RESUMEN

This unit describes a procedure for determining ability of mouse macrophages to lyse tumor cells in vitro. The Basic Protocol outlines a three-stage assay that includes: (1) culture of macrophages (freshly explanted from mice or grown from a cell line) with suspected activating reagents; (2) extensive washing of the cultured macrophages to remove residual reagents, followed by incubation with [(111)In]-labeled tumor cells to allow lysis to occur; and (3) collection of cell-free culture supernatants and measurement of cytolytic activity as a function of (111)In released from tumor cells destroyed by activated macrophages. The Support Protocol outlines a method for radiolabeling tumor cells with [(111)In]oxine.

Asunto(s)

Técnicas Inmunológicas , Macrófagos/inmunología , Neoplasias Experimentales/inmunología , Animales , Línea Celular , Línea Celular Tumoral , Pruebas Inmunológicas de Citotoxicidad , Activación de Macrófagos/inmunología , Ratones , Neoplasias Experimentales/terapia

RESUMEN

To evaluate the role of autocrine TNF-alpha signaling in macrophage activation, immortalized macrophages from normal mice (B6/J2) and from mice containing gene targeted disruptions of the type 1 and type 2 TNF-receptor genes (TRN) were stimulated under CD14-dependent or serum-free conditions. Although the B6/J2 and TRN clones mounted similar nitric oxide responses to LPS in the presence of serum, the TRN macrophages responded poorly when stimulated with LPS under serum free conditions. LPS stimulation of TRN and B6/J2 under serum-free conditions resulted in equivalent levels of IL-1beta, TNF-alpha, and iNOS gene expression. However, Western blot analysis revealed that iNOS protein production by TRN was 2-fold lower than that produced by B6/J2. These results indicate that autocrine TNF-alpha stimulation contributes to the signaling pathways initiated by ligation of LPS receptors in the absence of LBP and is involved in iNOS post-transcriptional regulation.

Asunto(s)

Citocinas/metabolismo , Lipopolisacáridos/farmacología , Macrófagos/efectos de los fármacos , Óxido Nítrico/biosíntesis , Receptores del Factor de Necrosis Tumoral/fisiología , Factor de Necrosis Tumoral alfa/fisiología , Animales , Western Blotting , Células Cultivadas , Medio de Cultivo Libre de Suero , Expresión Génica , Interleucina-1/análisis , Interleucina-1/genética , Interleucina-1/metabolismo , Receptores de Lipopolisacáridos/farmacología , Activación de Macrófagos , Macrófagos/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Óxido Nítrico Sintasa/genética , Óxido Nítrico Sintasa/metabolismo , Óxido Nítrico Sintasa de Tipo II , Receptores del Factor de Necrosis Tumoral/deficiencia , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transducción de Señal/efectos de los fármacos , Factor de Necrosis Tumoral alfa/genética

RESUMEN

Many tumor cells or their secreted products suppress the function of tumor-infiltrating macrophages. Tumor cells often produce abundant transforming growth factor beta1 (TGF-beta1), which in addition to other immunosuppressive actions suppresses the inducible isoform of NO synthase. TGF-beta1 is secreted in a latent form, which consists of TGF-beta1 noncovalently associated with latency-associated peptide (LAP) and which can be activated efficiently by exposure to reactive oxygen species. Coculture of the human lung adenocarcinoma cell line A549 and ANA-1 macrophages activated with IFN-gamma plus lipopolysaccharide resulted in increased synthesis and activation of latent TGF-beta1 protein by both A549 and ANA-1 cells, whereas unstimulated cultures of either cell type alone expressed only latent TGF-beta1. We investigated whether exposure of tumor cells to NO influences the production, activation, or activity of TGF-beta1.A549 human lung adenocarcinoma cells exposed to the chemical NO donor diethylamine-NONOate showed increased immunoreactivity of cell-associated latent and active TGF-beta1 in a time- and dose-dependent fashion at 24-48 h after treatment. Exposure of latent TGF-beta1 to solution sources of NO neither led to recombinant latent TGF-beta1 activation nor modified recombinant TGF-beta1 activity. A novel mechanism was observed, however: treatment of recombinant LAP with NO resulted in its nitrosylation and interfered with its ability to neutralize active TGF-beta1. These results provide the first evidence that nitrosative stress influences the regulation of TGF-beta1 and raise the possibility that NO production may augment TGF-beta1 activity by modifying a naturally occurring neutralizing peptide.

Asunto(s)

Precursores Enzimáticos/metabolismo , Regulación Neoplásica de la Expresión Génica , Proteínas de Neoplasias/biosíntesis , Óxido Nítrico/fisiología , Fragmentos de Péptidos , Precursores de Proteínas , Procesamiento Proteico-Postraduccional/efectos de los fármacos , Proteínas/metabolismo , Factor de Crecimiento Transformador beta/biosíntesis , Adenocarcinoma/metabolismo , Adenocarcinoma/patología , Animales , Técnicas de Cocultivo , Inducción Enzimática , Precursores Enzimáticos/genética , Humanos , Hidrazinas/farmacología , Procesamiento de Imagen Asistido por Computador , Interferón gamma/farmacología , Lipopolisacáridos/farmacología , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patología , Activación de Macrófagos/efectos de los fármacos , Macrófagos/efectos de los fármacos , Macrófagos/fisiología , Ratones , Proteínas de Neoplasias/genética , Óxido Nítrico/farmacología , Donantes de Óxido Nítrico/farmacología , Óxido Nítrico Sintasa/biosíntesis , Óxido Nítrico Sintasa/fisiología , Óxido Nítrico Sintasa de Tipo II , Óxidos de Nitrógeno , Estrés Oxidativo , Proteínas Recombinantes de Fusión/biosíntesis , Factor de Crecimiento Transformador beta/genética , Factor de Crecimiento Transformador beta/metabolismo , Factor de Crecimiento Transformador beta1

RESUMEN

Transforming growth factor-beta1 (TGF-beta) is secreted in a latent form consisting of mature TGF-beta noncovalently associated with its amino-terminal propeptide, which is called latency associated peptide (LAP). Biological activity depends upon the release of TGF-beta from the latent complex following extracellular activation, which appears to be the key regulatory mechanism controlling TGF-beta action. We have identified two events associated with latent TGF-beta (LTGF-beta) activation in vivo: increased immunoreactivity of certain antibodies that specifically detect TGF-beta concomitant with decreased immunoreactivity of antibodies to LAP. Macrophages stimulated in vitro with interferon-gamma and lipopolysaccharide reportedly activate LTGF-beta via cell membrane-bound protease activity. We show through dual immunostaining of paraformaldehyde-fixed macrophages that such physiological TGF-beta activation is accompanied by a loss of LAP immunoreactivity with concomitant revelation of TGF-beta epitopes. The induction of TGF-beta immunoreactivity colocalized with immunoreactive betaglycan/RIII in activated macrophages, suggesting that LTGF-beta activation occurs on the cell surface. Confocal microscopy of metabolically active macrophages incubated with antibodies to TGF-beta and betaglycan/RIII prior to fixation supported the localization of activation to the cell surface. The ability to specifically detect and localize LTGF-beta activation provides an important tool for studies of its regulation.

Asunto(s)

Macrófagos/fisiología , Factor de Crecimiento Transformador beta/fisiología , Animales , Línea Celular , Humanos , Inmunohistoquímica , Luciferasas/biosíntesis , Luciferasas/genética , Macrófagos/citología , Ratones , Visón , Proteínas Recombinantes/biosíntesis , Transfección , Factor de Crecimiento Transformador beta/biosíntesis , Factor de Crecimiento Transformador beta/genética , Células Tumorales Cultivadas

RESUMEN

The activation of Janus kinases (JAKs) is crucial for propagation of the proliferative response initiated by many cytokines. The proliferation of various cell lines, particularly those of hematopoietic origin, is also modulated by mediators of oxidative stress such as nitric oxide and thiol redox reagents. Herein we demonstrate that nitric oxide and other thiol oxidants can inhibit the autokinase activity of rat JAK2 in vitro, presumably through oxidation of crucial dithiols to disulfides within JAK2. The reduced form of JAK2 is the most active form, and the oxidized JAK2 form is inactive. Nitric oxide pretreatment of quiescent Ba/F3 cells also inhibits the interleukin 3-triggered in vivo activation of JAK2, a phenomenon that correlates with inhibited proliferation. Furthermore, we observed that the autokinase activity of JAK3 responds in a similar fashion to thiol redox reagents in vitro and to nitric oxide donors in vivo. We suggest that the thiol redox regulation of JAKs may partially explain the generally immunosuppressive effects of nitric oxide and of other thiol oxidants.

Asunto(s)

Óxido Nítrico/farmacología , Proteínas Tirosina Quinasas/metabolismo , Proteínas Proto-Oncogénicas , Reactivos de Sulfhidrilo/farmacología , Animales , Baculoviridae , Células Cultivadas , Ditiotreitol/farmacología , Activación Enzimática , Inhibidores Enzimáticos/farmacología , Humanos , Yodobenzoatos/farmacología , Janus Quinasa 1 , Janus Quinasa 2 , Janus Quinasa 3 , Ratones , Oxidación-Reducción , Ratas , Spodoptera

RESUMEN

Bryostatin-1 (Bryo) is a nontumor-promoting protein kinase C modulator that has been shown to have both in vitro and in vivo activity against several murine and human tumors. In this study, we investigated the effects of Bryo on nitric oxide production, measured as accumulated nitrite (NO2-) in culture supernatant, and inducible nitric oxide synthase (iNOS) gene expression in the murine macrophage cell line ANA-1. ANA-1 macrophages did not produce NO2- or iNOS mRNA constitutively, and very little or no NO2- or iNOS mRNA were detectable upon exposure to IFN-gamma. Bryo, although ineffective alone, and IFN-gamma synergized to produce high levels of NO2- and iNOS mRNA. The activity of Bryo was evident at a concentration of 0.1 ng/ml and reached its maximum at 1 ng/ml. The effects of Bryo were time dependent because expression of iNOS mRNA was detectable as early as 6 h and increased through 24 h. Analyses of the molecular mechanisms involved indicate that Bryo and IFN-gamma mainly regulate iNOS gene expression posttranscriptionally through stabilization of iNOS mRNA. Experiments designed to investigate the role of tumor necrosis factor alpha (TNF-alpha) in NO2- production by Bryo- and IFN-gamma-activated macrophages revealed that ANA-1 macrophages expressed low levels of TNF-alpha mRNA constitutively that were not augmented in the presence of IFN-gamma. However, Bryo alone augmented the TNF-alpha mRNA expression, which was only slightly increased with the addition of IFN-gamma. A polyclonal antibody to TNF-alpha was able to completely neutralize TNF-alpha secreted in either medium or Bryo plus IFN-gamma-treated cultures. Neutralizing concentrations of anti-TNF-alpha antibody suppressed the Bryo plus IFN-gamma-induced NO2- production approximately by 50%, suggesting that NO2- produced by Bryo plus IFN-gamma-treated ANA-1 macrophages may involve both TNF-alpha-dependent and TNF-alpha-independent mechanisms. Overall, these findings provide the first evidence that Bryo and IFN-gamma can synergize for the induction of NO2- production as well as iNOS gene expression and show the involvement of posttranscriptional mechanisms in the induction of iNOS mRNA.

Asunto(s)

Regulación Enzimológica de la Expresión Génica , Interferón gamma/farmacología , Lactonas/farmacología , Macrófagos/metabolismo , Mitógenos/farmacología , Óxido Nítrico Sintasa/metabolismo , Óxido Nítrico/metabolismo , Animales , Brioestatinas , Células Cultivadas , Relación Dosis-Respuesta a Droga , Activación Enzimática , Macrólidos , Macrófagos/efectos de los fármacos , Ratones , Procesamiento Postranscripcional del ARN , ARN Mensajero/metabolismo , Proteínas Recombinantes/metabolismo , Factor de Necrosis Tumoral alfa/metabolismo

RESUMEN

We have previously reported that a 19-base pair element of the 5'-flanking region of the inducible nitric oxide synthase (iNOS) gene containing a sequence homology to a hypoxia-responsive enhancer (iNOS-HRE) mediates picolinic acid (PA)- or hypoxia-induced activation of the iNOS promoter in interferon-gamma (IFN-gamma)-treated murine macrophages. The iron chelator desferrioxamine (DFX) induces the activity of the human erythropoietin enhancer in Hep3B cells. We have investigated the influence of DFX on the activation of the iNOS promoter and iNOS gene expression in ANA-1 macrophages. We have found that DFX induced DNA-binding activity to the hypoxia-inducible factor 1 (HIF-1) consensus sequence of the iNOS promoter and activated the iNOS-HRE in murine macrophages. These activities of DFX were associated with a synergistic induction of iNOS mRNA expression and iNOS transcription in IFN-gamma-treated ANA-1 macrophages. Functional analysis of the 5'-flanking region of the iNOS gene demonstrated that IFN-gamma plus DFX activated the full-length iNOS promoter and that the iNOS-HRE was required for DFX-induced iNOS transcriptional activity. We also investigated the role of iron metabolism in the DFX- or PA-dependent induction of HIF-1 activity and iNOS expression. We demonstrate that addition of iron sulfate completely abolished DFX or PA induction of HIF-1 binding and iNOS-HRE activation and abrogated IFN-gamma plus either DFX- or PA-induced iNOS expression. These data establish that DFX is a co-stimulus for the transcriptional activation of the iNOS gene in IFN-gamma-treated macrophages, and they provide evidence that the iNOS-HRE is required for the DFX-dependent activation of the iNOS promoter. Furthermore, our results indicate that the iNOS-HRE is a regulatory element of the iNOS promoter responsive to iron chelation.

Asunto(s)

Hipoxia de la Célula , Deferoxamina/farmacología , Regulación Enzimológica de la Expresión Génica/efectos de los fármacos , Interferón gamma/farmacología , Macrófagos/enzimología , Óxido Nítrico Sintasa/biosíntesis , Óxido Nítrico Sintasa/genética , Regiones Promotoras Genéticas , Animales , Secuencia de Bases , Línea Celular , Núcleo Celular/metabolismo , Secuencia de Consenso , Inducción Enzimática , Compuestos Ferrosos/farmacología , Humanos , Ratones , Ratones Endogámicos C57BL , Proteínas Recombinantes de Fusión/biosíntesis , Secuencias Reguladoras de Ácidos Nucleicos , Transcripción Genética/efectos de los fármacos , Transfección

RESUMEN

Macrophages treated with IFN-gamma alone are stimulated to produce nitric oxide. The level of nitric oxide production can be enhanced significantly when IFN-gamma treatment is combined with other agents (e.g., LPS, TNF-alpha, IL-2, etc.). We tested the hypothesis that cAMP plays a role in the IFN-gamma-induced activation of macrophages. Our experiments indicate that factors that increase the concentration of cAMP in the murine macrophage cell line ANA-1 can also enhance IFN-gamma-induced production of nitric oxide. PGE2 and cholera toxin increased the production of nitrite (an indicator of nitric oxide production) in IFN-gamma-treated ANA-1 macrophages by at least twofold. These factors produced no increase in nitric oxide production in the absence of IFN-gamma treatment. The increase in nitric oxide production corresponded to an increase in the accumulation of nitric oxide synthase mRNA without a change in stability of mRNA. Dibutyryl cAMP and Sp-cAMPs (a selective activator of cAMP-dependent protein kinase I and II) also increased nitric oxide production in IFN-gamma-treated macrophages. However, at very high concentrations (i.e., >100 microM), the stimulatory effect was decreased. These studies indicate that elevation of intracellular cAMP causes a dose-dependent, biphasic alteration of IFN-gamma-induced nitric oxide production in murine macrophages. Moreover, they suggest that agents that affect nitric oxide synthesis may do so via modulation of the cAMP second messenger system.

Asunto(s)

AMP Cíclico/biosíntesis , AMP Cíclico/fisiología , Interferón gamma/farmacología , Macrófagos/efectos de los fármacos , Macrófagos/metabolismo , Óxido Nítrico/biosíntesis , Animales , Bucladesina/farmacología , Línea Celular , Toxina del Cólera/farmacología , AMP Cíclico/análogos & derivados , AMP Cíclico/farmacología , Dinoprostona/farmacología , Sinergismo Farmacológico , Inhibidores Enzimáticos/farmacología , Ratones , FN-kappa B/efectos de los fármacos , Óxido Nítrico Sintasa/biosíntesis , Óxido Nítrico Sintasa/genética , ARN Mensajero/biosíntesis , Tionucleótidos/farmacología

Asunto(s)

Interleucina-12/administración & dosificación , Interleucina-2/administración & dosificación , Neoplasias Experimentales/terapia , Animales , Protocolos de Quimioterapia Combinada Antineoplásica , Esquema de Medicación , Inmunoterapia , Ratones , Metástasis de la Neoplasia , Análisis de Supervivencia

RESUMEN

Simple tandem repeats of the trinucleotide sequence CAG encode homopolymeric stretches of glutamine. Although polyglutamine has been identified in diverse proteins, it is present predominantly in transcription factors. We observed that oncogene-immortalized mouse macrophages express several genes that contain a CAG repeat motif. Therefore, we attempted to clone a novel gene that contains a CAG repeat and is associated with cytokine activation of macrophages. Screening of a mouse macrophage cDNA library with a probe comprising 12 consecutive CAG triplets identified at least one unique clone. The cDNA encodes a protein (named GRP-1 or glutamine repeat protein-1) with 171 amino acids, a calculated molecular mass of 21.6 kDa, and a predicted pI of 10.67. Greater than two-thirds of GRP-1 are only two amino acids, namely glutamine (50%) and histidine (18%). There are four polyglutamine motifs interspersed with histidine-rich regions. There is also a putative nuclear localization signal flanked by sites for possible serine phosphorylation. GRP-1 mRNA was expressed constitutively in some macrophage cell lines and B and T cell lines. Interferon-gamma or lipopolysaccharide augmented GRP-1 mRNA expression in the mouse macrophage cell line ANA-1. Western blot analyses using an antipeptide serum revealed that GRP-1 was localized in the nucleus of ANA-1 macrophages and transfected 3T3 fibroblasts. Overexpression of GRP-1 decreased Sp1-driven chloramphenicol acetyltransferase gene expression in transient cotransfection experiments. Because polyglutamine motifs can cause protein oligomerization and can function as transcriptional activation domains, we suggest that GRP-1 may be a transcription factor associated with interferon-gamma- or lipopolysaccharide-induced activation of macrophages.

Asunto(s)

Macrófagos/metabolismo , Proteínas Nucleares/biosíntesis , Proteínas Nucleares/química , Proteínas Nucleares/genética , Transcripción Genética , Secuencia de Aminoácidos , Animales , Linfocitos B , Secuencia de Bases , Southern Blotting , Clonación Molecular , Cartilla de ADN , Biblioteca de Genes , Histidina , Humanos , Interferón gamma/farmacología , Interleucina-2/farmacología , Macrófagos/efectos de los fármacos , Ratones , Ratones Endogámicos C3H , Ratones Endogámicos C57BL , Datos de Secuencia Molecular , Péptidos , Reacción en Cadena de la Polimerasa , Biosíntesis de Proteínas , ARN Mensajero/biosíntesis , Proteínas Recombinantes/farmacología , Linfocitos T , Repeticiones de Trinucleótidos , Células Tumorales Cultivadas

RESUMEN

We recently reported that a hypoxia-responsive element mediates a novel pathway of transcriptional activation of the inducible nitric oxide synthase (iNOS) promoter in murine macrophages treated with IFN-gamma plus hypoxia (1% O2). In this study, we investigated the expression of NOS activity and the regulation of NOS induction in IFN-gamma treated ANA-1 murine macrophages or thioglycollate-elicited peritoneal macrophages cultured under hypoxic conditions. We found that murine macrophages stimulated with IFN-gamma plus hypoxia, despite a significant accumulation of iNOS mRNA, did not release nitrite into culture supernatant. However, cytosol from macrophages treated with IFN-gamma plus hypoxia contained significant levels of iNOS protein and enzymatic activity. Experiments in which cells were treated with IFN-gamma plus hypoxia and then cultured in normoxic conditions (20% O2) demonstrated that reoxygenation was required to achieve detectable accumulation of nitrite in the culture supernatant. Furthermore, we demonstrated that IL-4 inhibited IFN-gamma plus hypoxia-dependent induction of iNOS mRNA expression, iNOS protein, and enzymatic activity. Experiments in which ANA-1 macrophages were transfected transiently with the full-length iNOS promoter linked to a chloramphenicol acetyltransferase reporter gene demonstrated that IL-4 also down-regulated the IFN-gamma plus hypoxia-induced activation of the iNOS promoter. These data establish that hypoxia is a costimulus with IFN-gamma for the induction of iNOS activity in ANA-1 macrophages as well as in murine peritoneal macrophages, and they provide the first evidence that IL-4 inhibits hypoxia-inducible gene expression. In addition, our results suggest that hypoxia, which occurs in many pathologic conditions, may play an important role in the activation of murine macrophages.

Asunto(s)

Hipoxia/enzimología , Hipoxia/inmunología , Interferón gamma/farmacología , Macrófagos/efectos de los fármacos , Macrófagos/enzimología , Óxido Nítrico Sintasa/biosíntesis , Animales , Línea Celular , Regulación de la Expresión Génica/efectos de los fármacos , Regulación de la Expresión Génica/inmunología , Interleucina-4/farmacología , Macrófagos/citología , Macrófagos Peritoneales/efectos de los fármacos , Macrófagos Peritoneales/metabolismo , Ratones , Ratones Endogámicos C57BL , Óxido Nítrico Sintasa/antagonistas & inhibidores

RESUMEN

Interleukin-12 (IL-12) is a recently described immunoregulatory cytokine with potent therapeutic activity in various preclinical models of infectious or malignant disease. As part of our ongoing evaluation of potential mechanisms accounting for the potent antitumor activity of IL-12, we have investigated the influence of IL-12 administration on total serum nitrate/nitrite (NO(x)(-)) levels and the production of nitric oxide (NO) by peritoneal macrophages from normal and tumor-bearing mice. We report here that IL-12 administration to either normal or tumor-bearing mice for periods of time ranging from 7-19 days induced progressive increases in serum NO(x)(-) levels and primed peritoneal macrophages for NO production on subsequent exposure to lipopolysaccharide or IL-2 ex vivo. Treatment of resident peritoneal macrophages or the macrophage cell line ANA-1 with IL-12 alone or IL-12 in combination with various other stimuli failed to induce NO production, suggesting that the effects of IL-12 occurred via an indirect mechanism. Furthermore, we have shown that not only was the production of NO by macrophages from untreated long-term, tumor- bearing mice suppressed compared with control mice treated with vehicle or IL-12, but also that IL-12 administration overcame this suppression and delayed tumor growth. Lastly, we have shown that administration of weekly pulses of IL-2 in combination with IL-12 additively enhanced the priming of macrophages for NO production ex vivo and delayed tumor growth far more effectively than either agent alone. These observations and reports in the literature regarding the potential influence of NO on development of the immune response and on the regulation of tumor growth and vascularization suggest that NO may play a significant role in the antitumor activity of IL-12 and IL-2.

Asunto(s)

Interleucina-12/farmacología , Interleucina-2/farmacología , Macrófagos Peritoneales/metabolismo , Neoplasias Experimentales/metabolismo , Óxido Nítrico/biosíntesis , Animales , Células Cultivadas , Macrófagos Peritoneales/patología , Ratones , Ratones Endogámicos BALB C , Neoplasias Experimentales/patología , Óxido Nítrico/sangre

RESUMEN

A panel of cytokines was tested for inhibitors of interleukin-6 (IL-6)-dependent cell proliferation. Murine type I and II interferons (mIFNs) strongly inhibited proliferation of IL-6-dependent B9 and 7TD1 cells in a dose-dependent manner. Human tumor necrosis factor-alpha (hTNF-alpha) and human transforming growth factor-beta (hTGF-beta) potently inhibited B9 and to a lesser extent 7TD1 cells, while hIL-11, human oncostatin M (hOSM), and human leukemia inhibitory factor (hLIF) had no inhibitory effects on IL-6-dependent growth. Conversely, IL-11 and OSM but not LIF stimulated B9 and 7TD1 cell growth. However, compared with IL-6, up to 1000-fold higher IL-11 and OSM concentrations were required to induce maximal cell proliferation. Increasing concentrations of IL-6 (up to 100 ng/ml) could not overcome the antiproliferative effects of mIFNs, hTNF-alpha and hTGF-beta. Supernatants from mIFN-gamma and lipopolysaccharide (LPS)-treated mouse macrophages (ANA-1 cell line) were tested in B9 cell assays to identify cytokines among stimulatory and inhibitory biological activities that can inhibit IL-6-dependent proliferation. Undiluted or relatively concentrated supernatants from ANA-1 macrophages treated with mIFN-gamma and/or LPS did not contain detectable IL-6 bioactivity. However, diluted samples contained considerable amounts of detectable IL-6 bioactivity (nanogram levels). Testing the same samples for IL-6 immunoreactivity using enzyme-linked immunoabsorbent assay revealed comparable levels of mIL-6. We conclude that IFNs, TNF-alpha, and TGF-beta and possibly other factors are potent, dominant inhibitors of IL-6-dependent plasmacytoma/hybridoma growth in vitro.

Asunto(s)

Citocinas/farmacología , Inhibidores de Crecimiento/farmacología , Hibridomas/efectos de los fármacos , Interleucina-6/farmacología , Activación de Linfocitos/efectos de los fármacos , Plasmacitoma/inmunología , Animales , División Celular/efectos de los fármacos , División Celular/inmunología , Hibridomas/citología , Interferones/farmacología , Interleucina-6/antagonistas & inhibidores , Ratones , Plasmacitoma/patología , Factor de Crecimiento Transformador beta/farmacología , Factor de Necrosis Tumoral alfa/farmacología

RESUMEN

Picolinic acid, a catabolite of L-tryptophan, activates the transcription of the inducible nitric oxide synthase gene (iNOS) in IFN-gamma-treated murine macrophages. We performed functional studies on the 5' flanking region of the iNOS gene linked to a CAT reporter gene to identify the cis-acting element(s) responsible for the activation of iNOS transcription by picolinic acid. Transient transfection assays showed that the full-length iNOS promoter in the murine macrophage cell line ANA-1 was activated by the synergistic interaction between IFN-gamma and picolinic acid. Deletion or mutation of the iNOS promoter region from -227 to -209, containing a sequence homology to a hypoxia-responsive enhancer (iNOS-HRE), decreased picolinic acid- but not LPS-induced CAT activity by more than 70%. Functional studies using a tk promoter-CAT reporter gene plasmid demonstrated that the iNOS-HRE was sufficient to confer inducibility by picolinic acid but not by IFN-gamma or LPS. Electrophoretic mobility shift assays confirmed that picolinic acid alone induced a specific binding activity to the iNOS-HRE. Furthermore, we found that the iNOS-HRE activity was inducible by hypoxia and that hypoxia in combination with IFN-gamma activated the iNOS promoter in transient transfection assays and induced iNOS transcription and mRNA expression. These data establish that the iNOS-HRE is a novel regulatory element of the iNOS promoter activity in murine macrophages and provide the first evidence that iNOS is a hypoxia-inducible gene.

Asunto(s)

Regulación Enzimológica de la Expresión Génica , Hipoxia/genética , Interferón gamma/administración & dosificación , Óxido Nítrico Sintasa/genética , Ácidos Picolínicos/administración & dosificación , Regiones Promotoras Genéticas , Animales , Secuencia de Bases , Células Cultivadas , Proteínas de Unión al ADN/metabolismo , Sinergismo Farmacológico , Inducción Enzimática , Macrófagos/enzimología , Ratones , Datos de Secuencia Molecular , ARN Mensajero/genética , Transcripción Genética

RESUMEN

Previous studies have demonstrated that the combination of the T helper cell 1 (Th1)-derived cytokines interleukin (IL)-2 and interferon (IFN)-gamma induces nitric oxide (NO) production and tumor cytolysis by mouse peritoneal macrophages and the mouse macrophage cell line ANA-1 in vitro. Conversely, the Th2-derived cytokine IL-4 inhibits IL-2 and IFN-gamma-induced NO production and tumor cytolysis by ANA-1 macrophages. To examine the paracrine regulatory effects of Th1 and Th2 cells on macrophages, various mouse T cell clones were tested for their ability to regulate NO production by mouse peritoneal macrophages or ANA-1 macrophages. Antigen, superantigen, and mitogen stimulated Th1 cells but not Th2 cells induced NO production by macrophages. Supernatants from these activated Th1 clones also induced NO production by peritoneal macrophages and ANA-1 macrophages. Neutralization analysis using monoclonal anticytokine antibodies revealed that both IL-2 and IFN-gamma production by activated Th1 cells were required for the production of NO by macrophages. Co-culture studies using a panel of Th2 cell clones that share the same antigen specificity revealed that these cells suppressed Th1-mediated macrophage activation. The Th2-mediated impairment of Th1-induced NO production was primarily due to the secretion of IL-4. IL-4 appeared to have a direct effect on macrophage activation because neither mitogen-induced proliferation of Th1 cells nor cytokine production by Th1 cells were affected by IL-4. Overall, these results suggest that a potent paracrine regulatory network involving Th1 cells and Th2 cells may control the activation of macrophages for NO production and antitumor cytotoxicity.

Asunto(s)

Activación de Macrófagos , Macrófagos Peritoneales/metabolismo , Óxido Nítrico/biosíntesis , Células TH1/fisiología , Células Th2/fisiología , Animales , Línea Celular , Células Clonales , Citocinas/metabolismo , Técnicas In Vitro , Interleucina-4/farmacología , Activación de Linfocitos , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C3H , Superantígenos/inmunología , Factor de Necrosis Tumoral alfa/biosíntesis

RESUMEN

This study was performed to determine the effects of nitric oxide (NO) on the expression of inducible NO synthase (iNOS) in mouse macrophages. We used the NO donor diethylamine dinitric oxide (DEA/NO) and the mouse macrophage cell line ANA-1 in these experiments. ANA-1 macrophages did not express iNOS mRNA either constitutively or following exposure to 100 U/ml IFN-gamma alone, to 10 ng/ml LPS alone, or to 200 microM DEA/NO alone. Similarly, ANA-1 macrophages did not express detectable levels of iNOS mRNA following treatment with 100 U/ml IFN-gamma plus 200 microM DEA/NO. However, IFN-gamma (100 U/ml) plus LPS (10 ng/ml) induced high levels of iNOS mRNA in ANA-1 macrophages after 6 h. Low concentrations of DEA/NO (approximately 1 to 12 microM) caused up to a 2.5-fold augmentation of IFN-gamma plus LPS-induced iNOS mRNA expression. In contrast, 200 microM DEA/NO suppressed IFN-gamma plus LPS-induced iNOS mRNA expression (60% decrease). The effects of DEA/NO were gene-specific because DEA/NO did not affect the IFN-gamma plus LPS-induced expression of TNF-alpha mRNA. Moreover, the biphasic effects of DEA/NO were specifically due to released NO. Diethylamine and nitrite were unable to regulate IFN-gamma plus LPS-induced gene expression in ANA-1 macrophages. Time-response experiments suggested that the effects of NO were short-lived and occurred early during the induction of iNOS gene expression. The effects of NO were not limited to iNOS mRNA expression but were apparent at the level of iNOS protein expression and enzymatic activity. Overall, these results suggest that NO has immunoregulatory effects and may control the extent and duration of cytokine- and/or endotoxin-induced iNOS expression in macrophages.

Asunto(s)

Aminoácido Oxidorreductasas/biosíntesis , Aminoácido Oxidorreductasas/genética , Hidrazinas/farmacología , Interferón gamma/efectos de los fármacos , Lipopolisacáridos/farmacología , Macrófagos/enzimología , Aminoácido Oxidorreductasas/efectos de los fármacos , Animales , Línea Celular , Relación Dosis-Respuesta a Droga , Inducción Enzimática/efectos de los fármacos , Regulación de la Expresión Génica/efectos de los fármacos , Procesamiento de Imagen Asistido por Computador , Lipopolisacáridos/metabolismo , Ratones , Óxido Nítrico Sintasa , Óxidos de Nitrógeno , ARN Mensajero/biosíntesis , Factor de Necrosis Tumoral alfa/genética

RESUMEN

We examined the ability of nitric oxide (NO) to stimulate the ADP-ribosylation of proteins from the mouse macrophage cell line ANA-1. To demonstrate a specific effect of NO, we used a novel compound named diethylamine dinitric oxide (DEA/NO; 1,1-diethyl-2-hydroxy-2-nitrosohydrazine, sodium salt; [Et2NN(O)NO]Na), which releases NO in aqueous solution at neutral pH. DEA/NO stimulated the ADP-ribosylation of at least three cytosolic proteins (M(r) = 28,000, 33,000 and 39,000) from ANA-1 macrophages. The effect of DEA/NO on the ADP-ribosylation of the predominant target p39 was dose dependent (EC50 = 80 microM). Moreover, the effect of DEA/NO was attributed specifically to released NO rather than diethylamine or nitrite. Sodium nitroprusside (SNP) also stimulated the ADP-ribosylation of cytosolic proteins from ANA-1 mouse macrophages. However, SNP exhibited different time- and dose-dependent effects on the modification of p39. NO synthesized via the activity of interferon-gamma plus lipopolysaccharide-induced NO synthase also enhanced the ADP-ribosylation of p39, confirming that the effects of DEA/NO and SNP could be attributed to NO or reactive nitrogen oxide species. Neither pertussis toxin nor cholera toxin stimulated the ADP-ribosylation of p39; however, cholera toxin stimulated the ADP-ribosylation of proteins with approximate molecular weight of 28,000 and 33,000. These data suggest that the induced expression of NO synthase in tumoricidal macrophages may be associated with autocrine and paracrine effects of NO that include the ADP-ribosylation of various proteins. Moreover, these results indicate that DEA/NO and related compounds may be useful as pharmacologic tools for investigating the effects of NO and reactive nitrogen oxide species on macrophages.

Asunto(s)

Adenosina Difosfato Ribosa/metabolismo , Hidrazinas/farmacología , Macrófagos/metabolismo , Óxido Nítrico/metabolismo , Nitroprusiato/farmacología , Aminoácido Oxidorreductasas/metabolismo , Animales , Arginina/metabolismo , Northern Blotting , Fraccionamiento Celular , Línea Celular , Toxina del Cólera/farmacología , Citosol/metabolismo , Relación Dosis-Respuesta a Droga , Electroforesis en Gel de Poliacrilamida , Hidrazinas/metabolismo , Macrófagos/citología , Macrófagos/efectos de los fármacos , Ratones , Óxido Nítrico/farmacología , Óxido Nítrico Sintasa , Óxidos de Nitrógeno , Toxina del Pertussis , Factores de Virulencia de Bordetella/farmacología

Asunto(s)

Encéfalo/fisiología , Comunicación Celular , Neuronas/fisiología , Óxido Nítrico/fisiología , Transducción de Señal , Sinapsis/fisiología , Animales , Células Cultivadas , Dopamina/metabolismo , Radicales Libres/metabolismo , Humanos , Mesencéfalo/fisiología , NAD/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Óxido Nítrico/farmacología , Nitroprusiato/farmacología

RESUMEN

In the present study, we compared four macrophage (M phi) cell lines from different anatomical origins for functional and secretory activities against the two morphogenetic forms of the fungus Candida albicans. We show that all the cell lines actively phagocytize the yeast and exert antimicrobial activity against both forms of Candida, although M phi of microglial origin are the most effective. When assessed for secretory properties, microglial M phi exhibit a peculiar pattern with respect to other M phi populations under either basal or stimulated conditions. In particular, only microglial M phi fail to respond to the hyphal form of the fungus (H-Candida), which instead acts as a potent tumor necrosis factor inducer in the other M phi cell lines. When exposed to H-Candida, microglial M phi are indistinguishable from other M phi in their ability to modulate specific surface adhesion molecules. In addition to strengthening the knowledge on functional heterogeneity among M phi, our data provide evidence on the peculiar behavior of microglial M phi. To what extent M phi heterogeneity may be related to tissue homeostasis is discussed.

Asunto(s)

Candida albicans/inmunología , Macrófagos/inmunología , Macrófagos/metabolismo , Animales , Northern Blotting , Médula Ósea/inmunología , Encéfalo/inmunología , Línea Celular/inmunología , Línea Celular/metabolismo , Línea Celular/microbiología , Citometría de Flujo , Lipopolisacáridos/farmacología , Pulmón/inmunología , Macrófagos/microbiología , Ratones , Ratones Endogámicos C57BL , Especificidad de Órganos , Cavidad Peritoneal/citología , Fagocitosis/fisiología , ARN/aislamiento & purificación , Factor de Necrosis Tumoral alfa/análisis

RESUMEN

The critical regulatory function of nitric oxide (NO) in many physiologic processes is well established. However, in an aerobic aqueous environment NO is known to generate one or more reactive and potentially toxic nitrogen oxide (NOx) metabolites. This has led to the speculation that mechanisms must exist in vivo by which these reactive intermediates are detoxified, although the nature of these mechanisms has yet to be elucidated. This report demonstrates that among the primary bioorganic products of the reaction of cellular constituents with the intermediates of the NO/O2 reaction are S-nitrosothiol (S-NO) adducts. Anaerobic solutions of NO are not capable of nitrosating cysteine or glutathione, while S-NO adducts of these amino acids are readily formed in the presence of O2 and NO. Investigation of the kinetics for the formation of these S-NO adducts has revealed a rate equation of d[RSNO]/dt = kSNO[NO]2[O2], where kSNO = (6 +/- 2) x 10(6) M-2S-1, a value identical to that for the formation of reactive intermediates in the autoxidation of NO. Competition studies performed with a variety of amino acids, glutathione, and azide have shown that cysteine residues have an affinity for the NOx species that is 3 orders of magnitude greater than that of the nonsulfhydryl amino acids, and > 10(6) times greater than that of the exocyclic amino groups of DNA bases. The dipeptide alanyltyrosine reacts with the intermediates of the NO/O2 reaction with an affinity 150 times less than that of the sulfhydryl-containing compounds. Furthermore, Chinese hamster V79 lung fibroblasts depleted of glutathione display enhanced cytotoxicity on exposure to NO.(ABSTRACT TRUNCATED AT 250 WORDS)

Asunto(s)

Cisteína/química , Glutatión/química , S-Nitrosotioles , Animales , Supervivencia Celular , Cricetinae , Cricetulus , Cisteína/análogos & derivados , Cisteína/síntesis química , Fibroblastos/efectos de los fármacos , Fibroblastos/metabolismo , Concentración de Iones de Hidrógeno , Cinética , Óxido Nítrico/química , Óxido Nítrico/metabolismo , Nitrosación , Oxígeno/química , Oxígeno/metabolismo , Tirosina/química