Cytokines secreted by lymphokine-activated killer cells induce endogenous nitric oxide synthesis and apoptosis in DLD-1 colon cancer cells

IL-2-activated killer lymphocytes (LAK cells) secrete inflammatory cytokines such as interferon-gamma (IFN-gamma) and tumor necrosis factor alpha (TNFalpha) that can induce nitric oxide (NO) synthesis. We evaluated whether LAK cells could activate NO synthesis in human cancer cells. LAK cells and their culture supernatants induced NO synthesis in DLD-1 colon cancer cells in a dose-dependent manner. NO synthesis was inhibited completely by blocking antibodies to IFN-gamma, demonstrating a key role for this LAK cell cytokine in regulating NO synthesis. The addition of TNFalpha antibodies resulted in partial inhibition. Induction of iNOS mRNA and protein expression in DLD-1 cells was detected. Endogenous NO production inhibited DLD-1 cell proliferation and induced apoptosis, processes that were inhibitable by the NO synthase inhibitor N(G)-monomethyl-l-arginine. Our study has identified a novel, non-contact-dependent LAK cell cytotoxic mechanism: induction of growth inhibition and programmed cell death due to endogenous NO synthesis in susceptible human cancer cells.     

Ionizing radiation potentiates the induction of nitric oxide synthase by interferon-gamma (Ifn-gamma) or Ifn-gamma and lipopolysaccharide in bnl cl.2 murine embryonic liver cells: role of hydrogen peroxide

The effects of ionizing irradiation on the nitric oxide (NO) production in murine embryonic liver cell line, BNL CL.2 cells, were investigated. Various doses (5-40 Gy) of radiation made BNL CL.2 cells responsive to interferon-gamma alone for the production of NO in a dose-dependent manner. Small amounts of lipopolysaccharide (LPS) or tumor necrosis factor-alpha (TNF-alpha) synergized with IFN-gamma in the production of NO from irradiated BNL CL.2 cells, even though LPS or TNF-alpha alone did not induce NO production from the same cells. Immunoblots showed parallel induction of inducible nitric oxide synthase (iNOS). NO production in irradiated BNL CL.2 cells by IFN-gamma or IFN-gamma plus LPS was decreased by the addition of catalase, suggesting that H(2)O(2) produced by ionizing irradiation primed the cells to trigger NO production in response to IFN-gamma or IFN-gamma plus LPS. Furthermore, the treatment of nongamma-irradiated BNL CL.2 cells with H(2)O(2) made the cells responsive to IFN-gamma or IFN-gamma plus LPS for the production of NO. This study shows that ionizing irradiation has the ability to induce iNOS gene expression in responsive to IFN-gamma via the formation of H(2)O(2) in BNL CL.2 murine embryonic liver cells.     

Interleukin-10 (IL-10) inhibits the induction of nitric oxide synthase by interferon-gamma in murine macrophages.

A murine macrophage cell line, J774, expresses high levels of the enzyme nitric oxide synthase (NOS) and produces large amounts of nitric oxide (NO) when activated with recombinant interferon (IFN)-gamma and a low concentration of LPS (10 ng/ml). Both the expression of NOS and the production of NO were inhibited by recombinant IL-10 in a dose-dependent manner. The inhibition was effective only when the cells were pretreated with IL-10; addition of IL-10 at the same time or after IFN-gamma activation was without effect. These results demonstrate that IL-10, a product of Th2 (helper T lymphocyte 2) cells, can antagonise the function of IFN-gamma, a product of Th1 cells, by modulating the mechanism of synthesis of nitric oxide in the macrophages.     

Inhibitory effects of phloroglucinol derivatives from Mallotus japonicus on nitric oxide production by a murine macrophage-like cell line, RAW 264.7, activated by lipopolysaccharide and interferon-gamma.

An aqueous acetone extract of the pericarps of Mallotus japonicus (MJE) inhibited nitric oxide (NO) production by a murine macrophage-like cell line, RAW 264.7, which was activated by lipopolysaccharide (LPS) and interferon-gamma (IFN-gamma). Seven phloroglucinol derivatives isolated from MJE exhibited inhibitory activity against NO production. Among these phloroglucinol derivatives, isomallotochromanol exhibited strong inhibitory activity toward NO production, exhibiting an IC(50) of 10.7 microM. MJE and the phloroglucinol derivatives significantly reduced both the induction of inducible nitric oxide synthase (iNOS) protein and iNOS mRNA expression. NO production by macrophages preactivated with LPS and IFN-gamma for 16 h was also inhibited by MJE and the phloroglucinol derivatives. Furthermore, MJE and the derivatives directly affected the conversion of L-[(14)C]arginine to L-[(14)C]citrulline by the cell extract. These results suggest that MJE and the phloroglucinol derivatives have the pharmacological ability to suppress NO production by activated macrophages. They inhibited NO production by two mechanisms: reduction of iNOS protein induction and inhibition of enzyme activity.     

Nitric oxide synthesis and TNF-alpha secretion in RAW 264.7 macrophages: mode of action of a fermented papaya preparation

Macrophage inducible nitric oxide synthase is able to generate massive amounts of nitric oxide (NO) which contributes to the host immune defense against viruses and bacteria. Monocyte-macrophages stimulated with the bacterial wall component lipopolysaccharide (LPS) and cytokines such as interferon-gamma (IFN-gamma) express the inducible form of nitric oxide synthase (iNOS). Furthermore, tumor necrosis factor-alpha (TNF-alpha) is one of the central regulatory cytokines in macrophage antimicrobial activity and synergizes with IFN-gamma in the induction of NO synthesis. Because of its pivotal role in both antimicrobial and tumoricidal activities of macrophages, a significant effort has focused on developing therapeutic agents that regulate NO production. In the present study fermented papaya preparation (FPP) is shown to exert both immunomodulatory and antioxidant activity in the macrophage cell line RAW 264.7. Interestingly, a low and a high molecular weight fraction (LMF and HMF, respectively) of FPP exhibited different activity patterns. FPP fractions alone did not affect NO production. However in the presence of IFN-gamma, both LMF and HMF significantly increased iNOS activity and nitrite as well as nitrate accumulation. NO radical formation measured in real-time by electron paramagnetic resonance spectroscopy was higher in the presence of LMF and IFN-gamma. On the contrary, iNOS mRNA levels were enhanced further with HMF than with LMF. Moreover, LMF displayed a stronger superoxide anion scavenging activity than HMF. In the presence of IFN-gamma, both FPP fractions stimulated TNF-alpha secretion. However in non-stimulated macrophages, TNF-alpha secretion was enhanced by HMF only. Since water-soluble FPP fractions contained no lipid A, present data indicate that FPP is a macrophage activator which augments nitric oxide synthesis and TNF-alpha secretion independently of lipopolysaccharides.     

Nitric oxide production in murine spleen cells: role of interferons and prostaglandin E(2) in the generation of cytotoxic activity

The production of nitric oxide (NO) was measured in cultures of spleen cells stimulated by lipopolysaccharide (LPS), IL-2 or LPS + IL-2. We observed that NO synthesis is increased by IFN-gamma but inhibited by IFN-alpha/beta. This is not the case when IL-2 is present in the cultures, since interferons play a minor role in the regulation of the NO production. When IL-2 and LPS were associated in the cultures, the IFN-alpha/beta role seems more important than that of IFN-gamma. PGE(2) inhibits NO production in LPS supplemented cultures but has a slight effect in the presence of IL-2 and no effect with IL-2 + LPS. 3-isoButyl-1-methylxanthine (IBMX), an inhibitor of phosphodiesterases, induces a decrease of IFN production. In the presence of H-7, an inhibitor of protein kinase C (PKC), NO production is reduced when the cultures are supplemented by LPS or IL-2 but not when IL-2 and LPS are both added. H-7 also reduced IFN production. In the presence of N(G)-monomethyl-L-arginine (N-MMA), an inhibitor of NO synthesis, IFN production was increased, with no change in the cytotoxic activity. Hence, interferons regulate NO production by mouse spleen cells and, in return, NO modulates the generation of IFN.     

Role of TNF-alpha in the induction of fungicidal activity of mouse peritoneal exudate cells against Cryptococcus neoformans by IL-12 and IL-18

We have recently demonstrated that two IFN-gamma-inducing cytokines, interleukin (IL)-12 and IL-18, synergistically induced the fungicidal activity of mouse peritoneal exudate cells (PEC) against Cryptococcus neoformans through NK cell production of interferon (IFN)-gamma and nitric oxide (NO) synthesis. In the present study, we further dissected these effects by examining the involvement of tumor necrosis factor (TNF)-alpha in the induction of IL-12/IL-18-stimulated PEC fungicidal activity. The addition of neutralizing anti-TNF-alpha mAb significantly suppressed IL-12/IL-18-stimulated PEC anticryptococcal activity. This effect was ascribed to the inhibition of macrophage NO synthesis, but not of IFN-gamma production by NK cells, because the same treatment inhibited the former response, but not the latter one. On the other hand, combined treatment with IL-12 and IL-18 synergistically induced the production of TNF-alpha by PEC and this effect was almost completely abrogated by neutralizing anti-IFN-gamma mAb. The cell type producing TNF-alpha among PEC was mostly macrophage. TNF-alpha significantly promoted macrophage NO production and anticryptococcal activity induced by IFN-gamma, and furthermore anti-TNF-alpha mAb partially inhibited these responses. Considered together, our results indicated that TNF-alpha contributed to the potentiation of IL-12/IL-18-induced PEC fungicidal activity against C. neoformans through enhancement of IFN-gamma-induced production of NO by macrophages, but not through increased production of IFN-gamma by NK cells.     

Production of nitric oxide by murine bone marrow cells. Inverse correlation with cellular proliferation.

The present studies were designed to assess the ability of primary cultures of bone marrow cells to produce nitric oxide. We found that two inflammatory stimuli, IFN-gamma and LPS, were potent inducers of nitric oxide production by bone marrow cells. In addition, the CSF granulocyte-macrophage (GM)-CSF and IL-3 as well as TNF-alpha, while inactive by themselves, were synergistic with LPS and IFN-gamma in inducing nitric oxide production. Maximal effects were observed with combinations of GM-CSF and LPS. Nitric oxide production by bone marrow cells was found to be dependent on the presence of L-arginine in the culture medium and inhibitable by NG-monomethyl-L-arginine and L-canavanine, two nitric oxide synthase inhibitors. Nitric oxide produced by the cells was also suppressed by TGF-beta 1 and the tumor promoter 12-O-tetradecanoyl-phorbol-13-acetate. Separation of bone marrow cells by density gradient centrifugation and flow cytometry revealed that the granulocyte-containing fraction was largely responsible for nitric oxide production. In additional experiments we found that treatment of bone marrow cells with GM-CSF significantly stimulated bone marrow cell growth. In contrast, the combination of GM-CSF and LPS or IFN-gamma markedly suppressed cellular proliferation. This suppression was completely reversed by treatment of the cells with NG-monomethyl-L-arginine. Taken together, these data demonstrate that various inflammatory stimuli and cytokines induce nitric oxide production by primary cultures of bone marrow cells and that this mediator may play a role in the regulation of bone marrow cell growth and development.     

Antigen presentation to Th1 but not Th2 cells by macrophages results in nitric oxide production and inhibition of T cell proliferation: interferon-gamma is essential but insufficient

The induction and role of nitric oxide (NO) during antigen presentation by macrophages to T helper (Th) cell subsets was examined. When cultured with Th1 clones, macrophage APC produced NO only in the presence of cognate Ag, which in turn suppressed T cell proliferation. IFN-gamma production by the activated Th1 cells was essential for the induction of NO. Th2 cells presented with the same cognate Ag did not induce NO production and proliferated uninhibited. Coactivation of Th1 and Th2 cells specific for the same Ag indicated that Th2 cells did not inhibit NO production, but were sensitive to NO induced by stimulated Th1 cells. Antigenic activation of Th2 cells in the presence of rIFN-gamma resulted in NO-mediated inhibition of proliferation. Th2 cells provided only a cell-associated cofactor, whereas Th1 cells secreted a soluble cofactor for IFN-gamma as well, i.e., TNF-alpha. Finally, a role for IFN-gamma and NO during immune responses was studied in spleen cells obtained from immunized IFN-gamma(-/-) mice. NO production and subsequent inhibition of Ag-specific proliferation ex vivo was observed only after the addition of rIFN-gamma. These studies suggest an IFN-gamma-dependent regulatory role for NO during Ag-specific Th cell activation involving macrophages, with obvious implications for Th subset-dependent immune responses in general.     

Interferon-gamma plus lipopolysaccharide induction of delayed neuronal apoptosis in rat hippocampus

Interferon-gamma and lipopolysaccharide (IFN-gamma/LPS) induce expression of inducible nitric oxide synthase (iNOS) protein both in cells in vitro and in the brain in vivo. In cultured cells, excessive production of nitric oxide (NO) induces neuronal cell death. However, it is still unclear whether IFN-gamma and LPS might induce neuronal cell death in vivo. In this study, we examined the neuronal cell death and induction of major histocompatibility complex (MHC) antigens after microinjection of IFN-gamma/LPS into the rat hippocampus. Although microglia appeared morphologically ramified in the normal and vehicle-injected hippocampus, microinjection of IFN-gamma/LPS immediately induced the ameboid type. From days 1-7, iNOS was expressed in ameboid microglia surrounding the site of the microinjection. Terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL)-positive cells appeared among the granular neurons of the dentate gyrus on day 3 and peaked about 7 days after microinjection. When the NOS inhibitor N(G)-nitro-L-arginine (L-NA) was intraperitoneally administered prior to the microinjection, the number of TUNEL-positive neurons decreased in a L-NA dose-dependent manner. These results suggest that IFN-gamma/LPS induces delayed neuronal apoptosis in the hippocampus in vivo, and it possibly involves excessive NO production by iNOS. Thus, this animal model may be one of neurodegenerative with extensive inflammatory activation in the hippocampus.     

Dominant negative MyD88 proteins inhibit interleukin-1beta /interferon-gamma -mediated induction of nuclear factor kappa B-dependent nitrite production and apoptosis in beta cells

Insulin-dependent diabetes mellitus is an autoimmune disease in which pancreatic islet beta cells are destroyed by a combination of immunological and inflammatory mechanisms. In particular, cytokine-induced production of nitric oxide has been shown to correlate with beta cell apoptosis and/or inhibition of insulin secretion. In the present study, we investigated whether the interleukin (IL)-1beta intracellular signal transduction pathway could be blocked by overexpression of dominant negative forms of the IL-1 receptor interacting protein MyD88. We show that overexpression of the Toll domain or the lpr mutant of MyD88 in betaTc-Tet cells decreased nuclear factor kappaB (NF-kappaB) activation upon IL-1beta and IL-1beta/interferon (IFN)-gamma stimulation. Inducible nitric oxide synthase mRNA accumulation and nitrite production, which required the simultaneous presence of IL-1beta and IFN-gamma, were also suppressed by approximately 70%, and these cells were more resistant to cytokine-induced apoptosis as compared with parental cells. The decrease in glucose-stimulated insulin secretion induced by IL-1beta and IFN-gamma was however not prevented. This was because these dysfunctions were induced by IFN-gamma alone, which decreased cellular insulin content and stimulated insulin exocytosis. These results demonstrate that IL-1beta is involved in inducible nitric oxide synthase gene expression and induction of apoptosis in mouse beta cells but does not contribute to impaired glucose-stimulated insulin secretion. Furthermore, our data show that IL-1beta cellular actions can be blocked by expression of MyD88 dominant negative proteins and, finally, that cytokine-induced beta cell secretory dysfunctions are due to the action of IFN-gamma.     

Tumor necrosis factor-alpha synergizes with IFN-gamma in mediating killing of Leishmania major through the induction of nitric oxide

CBA mice develop cutaneous lesions when infected with Leishmania major. The disease development was significantly reduced by injecting into the lesion a combination of rIFN-gamma and rTNF-alpha. The doses of IFN-gamma and TNF-alpha used were suboptimal in that either cytokine alone did not have any effect. The therapeutic effect of IFN-gamma and TNF-alpha in vivo is reflected in their ability to activate macrophages to kill the intracellular parasites in vitro. The macrophage leishmanicidal activity induced by TNF-alpha and IFN-gamma can be completely inhibited by a specific inhibitor (L-NG monomethyl arginine) of nitric oxide synthesis. There was a direct correlation between the intracellular killing of the parasites and the production of nitric oxide by the macrophages. In contrast, there was no correlation between leishmanicidal activity and superoxide production by macrophages.     

Macrophage-derived nitric oxide inhibits the proliferation of activated T helper cells and is induced during antigenic stimulation of resting T cells

To examine how macrophage-derived nitric oxide (NO) affects T helper (Th) cell activity, T cell clones representing Th1 and Th2 subsets were activated before exposure to stimulated peritoneal macrophages or microglia. Both Th subsets were similarly sensitive to inhibition by NO, indicating that macrophage-derived NO regulates the proliferation of activated Th1 and Th2 cells equally well. Since IFN-gamma production remained intact in NO-treated Th1 cells, we studied whether NO was produced during antigen-specific activation of Th1 cells by unstimulated macrophages. Indeed, T cell proliferation only occurred when a NO synthase inhibitor was included, while IFN-gamma was essential for the induction of NO. These studies demonstrate that macrophages produce NO following antigen presentation to Th1 cells and that macrophage-derived NO inhibits Th1 and Th2 cell proliferation without inhibiting cytokine production.     

The catalytic RNA of RNase P from Escherichia coli cleaves Drosophila 2S ribosomal RNA in vitro: a new type of naturally occurring substrate for the ribozyme

The production of superoxide and nitric oxide induced in U87 glioma treated with lipopolysaccharide (LPS) and interferon-gamma (IFN-gamma) was examined by electron spin resonance (ESR) spectroscopy using a newly designed flow-type quartz cuvette without detaching cells from the culture plate. ESR spectra of 2,2,6, 6-tetramethyl-4-hydroxy-1-piperidinyloxy (TEMPOL) with U87 cells on a quartz culture plate were measured at 15 min intervals. The signal intensity of TEMPOL decreased in the presence of U87 cells at the pseudo-first order rate. The signal decay was accelerated in the U87 cells treated with LPS/IFN-gamma for 24 h, and was suppressed in the presence of superoxide dismutase and catalase. By the spin-trapping method, nitric oxide from U87 cells pretreated with LPS/IFN-gamma for 24 h was measured by the ESR, but only a weak signal of nitric oxide adducts was detected. Further, the nitrite and nitrate levels in the medium did not increase for 24 h. By the ESR measurement of cells on culture plates without detachment stress, it was found that the production of superoxide was induced by LPS/IFN-gamma, but that of nitric oxide was not, in U87 glioma cells.     

Ascorbate enhances iNOS activity by increasing tetrahydrobiopterin in RAW 264.7 cells

Studies on the effect of ascorbic acid on inducible nitric oxide synthase (iNOS) activity are few and diverse, likely to be dependent on the species of cells. We investigated a role of ascorbic acid in iNOS induction and nitric oxide (NO) generation in mouse macrophage cell line RAW 264.7. Although interferon- (IFN-) gamma alone produced NO end products, ascorbic acid enhanced NO production only when cells were synergistically stimulated with IFN-gamma plus Escherichia coli lipopolysaccharide (LPS). Ascorbate neither enhanced nor decreased the expression of iNOS protein in RAW 264.7 cells, in contrast to the reports that ascorbic acid augments iNOS induction in a mouse macrophage-like cell line J774.1 and that ascorbate suppresses iNOS induction in rat skeletal muscle endothelial cells. Intracellular levels of tetrahydrobiopterin (BH4), a cofactor for iNOS, were increased by ascorbate in RAW 264.7 cells. However, ascorbate did not increase GTP cyclohydrolase I mRNA, the main enzyme at the critical steps in the BH4 synthetic pathway, expression levels and activity. Sepiapterin, which supplies BH4 via salvage pathway, more efficiently enhanced NO production if ascorbate was added. These data suggest that enhanced activation of iNOS by ascorbic acid is mediated by increasing the stability of BH4 in RAW 264.7 cells.