Effects of C5a and FMLP on Interleukin-8 Production and Proliferation of Human Umbilical Vein Endothelial Cells

Interleukin-8 (IL-8), C5a and N-formyl-methionyl-leucyl-phenylalanine (fMLP) are chemotactic peptides with predominant effects on leukocytes during inflammation. With emphasis on C5a we studied the regulation of the production of IL-8 by human umbilical vein endothelial cells (HUVEC) in vitro. Primary HUVEC cultures were incubated with IL-1, TNF, C5a and fMLP for 24 h and 48 h prior to measurement of IL-8 in supernatants of the cells by an enzyme immunoassay. Whereas IL-1 and TNF significantly increased the levels of IL-8, C5a decreased the IL-8 production after 48 h. In addition, the ability of IL-1, TNF, C5a, fMLP and IL-8 to induce cell proliferation was compared by means of a ³H-thymidine incorporation assay. In contrast with IL-1 and TNF, both C5a and fMLP increased cell proliferation of HUVEC. This increase occurred with increasing concentrations of C5a contrary to IL-8, which showed increased cell proliferation at low, but not high IL-8 concentrations.     

A comparative immunohistological study of cerebellar enolases

Summary A comparative immunohistological study of the neurone-specific enolase and enolase, demonstrates the exclusive neuronal localization of enolase and its absence from glial cells. In contrast, enolase is located in astroglial cells. The validity of enolase as a neuronal marker and enolase as an astrocytic marker, is confirmed both by a double labelling technique, using antibodies to and to revealed with fluorescence or peroxidase in the same tissue sections, and by immunoelectronmicroscopy.     

Monokine Production Following in Vitro Stimulation of the THP-1 Human Monocytic Cell Line with Pertussis Vaccine Components

Whole-cell pertussis found in diphtheria–tetanus–pertussis (DTP) vaccine can produce symptoms reminiscent of biological responses to circulating proinflammatory monokines such as IL-6, IL-1, and TNF. Therefore the ability of pertussis-containing vaccines and several heat-killed Bordetella pertussis preparations to stimulate cytokine production in a human monocytic cell line, THP-1, were examined. The whole-cell pertussis vaccine induced significantly more IL-6, IL-1, and TNF production than did the acellular pertussis or diphtheria–tetanus-only vaccine. Polymyxin B was able to inhibit most of the IL-6 induced by pertussis endotoxin and a heat-killed preparation of B. pertussis containing a null mutation in bvgAS, a regulatory locus required for expression of all known protein virulence factors synthesized by this organism. However, it only partially inhibited IL-6 production induced by other pertussis-containing preparations, including DTP vaccine. These results indicate that in vitro whole-cell vaccine is a potent stimulator of IL-6, IL-1, and TNF. They also suggest that although endotoxin is a major inducer of IL-6, other components of B. pertussis also contribute to IL-6 production by monocytes.     

Potential roles for tumour necrosis factorα during embryonic development

This paper reviews the evidence indicating possible roles for tumour necrosis factor-alpha (TNF) in development. It is proposed that TNF may have essentially three major roles during embryonic development, which may be analogous to its roles in the immune system and during inflammation: a role in programmed cell death; a role as a cellular growth and differentiation factor; and also a role in the remodelling of extracellular matrix, and the regulation of cell adhesion molecules and integrins. The concept of the existence of a cytokine array during embryogenesis, analogous to that occurring in inflammation, is discussed, as well as potential roles for TNF in the induction of ubiquitin; protective mechanisms embryonic cells may employ against TNF-mediated cytotoxicity; and a consideration of the role TNF may play in a free radical theory of development.     

Acute ethanol consumption synergizes with trauma to increase monocyte tumor necrosis factor α production late postinjury

The hypothesis that acute ethanol uptake plus trauma can synergize to increase immunosuppression was tested. We found that, unlike non-alcohol-exposed patients, patients with acute alcohol use prior to trauma have a transient decrease in monocyte tumor necrosis factor (TNF) production during the very early postinjury (0–3 days) period. However, TNF production by these alcoholexposed patients' monocytes (MØ) became hyperelevated late postinjury (>9 days). Consequently, these massively elevated MØ TNF levels can contribute to posttrauma immunosuppression after acute alcohol use. We also demonstrate that normal monocyte activation with the superantigen,Staphylococcus enterotoxin B (SEB), results in a preferential induction of cellassociated MØ TNF production, described as characteristic of immunosuppressed trauma patients. Acutein vitro ethanol treatment down-regulated the elevated TNF production by trauma patients' MØ after either SEB, muramyl-dipeptide (MDP), interferon- plus MDP, or lipopolysaccharide (LPS) stimulation. Both SEB- and LPS-induced TNF mRNA induction was inhibited by acute alcohol treatment in normal MØ, indicating that ethanol can regulate cytokine gene expression. An additional immunosuppressive effect of acute ethanol's stimulation was suggested by its induction of elevated transforming growth factor production in trauma patients' activated MØ.     

Synergistic effects of tumour necrosis factor-α and interferon-γ on feline haemopoietic progenitors

Pathogenic mechanisms that underlie feline leukaemia virus subgroup-C (FeLV-C) induced erythroid aplasia are unknown. FeLV-C infection is associated with higher serum levels of interferon- (IFN-) and tumour necrosis factor- (TNF-), which may act synergistically to cause haemopoietic suppression. In the present studies, the synergistic effects of TNF- and IFN- on feline bone marrow progenitors in vitro were evaluated. Bone marrow mononuclear cells from specific-pathogen-free cats were exposed to TNF- (100 and 200 pg/ml) and IFN- (100 or 200 units/ml), alone or in combination, for 2 h before plating for clonal assays of colony forming units. Our results show that TNF- and IFN- in combination caused marked suppression of feline colony forming units-erythroid (CFU-E), burst forming units-erythroid (BFU-E), and colony forming units-fibroblasts (CFU-F), whereas colony forming units-granulocyte/macrophage (CFU-GM) were minimally affected. The same concentrations of TNF- and IFN- alone had minimal effects on CFU-E, BFU-E and CFU-F. These results suggest that TNF- and IFN- may play a significant role in regulating haemopoiesis in cats and may be involved in the pathogenesis of erythroid aplasia in cats infected with feline leukaemia virus.     

Inhibition of PAF-, LPS-, and cytokine-induced granulocyte accumulation in guinea pig lung by dexamethasone: Evidence that inhibition of IL-5 release is responsible for the selective inhibition of eosinophilia by glucocorticoids in guinea-pigs

The potency of dexamethasone has been determined as an inhibitor of intratracheally administered platelet activating factor- (PAF) or interleukin (IL)-5-induced eosinophilia, and of lipopolysaccharide-(LPS), tumour necrosis factor -(TNF) or cytokine-induced neutrophil chemoattractant- (CINC) induced neutrophilia in guinea-pig lungs. Dexamethasone was a potent inhibitor of PAF- induced eosinophil accumulation, but higher doses of dexamethasone were required to inhibit IL-5-induced eosinophilia. LPS-induced neutrophilia was less sensitive to the inhibitory effects of dexamethasone, than PAF-induced eosinophilia. Both LPS- and TNF-induced neutrophilia were inhibited by the same doses of dexamethasone. In contrast, higher doses of dexamethasone were required to inhibit CINC-induced neutrophilia. Since data in the literature show that PAF-induced eosinophilia in guinea-pig lungs is dependent on the generation of IL-5, it is concluded that inhibition of this response, by dexamethasone, is due to inhibition of release of IL-5. Similarly, although data in the literature show that LPS-induced neutrophilia is dependent on the generation of TNF, it is concluded that inhibition of this response, by glucocorticoids, is due to an action on an event which occurs after the release of TNF, possibly through inhibition of chemokine release.accepted by G. Bowen     

NADPH oxidase priming and p47phox phosphorylation in neutrophils from synovial fluid of patients with rheumatoid arthritis and spondylarthropathy

Superoxide anion (O2°-)production by neutrophil NADPH oxidase participates in arthritic joint lesion formation. Proinflammatory cytokines such as tumor necrosis factor (TNF), interleukin 8 (IL-8) and granulocyte/macrophage-colony stimulating factor (GM-CSF) have a priming effect on neutrophil NADPH oxidase activity. NADPH oxidase activation is dependent on phosphorylation of p47phox, a cytosolic component of the enzyme. We studied O2°-production and p47phox phosphorylation in synovial fluid (SF) from patients with rheumatoid arthritis (RA) and spondylarthropathy (SpA) according to TNF, IL-8 and GM-CSF levels. O2°-production by neutrophils isolated from SF of all the arthritis patients (RA and SpA) was higher than that of circulating resting neutrophils and when stimulated with fMLP or PMA. In addition, p47phox was partially phosphorylated in SF neutrophils compared to circulating neutrophils. High levels of TNF and IL-8 (but not GM-CSF) are detected in patient's SF (compared to circulating blood levels). TNF levels were significantly higher in RA than in SpA SF. These results suggest that increased NADPH oxidase activity could be involved in arthritic joint inflammation through increased p47phox phosphorylation. This could be the result of the presence of high levels of priming agents such as TNF and IL-8 but not GM-CSF.     

Defective expression of FasL and Bax in human lung cancer

Abstract. The present studies investigated whether FasL and Bax genes are expressed in pleuro-pulmonary biopsies from patients with lung cancer. FasL, Bax, and TNF mRNAs were detected in 19 biopsies of primary or metastasic lung cancer by fluorescent in situ hybridization assays. Fluorescent probes were produced by polymerase chain reaction using a human spleen lambda gt11 library and specific primers for FasL, Bax, and TNF. Proteins were detected by immunohistochemistry using monoclonal anti- FasL, anti-Bax, and anti-TNF antibodies. Chromatin fragmentation was detected by TUNEL. Seven negative samples from subjects without lung pathology were obtained during legal autopsies and 12 positive control biopsies from patients with lung infections were also included. Sixty-eight percent of lung cancer biopsies exhibited FasL; Bax was expressed in 68% and TNF in 63%. FasL protein was detected in 21%, Bax protein in 26%, and TNF was present in 31% of cancer biopsies. A low degree of apoptosis in lung cancer was demonstrated by TUNEL assays. A defect in FasL, Bax, and TNF gene expression was found in lung cancer biopsies. Some tumors normally expressed the mRNA of FasL, Bax, or TNF, but their proteins were absent, or were non-functional, since TUNEL assays were negative. Such a failure would contribute to cancer cell survival and dissemination.     

Expression of matrix metalloproteinase 9 (92-kDa gelatinase/type IV collagenase) induced by tumour necrosis factor α correlates with metastatic ability in a human osteosarcoma cell line

We have examined the correlation between matrix metalloproteinase (MMP) expression and metastatic properties of a low metastatic osteosarcoma cell line, osteosarcoma takase (OST), under stimulation by tumour necrosis factor (TNF). In vivo, OST cells exhibited significantly increased colonization in the lungs of nude mice in a dose-dependent manner when they were treated by TNF prior to injection. In vitro, TNF enhanced tumour cell invasion through the reconstituted basement membrane in a transwell chamber up to 2.5-fold. Gelatin zymography and sandwich enzyme immunoassays demonstrated marked production of MMP-9 [92-kDa gelatinase/type IV collagenase (gelatinase B)] but not MMP-2 [72-kDa gelatinase/type IV collagenase (gelatinase A)], MMP-3 (stromelysin-1) or MMP-7 (matrilysin). Motility of the tumour cells and adhesion to cultured endothelial cells were slightly increased by the TNF treatment up to 1.6-fold and 1.4-fold, respectively, while the growth rate was decreased. These results suggest that upregulation of MMP-9 together with enhanced motility and endothelial adhesion contribute to the increased metastatic ability of OST cells induced by TNF treatment.     

Acute and chronic inflammatory responses to local administration of recombinant IL-1α, IL-β, TNFα, IL-2 and Ifnγ in mice

The contention that cytokines are important mediators of inflammation prompted the present studies which were designed to compare acute and chronic pathological effects of locally-administered recombinant (r) IL-1, IL-1, TNF, IL-2 and Ifn. Acute (6 hr), resolving (48 hr) inflammation was induced by the following, in order of potency: rIL-1>rIL-1>rTNF>rIfn=BSA (control) following a single sc. injection. However, only rIL-1 and rIL-2 initiated and maintained chronic granulomatous reactions when delivered locally from a sc. ethylene vinyl acetate (EVA) slow-release polymer. The predominance of macrophages in EVA-rIL-1 lesions contrasted with the proliferative lymphoid granulomata induced by EVA-rIL-2 implants. These in vivo observations reinforce, the roles of both IL-1 and IL-2 as potent mediators of chronic immunoinflammatory disease.     

Rho protein inhibition blocks cyclooxygenase-2 expression by proinflammatory mediators in endothelial cells

Rho proteins participate in the regulation of inflammatory gene expression in endothelial cells. We made use of Clostridium difficile toxin B-10643 (TcdB-10463) which inhibites RhoA/Rac1/Cdc42 to analyze their role in expression and regulation of cyclooxygenase-2 (COX-2) in endothelial cells (EC). Pretreatment of EC with TcdB-10643 prevented lipopolysaccharide (LPS)- or tumor necrosis factor- (TNF )-related COX-2 expression but had no effect on COX-1 protein levels. TcdB-10463 preincubation suppressed LPS-dependent nuclear factor-B activation (NF- B). Rho inhibition did not affect COX-1 activity. Inactivation of Rho proteins before LPS stimulation blocked arachidonic acid (AA)-, thrombin-, and Escherichia coli hemolysin (HlyA)-dependent release of COX-2-related 6-ketoprostaglandin F₁ (6k-PGF₁). In contrast, Rho inhibition did not affect COX-2-dependent 6k-PGF₁ liberation when TcdB-10643 was added 10 h after LPS or TNF stimulation of EC. Therefore, RhoA/Rac1/Cdc42 contribute to NF- B-dependent LPS- and TNF-induced expression of PGHS-2 in EC but had no effect on the activity of expressed COX-1 and COX-2.     

Enhancement by TNF-α of reactivation and replication of latent herpes simplex virus from trigeminal ganglia of mice

Summary The influence of tumor-necrosis-factor- (TNF-), granulocytemacrophage colony-stimulating factor (GM-CSF), interleukine-1 (IL-1) and IL-3 on the in vitro reactivation frequency and replication rate of trigeminal ganglia of mice latently infected with herpes simplex virus (HSV) strain KOS was studied. It could be demonstrated that TNF- and possibility GM-CSF, but not IL-1 and IL-3, enhanced the reactivation frequency and replication of HSV. Interferon / (IFN/) prevented reactivation and replication.     

Tumor necrosis factor alpha production in schistosomiasis with carcinoma of urinary bladder

Schistosomiasis parasitic infection (Schistosoma haematobium) is associated in some patients with bladder cancer. The production of cytokines such as tumor necrosis factor alpha (TNF) is a key event of inflammation in human infectious disease and malignancy. TNF has not been previously investigated from schistosomiasis infection and bladder malignancy. In this report we demonstrate that serum levels of TNF are highly elevated in patients with schistosomiasis of urinary bladder (SB), schistosomiasis with carcinoma of urinary bladder (SCB), and carcinoma of urinary bladder without schistosomiasis (CB). Purified monocytes from bladder malignancy (SCB and CB) cultured without exogeneous stimuli release TNF in the culture supernatants. However, lipopolysaccharides and concanavalin A stimulation of monocytes from these patients produced highly elevated levels of TNF compared with normal controls. The findings that monocytes are the potent producers of TNF in this malignancy may be a key observation implicating these cells in the pathophysiology of this disease. Furthermore, it was shown that serum TNF levels correlated with the clinical staging of disease in both SCB and CB, with higher levels in T3 and T4 advanced-stage patients and low levels in T1 and T2 early-stage patients. These results suggest that monocyte abnormality and serum TNF levels might be one of the factors contributing to the progression of disease.     

Down-regulation of tumor necrosis factor α activity by acute ethanol treatment in human peripheral blood monocytes

As the most commonly used drug that can modulate both metabolic and immune pathways, ethanol is evaluated in this report as a regulator of tumor necrosis factor (TNF) production in human peripheral blood monocytes (M) in combination with a variety of stimuli. While acute ethanol treatment did not induce TNF in M, it was a potent down-regulator of M TNF production whether induced by the combination of interferon- plus muramyl dipeptide (MDP) (P<0.001), lipopolysaccharide (LPS) alone (P<0.01), or interferon- plus LPS. Down-regulation of M TNF by ethanol was dose dependent and statistically significant in the biologically relevant, 25–150 mM, ethanol concentration range. We also demonstrate that these ethanol concentrations did not affect M viability. TNF down-regulation by ethanol was most effective when ethanol was administered 4 hr prior to MDP stimulation; however, it was also effective—though to a lesser extent—if it was added at the time of MDP stimulation. Furthermore, ethanol also down-regulated TNF production of thein vivo preactivated M of trauma patients, which produce hyperelevated levels of TNF. We have previously shown that the majority of posttrauma elevated M TNF is produced by the M subpopulation expressing high-affinity type I Fc receptors (FcRI). When the FcRI cross-linking-stimulated M subpopulation was treated with acute ethanol, TNF production was suppressed again both inin vivo preactivated M of trauma patients and in M of normal controls. In experiments utilizing cyclooxygenase inhibitor, we also demonstrate that ethanol has a direct, prostaglandin E₂-independent, effect on M TNF production. These results demonstrate that acute ethanol exposure has the potential to down-regulate M production of TNF significantly regardless of the TNF-inducing stimulus. Decreased capacity of M to produce TNF might, therefore, contribute to the immunological and metabolic abnormalities described after ethanol uptake.     

Genetic characterization of an α-specific gene responsible for sexual agglutinability in Saccharomyces cerevisiae: mapping and gene dose effect

Summary A recessive ag1 mutation leads to specific defect in sexual agglutinability specifically in cells of the yeast Saccharomyces cerevisiae. The cryptopleurine resistance gene cry^R 1, closely linked to the mating type locus, was used to select / strains which emerged from / strains by mitotic nonreciprocal recombination, to genetically analyse ag1, since ag1 is expressed only in mating type. The ag1 gene was found to be linked to the centromere tightly, to met3 at 4.4 cM, and to ilv3 at 12 cM on chromosome X. Sexual agglutinability of cells was shown to be dependent on the dose of the AG1 gene, using / isogenic strains carrying AG1/AG1, AG1/ag1 or ag1/ag1. The sst2-1 mutation did not suppress the ag1 mutation. Based on these results, function of the AG1 gene is discussed.Abbreviations cM centimorgan - FDS first division segregation - NPD nonparental ditype - PD parental ditype - SDS second division segregation - TT tetratype     

Dysregulated Cytokine Production in Human Cystic Fibrosis Bronchial Epithelial Cells

Although pulmonary inflammation is an important pathologic event in cystic fibrosis (CF), the relationship between expression of the CF gene and the inflammatory response is unclear. We studied tumor necrosis factor (TNF) and IL-1 stimulated production of IL-6 and IL-8 by CF, corrected CF, and normal human bronchial epithelial cells in culture. During the first 24 hours of TNF stimulation, CF cells produced significantly more IL-8 than normal or corrected CF cells. In the second 24 hours of TNF stimulation, IL-6 and IL-8 generation ceased in normal and corrected CF cells but accelerated in CF cells, resulting in marked IL-6 and IL-8 accumulation in CF cells. Similar results were found when cells were stimulated with IL-1. Finally, when CF cells were grown at 27°C (a culture condition which results in transport of CF transmembrane conductance regulator, CFTR, to the cell membrane and normalization of chloride conductance) TNF-stimulated production of IL-6 and IL-8 reverted to normal. We conclude that dysregulation of cytokine generation by CF bronchial epithelial cells is directly related to expression of mutant CFTR and these observations provide a potential mechanism for persistence of airway inflammation in CF.     

Immunohistochemical studies on S-100 cells in the anterior pituitary gland of Sprague Dawley rats and spontaneous dwarf rats

The S-100 cells in the pituitary glands of adult male Sprague Dawley rats (SDs) and spontaneous dwarf rats (SDRs) were immunohistochemically examined using anti-S-100 and anti-S-100 monoclonal antibodies. The immunoreactive cells against S-100 protein were divided into three subtypes on the basis of their immunore-activity against subunits of S-100 protein: S-100 dominant type (the -type cell), S-100 dominant type (the \-type cell) and immunoreactive against both S-100 and S-100 (the -type cell). In the SD, -type cells represented 26% of the total S-100 immunoreactive cells (S-100 cells) and were localized in the peripheral area of the ventral region of the pituitary gland. This type of cell was observed forming clusters, with more abundant cytoplasm than the -type cell. The proportion of -type cells was 53%. They were diffusely distributed throughout the gland, and their processes were thicker than those of the -type cell. In the SDR, the proportion of -type cells was 55%, and they were observed throughout the gland. In contrast, -type cells totalled 12% and were localized in small areas of the central and peripheral region of the gland. The proportion of -type cells was 21% in the SD and 33% in the SDR and they were observed forming small clusters in both animal groups. The proportion of -type cells compared with the total of S-100-immunoreactive cells was significantly higher (P < 0.05) in the SDR than in the SD, while the proportion of -type cells was markedly lower (P < 0.05).     

Protection of human cerebral neurons from neurodegenerative insults by gene delivery of soluble tumor necrosis factor p75 receptor

Apoptosis plays an important role in neuronal cell death in both chronic and acute human neurodegenerative diseases, including amyotrophic lateral sclerosis, Huntingtons disease, cerebral ischemia, and human immunodeficiency virus (HIV) encephalopathy. We evaluated the ability of the extracellular binding domain of a dimeric tumor necrosis factor receptor (p75TNFR) to prevent neurotoxicity and death of human fetal cerebral neurons that were exposed in vitro to toxic agents known to be implicated in human neurological disorders, including tumor necrosis factor (TNF) and the HIV proteins Tat and gp120. The extracellular domain of p75TNFR is capable of binding and neutralizing both soluble and transmembrane-anchored TNF. We efficiently transduced human neurons using adenoviral vectors expressing p75TNFR (Ad.p75TNFR) or a control gene (lacZ). Treatment of control cultures with the toxic agents TNF, TNF plus actinomycin D, or Tat and gp120, induced neurotoxic alterations and apoptotic death of neurons. By contrast, transduction of neurons with Ad.p75TNFR prevented apoptosis and cell death due to these agents. We conclude that viral vector transfer of the p75TNFR gene efficiently protects human neurons from TNF-, Tat- or gp120-induced apoptosis and cell death. These results suggest that p75TNFR transduction of neurons by viral vectors could be therapeutically useful in the treatment of many human neurodegenerative diseases.