of patients suffering from septic shock or SIRS, with a view to the development of strategies for the reduction of gastrointestinal damage through intraduodenal administration of cytokine inhibitors. Western blot analysis of human bile or intestinal fluid using anti-TNF-alpha antibodies resulted in the detection of a number of bands in samples from patients with septic shock or SIRS. However, these proteins differed antigenically from human recombinant TNF-alpha (rTNF-alpha) and showed no activity in a biological assay for TNF-alpha. Antibodies to IL-1 alpha and IL-1 beta detected several strong bands, some of which appeared to be identical to recombinant IL-1 alpha and IL-1 beta. It is concluded that proteins resembling several known inflammatory cytokines are present in the bile and intestine of septic shock patients, but it is suggested that further work is required to determine the nature and function of these molecules.

Construction of shuttle, expression vector of human tumor necrosis factor alpha (hTNF-α) gene and its expression in a cyanobacterium,Anabaena sp. PCC 7120

The construction of the shuttle, expression vector of human tumor necrosis factor alpha (hTNF-alpha) gene and its expression in a cyanobacterium Anabaena sp. PCC 7120 was reported. The 700-bp hTNF cDNA fragments have been recovered from plasmid pRL-rhTNF, then inserted downstream of the promoter PpsbA in the plasmid pRL439. The resultant intermediary plasmid pRL-TC has further been combined with the shuttle vector pDC-8 to get the shuttle, expression vector pDGTNF. The expression of the rhTNF gene in Escherichia coli has been analyzed by SDS-PAGE and thin-layer scanning, and the results show that the expressed TNF protein with these two vectors is 16.9 percent (pRL-TC) and 15.0 percent (pDC-TNF) of the total proteins in the cells, respectively, while the expression level of TNF gene in plasmid pRL-rhTNF is only 11.8 percent. Combined with the participation of the conjugal and helper plasmids, pDC-TNF has been introduced into Anabaena sp PCC 7120 by triparental conjugative transfer, and the stable transgenic strains have been obtained. The existence of the introduced plasmid pDC-TNF in recombinant cyanobacterial cells has been demonstrated by the results of the agarose electrophoresis with the extracted plasmid samples and Southern blotting with alpha-(32)P labeled hTNF cDNA probes, while the expression of the hTNF gene in Anabaena sp. PCC 7120 has been confirmed by the results of Western blotting with extracted protein samples and human TNF-alpha monoclonal antibodies. The cytotoxicity assays using the mouse cancer cell line L929 proved the cytotoxicity of the TNF in the crude extracts from the transgenic cyanobacterium Anabaena sp. PCC 7120.

Effects of Cerebrospinal Fluid From Patients With Parkinson Disease on Dopaminergic Cells

The pathogenesis of substantia nigra pars compacta neuronal injury in Parkinson disease (PD) remains unknown. Cerebrospinal fluid (CSF) has been reported to contain factors toxic to dopaminergic neurons. To determine whether the cytotoxic effects of CSF of PD patients are specific for dopaminergic neurons, dependent on prior levodopa therapy, and mediated by the cytokine tumor necrosis factor alpha (TNF-alpha). Specimens of CSF were evaluated in dopaminergic (MES 23.5) and nondopaminergic (N18TG2) cell lines for cytotoxicity by viability assay and by the inhibition of tyrosine hydroxylase. After specificity and time and dose response were established, CSF specimens were assayed in a blinded manner. The TNF-alpha levels in CSF were determined by enzyme-linked immunosorbent assay. The toxicity of TNF-alpha in MES 23.5 cells was determined. A university-based research facility. There were 4 groups of subjects: normal control subjects (n = 10), control subjects with neurologic disease (n = 8), PD patients treated with levodopa (n = 10), and untreated subjects with PD (n= 20). Specimens of CSF from 15 (50%) of 30 PD patients and 2 (11%) of 18 control subjects were cytotoxic to dopaminergic MES 23.5 cells and were nontoxic to the parental cell line N18TG2. There was no correlation between the degree of PD CSF cytotoxicity, levodopa therapy, or the severity and duration of PD. Terminal deoxynucleotidyl transferase-mediated biotin-deoxyuridine triphosphate nick-end labeling (TUNEL) for DNA fragmentation suggested the involvement of apoptotic mechanisms. The inhibition of tyrosine hydroxylase was an early effect of cell injury by PD CSF and correlated with the viability assay. The mean TNF-alpha level was 2.6-fold higher in CSF specimens from PD patients than in those of controls. The addition of recombinant human TNF-alpha equivalent to the highest level determined in PD CSF was not cytotoxic to MES 23.5 cultures. Blinded CSF specimens from PD patients, regardless of therapy, contain factors that cause specific dopaminergic neuronal cell injury. These factors are present in a substantial proportion of CSF specimens from patients with early PD, before the institution of medical therapy. Levels of TNF-alpha are elevated in the CSF of PD patients, but TNF-alpha is not responsible for the cytotoxicity.

Enhancing the immunotherapeutic potential of mycobacteria by transfection with tumour necrosis factor-alpha.

In an attempt to enhance the anti-tumour properties of mycobacteria we have developed recombinant forms of Mycobacterium smegmatis which express and secrete biologically active human tumour necrosis factor-alpha (TNF-alpha). This was achieved by transfecting M. smegmatis using shuttle plasmids incorporating the cDNA sequence for the human TNF-alpha mature peptide. In vitro experiments on a panel of human bladder tumour cell lines (EJ18, MGH-U1, RT4, RT112) indicate that our genetically modified mycobacteria are more effective than wild-type at inducing or up-regulating the expression of intracellular adhesion molecule-1 and the secretion of an array of proinflammatory cytokines [interleukin-1 (IL-1), IL-6, IL-8, granulocyte-macrophage colony-stimulating factor]. We have also demonstrated increased adhesion molecule and cytokine expression in response to mycobacteria transfected with vector containing no gene insert. However, this was not as pronounced as that observed following tumour cell stimulation by the TNF-alpha-transfected strain. In contrast, in three out of four tumour cell lines all M. smegmatis strains were found to down-regulate the secretion of the anti-inflammatory cytokine transforming growth factor-beta1. Our studies have also confirmed that M. smegmatis is a powerful inhibitor of bladder tumour cell growth and revealed that its antiproliferative potency is enhanced by transfecting with human TNF-alpha and, to a lesser extent, with vector alone. All M. smegmatis strains were effective in the activation of peripheral blood leucocyte cultures. However, no differences were observed in the ability of the TNF-alpha-transfected, mock-transfected and wild-type mycobacteria to induce tumour cell killing activity. These results suggest that the immunomodulatory effects of M. smegmatis can be enhanced by transfection with vectors which allow the secretion of human TNF-alpha, thus increasing mycobacterial immunotherapeutic potential.

Detection of macrophage inflammatory protein (MIP)-1alpha and MIP-1beta during experimental endotoxemia and human sepsis.

Macrophage inflammatory protein (MIP)-1alpha and MIP-1beta regulate leukocyte activation and trafficking. To assess the role of MIP-1alpha and MIP-1beta in human inflammation, healthy subjects were studied during experimental endotoxemia with prior administration of ibuprofen, a cyclooxygenase inhibitor, or dimeric p75 tumor necrosis factor (TNF)-alpha receptor, a TNF antagonist; septic patients were also studied. Following endotoxin, blood levels of both MIP-1 molecules rose acutely and fell to baseline by 6 h (P=. 001). While MIP-1 mediates fever in animals independent of cyclooxygenase blockade, in subjects given endotoxin and ibuprofen, MIP-1 levels increased and fever was suppressed. MIP-1 levels were not diminished by inhibiting circulating TNF-alpha in humans. In septic patients, elevated levels of MIP-1alpha and MIP-1beta were detected within 24 h of sepsis and fell in parallel with TNF-alpha and interleukin-6 (P<.01). MIP-1alpha and MIP-1beta increase during acute inflammation but are not associated with fever in endotoxemic humans during cyclooxygenase blockade.

Acute effect of tumor necrosis factor-alpha is minimal on mechanics but significant on energetics in blood-perfused canine left ventricles.

We hypothesized that tumor necrosis factor-alpha (TNF-alpha) acutely alters left ventricular mechanoenergetics in blood-perfused hearts. To test this hypothesis, we examined the relation between left ventricular mechanics and energetics, both before and after infusion of TNF-alpha. Prospective, experimental study. Research laboratory. Nine isolated, blood-perfused canine hearts. Recombinant human TNF-alpha (90 microg/min) was infused into the coronary circulation of the isolated hearts for 20 mins. In the isolated, cross-circulated, blood-perfused canine left ventricles, left ventricular contractility was assessed through measurement of end-systolic elastance (Ees). Energetics were examined in terms of the end-systolic pressure-volume area-myocardial oxygen consumption (MVo2) relation. TNF-alpha concentration in coronary venous blood was >1000 ng/mL throughout the experiments. Nevertheless, infusion of TNF-alpha barely affected contractility acutely, i.e., there was a minimal decrease during the infusion (8.1+/-2.8% at 10 mins, p < .01) and a minimal increase after the infusion (11.2+/-2.5% at 10 mins, p< .01). Neither did the TNF-alpha infusion affect the slope of the end-systolic pressure-volume area-MVo2 relation. This finding indicated that the chemomechanical conversion efficiency remained unchanged. However, TNF-alpha infusion significantly increased the oxygen cost of contractility by 40% (1.25+/-0.13 vs. 1.75+/-0.24 mL oxygen.mL/mm Hg/beat, p< .05), indicating that MVo2 for the excitation-contraction coupling increased. TNF-alpha minimally alters left ventricular mechanics, but significantly changes energetics. The latter effect may result from changes in intracellular calcium handling.

Effects of inflammatory cytokines induced by Helicobacter pylori infection on aminopyrine accumulation in parietal cells isolated from guinea pigs.

The effects of inflammatory cytokines induced by Helicobacter pylori infection on acid secretion have not been well defined. The purpose of this study was to investigate the direct effects of these cytokines on parietal cells isolated from guinea pigs. We examined the effects of human recombinant IL-1beta (0.05-100 ng/ml), IL-8 (2-256 ng/ml), and TNF-alpha (0.625-80 ng/ml) on acid secretion stimulated by three secretagogues (10(-4) M histamine, 10(-4) M carbachol, and 10(-5) M tetragastrin) and on basal acid secretion from isolated parietal cells, which was measured by the aminopyrine accumulation method. None of three cytokines showed any significant effects on stimulated or basal acid secretion from isolated guinea pig parietal cells. We concluded that inflammatory cytokines induced by Helicobacter pylori infection may affect acid secretion through mechanisms other than direct actions on parietal cells.

In vitro C3mRNA Expression in Pemphigus Vulgaris: Complement Activation is Increased by IL-1α and TNF-α

Pemphigus vulgaris (PV) is a potentially life-threatening disease, characterized immunohistologically by IgG deposits and complement activation on the surface of keratinocytes. Complement activation has been implicated in the pathogenesis with C3 deposits in about 90% of patients. In order to further elucidate the role of complement in PV and to define which cytokines play a role in C3 mRNA expression, we performed an in vitro study in human keratinocytes. Normal human epidermal keratinocytes (NHuK) were incubated with PV serum and C3 mRNA was measured. We previously had shown that IL-1alpha and TNF-alpha are expressed in PV in vivo and in vitro. Since cytokines are able to modulate complement activation, mRNA expression was evaluated in a similar experiment after pretreatment using antibodies against IL-1alpha and TNF-alpha. Incubation of NHuK with PV sera caused their detachment from the plates after 20-30 minutes with a complete acantholysis within 12 hours. An early C3 mRNA expression was seen after 30 minutes with a peak level after 1 hour. Blocking studies, using antibodies against human IL-1alpha and TNF-alpha in NHuK together with PV-IgG, showed reduction of in vitro induced acantholysis and inhibition of C3 mRNA expression. This study supports the hypothesis that complement C3 is important in PV acantholysis and that complement activation is increased by IL-1alpha and TNF-alpha.

Induction of rat uncoupling protein-2 gene treated with tumour necrosis factor alpha in vivo.

The cytokine tumour necrosis factor (TNF) alpha has been reported to induce metabolic abnormalities such as anorexia and thermogenesis. To investigate functional modulators of uncoupling protein-2 (UCP2) gene expression, we examined the effects of TNF-alpha on UCP2 mRNA expression in rats. Mature male Wistar King A (WKA) rats at 10-11 weeks of age were treated with recombinant human TNF-alpha at a dose of 0.6 nmol 100 g-1 body weight by intraperitoneal administration. TNF-alpha treatment induced an increase in UCP2 mRNA expression in broadly distributed tissues including brown adipose tissue (BAT), white adipose tissue (WAT) and skeletal muscle, and an elevation of ob gene mRNA expression in WAT. After the TNF-alpha treatment, an increase in plasma leptin concentration occurred in an ob gene-dependent manner, accompanied by an anorectic effect. The present results provide evidence for a new regulatory loop involving TNF-alpha and UCP2, and add novel insights into the regulatory mechanisms of energy homeostasis.

INTERLEUKIN-10 STABILIZES INHIBITORY kB-α IN HUMAN MONOCYTES

Interleukin-10 (IL-10) protects animals from lethal endotoxemia. This beneficial effect is mediated, in part, by inhibition of inflammatory cytokine production, including tumor necrosis factor-alpha (TNF-alpha). Evidence suggests that IL-10 may inhibit activation of the transcription factor nuclear factor-kappaB (NF-kappaB) through an unknown mechanism. NF-kappaB activation in response to inflammatory signals is dependent upon degradation of its associated inhibitory peptide, inhibitory kappaB-alpha (IkappaB-alpha). We hypothesized that IL-10 prevents human monocyte NF-kappaB activation and resultant TNF-alpha production by stabilization of IkappaB-alpha. The purpose of this study was to determine the effect of IL-10 on lipopolysaccharide (LPS)-induced human monocyte TNF-alpha production, NF-kappaB activation, and IkappaB-alpha degradation. Monocytes were isolated from human donors. Cells were stimulated with endotoxin (LPS, 100 ng/mL) with and without human IL-10 (10 ng/mL). Following stimulation, TNF-alpha was measured in cell supernatants by ELISA, NF-kappaB activity by electrophoretic mobility shift assay, and IkappaB-alpha levels by Western blot. We observed that after LPS stimulation of human monocytes, TNF-alpha increased to 798+/-67 pg/mL (p < .001 versus control). IL-10 attenuated LPS-stimulated TNF-alpha production (297+/-54; p < .001 versus LPS alone). After LPS stimulation in human monocytes, IkappaB-alpha protein levels decreased, and NF-kappaB DNA binding increased. IL-10 pretreatment prevented LPS-induced decreases in IkappaB-alpha protein levels and attenuated NF-kappaB DNA binding. IL-10 appears to prevent activation of NF-kappaB by preserving IkappaB-alpha protein levels, leading to a reduction in TNF-alpha release.

Activation of EGF receptor family members suppresses the cytotoxic effects of tumor necrosis factor-alpha.

Tumor necrosis factor (TNF)-alpha has a broad range of biological activities, which depend heavily on cell type and physiological condition. In a panel of human tumor cell lines we analyzed expression of the receptor tyrosine kinases EGFR, ErbB2 and ErbB3, and the response to TNF-alpha. Among the cell lines tested those resistant to TNF-alpha were found to express high levels of either EGFR, or ErbB2 and ErbB3. In TNF-sensitive breast and cervical carcinoma cells activation of EGFR or ErbB2 by the exogenous growth factors EGF and heregulin beta1 resulted in a significant increase in the number of cells surviving TNF-alpha treatment. In contrast, inhibition of EGFR activation in TNF-resistant breast carcinoma cells by the novel antagonistic anti-EGFR antibody 14E1 sensitized the cells to the cytotoxic effects of TNF-alpha. A bacterially expressed fusion protein consisting of a 14E1 single-chain (sc) Fv antibody fragment linked to human TNF-alpha retained TNF-alpha activity. This scFv(14E1)-TNF-alpha molecule localized specifically to EGFR on the surface of tumor cells and activated the NF-kappaB pathway in co-cultured T cells, as demonstrated by electrophoretic mobility shift assays.

Growth factors in cultured pterygium fibroblasts: immunohistochemical and ELISA analysis.

In order to study growth factors in the pathogenesis and recurrence of pterygium, we grew pterygium tissues in culture and compared fibroblasts from primary and from recurrent pterygia with reference to the fibroangiogenic growth factors basic fibroblast growth factor (b-FGF), platelet-derived growth factor (PDGF), transforming growth factor beta (TGF-beta) and tumor necrosis factor alpha (TNF-alpha). We used indirect immunohistochemical procedures against human b-FGF, PDGF, TGF-beta and TNF-alpha. As controls, we used cultured normal human conjunctival fibroblasts. A serum-free conditioned medium (CM) from confluent fibroblasts derived from primary and recurrent explants was assessed by enzyme-linked immunosorbent assay to determine the level of the above-mentioned growth factors. Immunoreactivity of b-FGF was stronger in recurrent than in primary pterygium fibroblasts. PDGF immunolabeling was stronger in primary than in recurrent pterygium fibroblasts. TGF-beta and TNF-alpha immunolabeling was weak in both pterygia. All these growth factors were very sparse in normal conjunctival fibroblasts. Basic-FGF and TGF-beta 1 were found in the CM from both primary and recurrent pterygium, while PDGF and TNF-alpha were not detectable. The strong immunoreactivity and the release of b-FGF in cultured fibroblasts of recurrent pterygia suggest that fibroblasts may play an important role in the recurrence of pterygium.

Cooperative Signaling by Tumor Necrosis Factor Receptors CD120a (p55) and CD120b (p75) in the Expression of Nitric Oxide and Inducible Nitric Oxide Synthase by Mouse Macrophages

Tumor necrosis factor-alpha (TNFalpha) is recognized by the cell-surface receptors CD120a (p55) and CD120b (p75). In the present study, we have investigated the role of these receptors in the expression of NO2-, a stable metabolite of nitric oxide, and inducible nitric oxide synthase (iNOS) by mouse macrophages. Specific antibody-mediated aggregation of CD120a (p55) induced NO2- accumulation in culture supernatants and iNOS mRNA expression in macrophage lysates, whereas cross-linking of CD120b (p75) had a minimal effect. In contrast, simultaneous cross-linking of both receptors led to a marked augmentation in NO2- and iNOS mRNA expression. Antibody-mediated blockade of CD120a (p55) completely inhibited NO2- expression in response to TNFalpha, whereas blockade of CD120b (p75) reduced NO2- accumulation by approximately 50%. Specific ligation of CD120a (p55) with either (i) human TNFalpha or (ii) by incubation with mouse TNFalpha following pretreatment of macrophages with blocking concentrations of anti-CD120b (p75) antibody resulted in a similar reduction in NO2- production in response to TNFalpha. Quantification of iNOS mRNA, protein, and NO2- expression during independent and co-ligation of CD120a (p55) and CD120b (p75) indicated that iNOS mRNA and protein expression was transient in nature when CD120a (p55) was cross-linked alone but was prolonged when both receptors were simultaneously cross-linked. In addition, cross-linking both receptors also led to a potentiation of NO2- accumulation in culture supernatants that was more pronounced at later time points. These findings suggest that while cross-linking of CD120a (p55) is necessary and sufficient for iNOS mRNA and NO2- expression, CD120b (p75) participates by (i) increasing the sensitivity of the cells to TNFalpha, probably by "passing" ligand to CD120a (p55), and (ii) initiating a signaling event that results in a more sustained induction of iNOS mRNA and protein and thereby augments the production of nitric oxide.

Tumour necrosis factor-alpha induces hyperreactivity in tracheal smooth muscle of the guinea-pig in vitro.

Recent studies have implicated a role for tumour necrosis factor-alpha (TNFalpha) in the development of the asthmatic reaction. In this study, we examined the influence of TNFalpha on isotonic contraction of tracheal smooth muscle of the guinea-pig in vitro in response to methacholine. Tracheal rings were incubated with recombinant human (rh)TNFalpha (3x10(-11) M) for 30 min, and concentration-response curves to methacholine before and after incubation with rhTNFalpha were compared with the control. The present study demonstrates that rhTNFalpha increases maximal isotonic contraction of tracheal smooth muscle to methacholine (mean+/-SEM 169.6+/-43%, p<0.005). This effect was observed only after a 30 min delay between incubation and methacholine challenge testing. Experiments with 10(-13) - 10(-10) M rhTNF-alpha yielded similar results at all concentrations used. The effects of rhTNFalpha (10(-11) M) on tracheal hyperreactivity could be completely inhibited by coincubation with dimeric rTNF-receptor-p80 construct (10(-10) M) (p<0.01). In order to analyse secondary mediator release, experiments using coincubation with indomethacin (10(-5) M) and WEB 2086 (10(-6) M), a specific platelet activating factor (PAF) antagonist, demonstrated that the effect of rhTNFalpha on tracheal rings was mediated by PAF, since WEB 2086 completely inhibited rhTNFalpha-induced hyperreactivity (p<0.05). In conclusion, this study demonstrates that recombinant human tumour necrosis factor-alpha induces hyperreactivity in tracheal smooth muscle in vitro, which was shown to be mediated by platelet activating factor. Our study emphasizes the role of tumour necrosis factor-alpha in the pathophysiology of bronchial hyperresponsiveness.

A novel human tumor necrosis factor alfa mutein, F4614, inhibits in vitro and in vivo growth of murine and human hepatoma: implication for immunotherapy of human hepatocellular carcinoma.

Although treatment for hepatocellular carcinoma (HCC) has recently improved, most patients still relapse and die from this disease. The development of new therapeutic and preventive strategies for HCC is, therefore, required. A novel mutant protein (mutein) of human tumor necrosis factor alfa (TNF-alpha mutein F4614, 1SSSRGDSD... 29V ... 155L) was developed to decrease several adverse effects of TNF-alpha. F4614 is known to lack hypotensive effects of human TNF-alpha without losing its anti-tumor effect in mice transplanted with Meth-A sarcoma. Our study investigated the anti-tumor effects of F4614 against hepatoma cells in vitro and in vivo. F4614 significantly inhibited growth of all four tumor cells in vitro. A murine hepatoma cell line, MH134, when incubated in the presence of F4614, exhibited upregulation of surface major histocompatibility complex (MHC) class-I, intercellular adhesion molecule-1 (ICAM-1) and B7-1 molecules, and a decreased proportion of cells in the G2/M phase of the cell cycle. In addition, F4614 induced apoptosis in a significant number of MH134 cells. TNF-alpha and F4614 (5 microg/mouse daily for 5 days) showed similar anti-tumor activities in syngeneic MH134-bearing mice and heterogeneic PLC/PRF/5-bearing athymic nude mice. Intratumoral injection of F4614 or TNF-alpha was more effective than intravenous injection. Immunohistochemical analysis of the tumors treated by F4614 revealed that tumors were surrounded with a large number of Mac-1+ cells and a small number of CD4+ and CD8+ T cells; that suggests that intratumoral injection of F4614 elicited host immunoreactions. Thus, F4614 may be a new strategy for immunotherapy of HCC.