Comparative analysis of the production of nitric oxide (NO) and tumor necrosis factor-α (TNF-α) from macrophages exposed to high virulent and low virulent strains of Edwardsiella tarda

Abstract Various functions of human phagocytes are modulated by nitric oxide (NO). We transfected the human U937 monoblastoid cell line with an expression vector containing human endothelial NO synthase (eNOS) or murine inducible NOS (iNOS) cDNA to study the regulatory role of NO without the nonspecific effects associated with exogenous NO sources. Western blot confirmed expression of eNOS or iNOS in respectively transfected cells, but not in naive or empty-vector transfected cells. Transfectants expressing iNOS, a calcium-independent enzyme, but not eNOS, a calcium-dependent enzyme, spontaneously produced NO (P < .001). The NO release from iNOS-transfected cells, as measured by nitrite and nitrate accumulation and by cyclic guanosine monophosphate (cGMP) increases in rat reporter cells, was inhibitable (P < .01 for both) with Nω-methyl-L-arginine (L-NMA), a NOS inhibitor. The eNOS transfectants were shown to contain functional enzyme by the conversion of L-arginine to L-citrulline in fractionated cells (P = .0001) and by exposing intact cells to calcium ionophore using the cGMP reporter cell assay (P = .0001). After differentiation with phorbol-12-myristate-13-acetate (PMA), iNOS transfectants produced more tumor necrosis factor-α (TNF-α) (124.9 ± 25.4 pg/5 × 105 cells per 24 hours) than did empty-vector transfected cells (21.9 ± 1.9 pg/5 × 105 cells per 24 hours; P = .02). This effect was inhibited by 500 μmol/L L-NMA (54.4 ± 3.1 pg/5 × 105 cells per 24 hours; P = .05). However, in the presence of high concentrations of lipopolysaccharide (1 μg/mL), which further increased NO production in iNOS transfected cells (P = .044), TNF-α production was similar comparing PMA-differentiated iNOS and empty-vector transfectants (12.2 ± 0.8 and 13.1 ± 1.7 ng/5 × 105 cells per 24 hours, respectively; P = .5). The results show that under certain conditions endogenously produced NO can upregulate TNF-α production in human phagocytes.

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Tumor Necrosis Factor-α (TNF-α), Interferon-γ, and Interleukin-6 but Not TNF-β Induce Differentiation of Neuroblastoma Cells: The Role of Nitric Oxide

Journal of Neurochemistry ◽

10.1046/j.1471-4159.1994.62041330.x ◽

2008 ◽

Vol 62 (4) ◽

pp. 1330-1336 ◽

Cited By ~ 37

Author(s):

M. Angeles Muñoz-Fernández ◽

Eva Cano ◽

Catherine A. O'Donnell ◽

Jackie Doyle ◽

F. Y. Liew ◽

...

Keyword(s):

Nitric Oxide ◽

Tumor Necrosis Factor ◽

Tumor Necrosis ◽

Tumor Necrosis Factor Α ◽

Neuroblastoma Cells ◽

Interferon Γ ◽

Tnf Α ◽

Factor Α ◽

Necrosis Factor

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Endogenously Produced Nitric Oxide Increases Tumor Necrosis Factor-α Production in Transfected Human U937 Cells

Blood ◽

10.1182/blood.v90.3.1160.1160_1160_1167 ◽

1997 ◽

Vol 90 (3) ◽

pp. 1160-1167

Author(s):

Liang Yan ◽

Shuibang Wang ◽

Steven P. Rafferty ◽

Robert A. Wesley ◽

Robert L. Danner

Keyword(s):

Nitric Oxide ◽

Tumor Necrosis Factor ◽

Tumor Necrosis ◽

Tumor Necrosis Factor Α ◽

Intact Cells ◽

Transfected Cells ◽

Empty Vector ◽

Tnf Α ◽

Factor Α ◽

Necrosis Factor

Various functions of human phagocytes are modulated by nitric oxide (NO). We transfected the human U937 monoblastoid cell line with an expression vector containing human endothelial NO synthase (eNOS) or murine inducible NOS (iNOS) cDNA to study the regulatory role of NO without the nonspecific effects associated with exogenous NO sources. Western blot confirmed expression of eNOS or iNOS in respectively transfected cells, but not in naive or empty-vector transfected cells. Transfectants expressing iNOS, a calcium-independent enzyme, but not eNOS, a calcium-dependent enzyme, spontaneously produced NO (P < .001). The NO release from iNOS-transfected cells, as measured by nitrite and nitrate accumulation and by cyclic guanosine monophosphate (cGMP) increases in rat reporter cells, was inhibitable (P < .01 for both) with Nω-methyl-L-arginine (L-NMA), a NOS inhibitor. The eNOS transfectants were shown to contain functional enzyme by the conversion of L-arginine to L-citrulline in fractionated cells (P = .0001) and by exposing intact cells to calcium ionophore using the cGMP reporter cell assay (P = .0001). After differentiation with phorbol-12-myristate-13-acetate (PMA), iNOS transfectants produced more tumor necrosis factor-α (TNF-α) (124.9 ± 25.4 pg/5 × 105 cells per 24 hours) than did empty-vector transfected cells (21.9 ± 1.9 pg/5 × 105 cells per 24 hours; P = .02). This effect was inhibited by 500 μmol/L L-NMA (54.4 ± 3.1 pg/5 × 105 cells per 24 hours; P = .05). However, in the presence of high concentrations of lipopolysaccharide (1 μg/mL), which further increased NO production in iNOS transfected cells (P = .044), TNF-α production was similar comparing PMA-differentiated iNOS and empty-vector transfectants (12.2 ± 0.8 and 13.1 ± 1.7 ng/5 × 105 cells per 24 hours, respectively; P = .5). The results show that under certain conditions endogenously produced NO can upregulate TNF-α production in human phagocytes.

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Inhibition of Nitric Oxide and Tumor Necrosis Factor-α (TNF-α) Production by Propenone Compound through Blockade of Nuclear Factor (NF)-κB Activation in Cultured Murine Macrophages

Biological and Pharmaceutical Bulletin ◽

10.1248/bpb.27.617 ◽

2004 ◽

Vol 27 (5) ◽

pp. 617-620 ◽

Cited By ~ 58

Author(s):

Eung-Seok Lee ◽

Hye Kyung Ju ◽

Tae Churl Moon ◽

Eunkyung Lee ◽

Yurngdong Jahng ◽

...

Keyword(s):

Nitric Oxide ◽

Tumor Necrosis Factor ◽

Tumor Necrosis ◽

Tumor Necrosis Factor Α ◽

Nuclear Factor ◽

Murine Macrophages ◽

Tnf Α ◽

Factor Α ◽

Necrosis Factor

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Nitric oxide-dependent downregulation of adipocyte UCP-2 expression by tumor necrosis factor-α

AJP Cell Physiology ◽

10.1152/ajpcell.2000.279.4.c1100 ◽

2000 ◽

Vol 279 (4) ◽

pp. C1100-C1106 ◽

Cited By ~ 31

Author(s):

Christelle Merial ◽

Anne Bouloumie ◽

Véronique Trocheris ◽

Max Lafontan ◽

Jean Galitzky

Keyword(s):

Nitric Oxide ◽

Tumor Necrosis Factor ◽

Blot Analysis ◽

Tumor Necrosis ◽

Tumor Necrosis Factor Α ◽

Uncoupling Protein ◽

Mitochondrial Protein ◽

Tnf Α ◽

Factor Α ◽

Necrosis Factor

Uncoupling protein-2 (UCP-2) is a mitochondrial protein expressed in adipocytes and has recently been involved in the control of energy dissipation. Because obesity is characterized by an imbalance between energy intake and expenditure and by an enhanced adipocyte-derived secretion of tumor necrosis factor-α (TNF-α), we asked whether TNF-α could directly influence UCP-2 expression in adipocytes. Experiments performed in differentiated 3T3F442A preadipocytes showed that TNF-α (10 ng/ml) induced a reduction of UCP-2 trancripts, assessed by Northern blot analysis. A significant decrease in UCP-2 expression (40%) was observed after 12 and 24 h of TNF-α stimulation of the cells. The characterization of the mechanisms responsible for the TNF-α effect on UCP-2 expression demonstrates an involvement of the TNF-α-induced inducible (i) nitric oxide synthase (NOS) expression. Cell treatment with the NOS inhibitor N G-nitro-l-arginine methyl ester (l-NAME; 1 mmol/l) significantly diminished the TNF-α-mediated sustained downregulation of UCP-2 expression, whereas cell treatment with a nitric oxide (NO) donor (10−3 mol/l S-nitroso-l-glutathione) mimicked the TNF-α effect on UCP-2 expression. Moreover, Western blot analysis clearly showed that TNF-α alone induces the expression of iNOS after 12–24 h treatment of differentiated 3T3F442A cells. These experiments demonstrate that TNF-α directly downregulates UCP-2 expression via NO-dependent pathways that involve the induction of iNOS expression.

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Countervailing Influence of Tumor Necrosis Factor-α and Nitric Oxide in Endotoxemia

Journal of the American Society of Nephrology ◽

10.1681/asn.v1261204 ◽

2001 ◽

Vol 12 (6) ◽

pp. 1204-1210

Author(s):

EDGAR A. JAIMES ◽

DOMINGO DEL CASTILLO ◽

MARK S. RUTHERFORD ◽

LEOPOLDO RAIJ

Keyword(s):

Nitric Oxide ◽

Tumor Necrosis Factor ◽

Knockout Mice ◽

Tumor Necrosis ◽

Tumor Necrosis Factor Α ◽

Wild Type ◽

Tnf Α ◽

Biologic Effects ◽

Factor Α ◽

Necrosis Factor

Abstract. Tumor necrosis factor-α (TNF-α), a crucial mediator in sepsis, elicits multiple biologic effects, including intravascular thrombosis and circulatory shock. TNF-α exerts its biologic effects through two distinct cell surface receptors, TNF-R1 and TNF-R2. The pathophysiologic interaction between TNF-α and nitric oxide (NO) in glomerular thrombosis caused by endotoxemia in rats and wild-type mice (C57BL6) as well as in knockout mice that are deficient in TNF-R1 (R1 —/—), TNF-R2 (R2 —/—), or both receptors (R1R2 —/—) was studied. Administration of lipopolysaccharide (LPS; Escherichia coli endotoxin) resulted in increased NO and TNF-α production but failed to induce glomerular thrombosis. Concomitant administration of LPS + NG-nitro-L-arginine methyl ester (L-NAME; an NO synthesis inhibitor) resulted in glomerular thrombosis in rats and in wild-type mice. Intraperitoneal administration of pentoxifylline before LPS inhibited TNF-α synthesis and prevented glomerular thrombosis in rats given LPS + L-NAME. In contrast to the results observed in rats and wild-type mice, administration of LPS + L-NAME did not result in glomerular thrombosis in knockout mice with either single or double TNF-α receptor deletion. Thus, during endotoxemia, (1) TNF-α fosters glomerular thrombosis if there is deficiency of NO synthesis and (2) both TNF-α receptors are necessary for TNF-α's prothrombogenic action. Clinically, these novel studies suggest that in gram-negative endotoxemia, inhibition of NO synthesis and selective blockade of TNF-α receptors may provide unique therapeutic approaches for mitigation of glomerular thrombosis and restitution of vascular tone.

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Nitric oxide and calcium signaling regulate myocardial tumor necrosis factor-α expression and cardiac function in sepsisThis article is one of a selection of papers published in this special issue on Calcium Signaling.

Canadian Journal of Physiology and Pharmacology ◽

10.1139/y09-097 ◽

2010 ◽

Vol 88 (2) ◽

pp. 92-104 ◽

Cited By ~ 19

Author(s):

Ting Zhang ◽

Qingping Feng

Keyword(s):

Nitric Oxide ◽

Tumor Necrosis Factor ◽

Calcium Signaling ◽

P38 Mapk ◽

Tumor Necrosis ◽

Tumor Necrosis Factor Α ◽

Myocardial Dysfunction ◽

Tnf Α ◽

Factor Α ◽

Necrosis Factor

Myocardial tumor necrosis factor-α (TNF-α), a proinflammatory cytokine, is a critical inducer of myocardial dysfunction in sepsis. The purpose of this review is to summarize the mechanisms through which TNF-α production is regulated in cardiomyocytes in response to lipopolysaccharide (LPS), a key pathogen-associated molecular pattern (PAMP) in sepsis. These mechanisms include Nox2-containing NAD(P)H oxidase, phospholipase C (PLC)γ1, and Ca2+ signaling pathways. Activation of these pathways increases TNF-α expression via activation of extracellular signal-regulated kinases 1 and 2 (ERK1/2) and p38 mitogen-activated protein kinase (MAPK). Conversely, activation of c-Jun NH2-terminal kinase 1 (JNK1) negatively regulates TNF-α production through inhibition of ERK1/2 and p38 MAPK activity. Interestingly, endothelial nitric oxide synthase (eNOS) promotes TNF-α expression by enhancing p38 MAPK activation, whereas neuronal NOS (nNOS) inhibits TNF-α production by reducing Ca2+-dependent ERK1/2 activity. Therefore, the JNK1 and nNOS inhibitory pathways represent a “brake” that limits myocardial TNF-α expression in sepsis. Further understanding of these signal transduction mechanisms may lead to novel pharmacological therapies in sepsis.

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