Regulation of ICAM-1 Expression in Mouse Macrophages

In a mouse model of silica (SI) induced lung injury, SI exposure increases expression of intercellular adhesion molecule-1 (ICAM-1) on lung (alveolar/interstitial) macrophages and alveolar type II epithelial cells. To investigate the regulation of SI induced ICAM-1 expression on mouse macrophages, freshly isolated macrophages (alveolar, peritoneal) and macrophage cell lines (MH-S, RAW 264.7) were evaluated for ICAM-1 expression elicited by the particle silica ( quartz; 20 g/ml; 6 g/cm²) or the inflammatory cytokine, TNF (20 ng/ml). TNF significantly increased ICAM-1 expression in all cell types whereas SI elicited an increase in peritoneal macrophages (PM) and the cell line, MH-S. This pattern of increased expression was confirmed by immunocytochemistry. To investigate the regulation of ICAM-1 expression, PM were incubated with SI, TNF or media concomitantly with anti-TNF antibody, the antioxidant, NAC, or the iNOS synthase inhibitor, L-NAME. Both anti-TNF and NAC, but not L-NAME, inhibited elicited (TNF, SI) as well as constitutive (media) ICAM-1 expression. These data demonstrate that both inflammatory cytokines and inorganic particles can increase ICAM-1 expression on mouse macrophages and that this expression is mediated, in part, by TNF and reactive oxygen species.     

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.     

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.     

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.     

Cytokine profile in systemic lupus erythematosus,rheumatoid arthritis,and other rheumatic diseases

We investigated serum levels of interleukin-6 (IL-6), interferon-gamma (IFN-), and tumor necrosis factor alpha (TNF) from patients with systemic lupus erythematosus (SLE) and its various clinical manifestations of disease and from patients with rheumatoid arthritis (RA) and other rheumatic diseases. The serum levels of IL-6 and IFN- were highly elevated from patients with SLE associated with lymphadenopathy (LN) or nephrotic syndrome (NS). On the contrary, the serum levels of TNF were elevated from most patients with SLE associated with thrombocytopenia (TP). However, serum levels of TNF were in the normal range from patients with SLE associated with NS, LN, or central nervous system disease. Of interest, patients with SLE associated with humoral immunodeficiency disorder, hypogammaglobulinemia, had highly elevated levels of serum IL-6. The concanavalin A-stimulated mononuclear cells (MNC) of patients with SLE associated with TP secreted highly elevated levels of TNF compared to other patient groups. We suggest that abnormal production of various cytokines in SLE is an intrinsic defect of MNC and the immune system that may be the key element for a variety of clinical manifestations of this 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.     

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.     

Modulation of the endothelial procoagulant response to lipopoly-saccharide and tumour necrosis factor-α in-vitro: The effects of dexamethasone,pentoxifylline, iloprost and a polyclonal anti-human IL-1α antibody

Endothelial expression of tissue factor (TF), a potent procoagulant molecule, is increased in response to inflammatory mediators such as lipopolysaccharide (LPS), tumour necrosis factor (TNF) and interleukin-1 (IL-1). We have examined the effects of three antiinflammatory agents and a polyclonal anti-human IL-1 antibody on the human endothelial TF response toE. coli 0111:B4 LPS and recombinant TNF (rTNF) in vitro. In contrast to the expected inhibitory effect, dexamethasone, pentoxyfilline and iloprost failed to block TF expression when administered simultaneously or 30 minutes prior to stimulation with either LPS or rTNF. Inhibition of procoagulant activity was demonstrated with the anti-IL-1 antibody, suggesting that endothelial derived IL-1 is partially responsible for the TF response to the agonists employed. The failure of the antiinflammatory agents to inhibit endothelial TF expression highlights the possibility that therapeutic agents that modulate the circulating monocyte response to LPS and TNF may not ameliorate the endothelial dysfunction that is also induced by these inflammatory mediators.     

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.     

Transient expression of myosin heavy chain MHCIα in rabbit muscle during fast-to-slow transition

The expression of an -cardiac-like myosin heavy chain, MHCI, was investigated at both the mRNA and protein levels in rabbit tibialis anterior muscle undergoing fast-to-slow transition by continuous chronic low-frequency stimulation (CLFS). According to sequence analyses of the PCR product, the MHCI isoform was found to be identical to the -cardiac MHC expressed in rabbit atrium. In muscles at different degrees of transformation, the upregulation of MHCI mRNA preceded that of the MHCI mRNA. At more advanced stages of the transformation, MHCI mRNA decayed while MHCI mRNA persisted at high levels. The expression of MHCI, therefore, was transitory. Studies at the protein level were based on immunoblotting using a monoclonal antibody (F88 12F8,1), characterized to be specific to MHCI in rabbit muscle. These studies revealed a similar relationship between initial increase and successive decline of the MHCI protein as seen at themRNA level. Immunohistochemistry of 30-day stimulated muscle revealed that up to 65% of the fibres expressed the MHCI isoform in combination with other adult MHC isoforms. The most frequent patterns of coexistence were MHCIIa+MHCI + MHCI (28%), MHCI+MHCI (18%), and MHCIIa + MHCI (11%). According to these combinations, the upregulation of MHCI may be assigned as an intermediate step in the transformation of existing fibres during theMHCIIa MHCI transition. A small fraction of fibres contained, in addition to the MHCI + MHCI and MHCIIa + MHCI combinations, developmental myosin, suggesting that MHCI was also expressed in regenerating fibres originating from satellite cell-derived myotubes.     

Antitumor effects of liposomal IL1α and TNFα against the pulmonary metastases of the B16F10 murine melanoma in syngeneic mice

Interleukin 1 alpha (IL1) and tumor necrosis factor alpha (TNF) have been successfully incorporated into specific phosphatidylcholine (PC) and phosphatidylserine (PS) multilamellar vesicle (MLV) liposomes by modifying the concentration of calcium ion and pH of the encapsulation buffer. Under these conditions, some of the cytokines may attach to the exterior surface of the MLV and therefore be readily accessible to target cells for receptor binding and signal transduction. These cytokine-associated liposomes are stable for up to 2 weeks in serum-free buffer, and leakage of cytokines into medium containing 10% fetal bovine serum was about 50% at the end of a 3-day incubation period at 37°C. The biological activities mediated by liposomal IL1 and TNF were specific: the stimulation of thymidine uptake in T-helper D10 lymphocytes and the cytolysis of TNF-sensitive L929 target cells could be blocked by specific neutralizing antibodies in a dose-dependent fashion. When administered intravenously into C57BL/6 mice bearing the syngeneic B16F10 murine melanoma cells, dual entrapment of liposomal IL1 and TNF significantly reduced the number of metastatic tumor nodules in the lungs and prolonged the life span of the animals. Thus, liposomal IL1 and TNF displayed significant in vivo antitumor activity against the IL1- and TNF-resistant B16F10 metastatic murine melanoma.     

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.     

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.     

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.     

Recombinant interleukin-1 and tumor necrosis factor induce neutrophil migration “in vivo” by indirect mechanisms

The and forms of recombinant interleukin-1 (IL-1 and IL-1) and of recombinant Tumor Necrosis Factor (TNF and TNF) induced dose-dependent neutrophil migration into rat peritoneal cavities. Migration induced by both IL-1s showed a bell-shaped dose-response curve and IL-1 was 3-fold more potent than IL-1. Pretreatment of the animals with dexamethasone or depletion of the peritoneal macrophage population, abolished the neutrophil migration induced by the four cytokines. In vitro stimulation of macrophage monolayers with IL-1 and the TNFs released a factor into the supernatant which, unlike these cytokines, induced neutrophil migration in dexamethasone pretreated animals. These results suggest that the neutrophil migration induced by IL-1, IL-1 and TNF is not due to a direct effect on neutrophils, but occurs via the release of a chemotactic factors(s) from resident macrophages.     

Effects of brain histamine depletion on stimulated prolactin release in rats

We examined the effects of an irreversible inhibitor of brain histamine (HA) synthesis, -fluoromethyl-histidine (-FMH), on prolactin (PRL) release induced by an opiate agonist (morphine, M) or by a serotonergic agonist (MK212). -FMH was administered intracerebroventricularly (i.c.v., 200 g/rat) into freely moving rats with indwelling catheters in the carotid. M (6 mg/kg, intracarotid, i.a.) was administered simultaneously with or 3 h after -FMH. MK212 (2.5 mg/kg, i.a.) was administered 3 h after -FMH. Blood samples for assay for PRL were drawn at 0, 10, 20, 40 min after M or MK212 administration. -FMH (3 h before) significantly reduced the PRL-releasing effect of M and MK212 but did not modify PRL release by M when administered simultaneously. The present results showing that the facilitatory actions of the opiate and serotonergic systems on PRL are impaired when brain HA synthesis is reduced, suggest that there is an HA-dependent step in opiate and serotonergic control of PRL.     

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.     

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.     

Integrins and extracellular matrix-proteins in the different components of the Wilms' tumour

The Wilms' tumour (WT) is composed of blastema, epithelium and mesenchyme; the epithelium and possibly also the mesenchyme develop from the blastema, parallel to embryonal development. Since interactions between cell adhesion receptors and extracellular matrix (ECM) proteins play an important role in tissue maturation, we examined the expression of the integrin subunits 1–6, 1 and 4, and of the ECM proteins fibronectin, laminin and collagen I and IV, in 20 frozen WT samples and in 5 fetal and 2 adult kidneys. The integrin and ECM protein distribution in tumour epithelium and mesenchyme showed strong similarities to that in their fetal counterparts, whereas the tumour blastema differed strongly from the fetal blastema. In the WT blastema different components were recognized. Undifferentiated blastema, characterized by expression of 3 and 6 and the virtual absence of ECM proteins. Blastema with epithelial commitment, showing increased expression of 3 and 6 and the appearance of 2 and, as a very early phenomenon, production of laminin. Blastema with mesenchymal commitment, with loss of 3 and 6 and expression of 1, 4 and 5 and presence of ECM proteins. It is speculated that the inability of the (undifferentiated) blastema to produce ECM proteins is related to its relatively high metastatic potential when compared with epithelium and mesenchyme.