RESUMEN

Interleukin-18 (IL-18) is a recently identified immunoregulatory cytokine that shares biochemical features with IL-1beta and acts in part by inducing interferon-gamma (IFN-gamma). Endotoxic bacterial lipopolysaccharide (LPS) (1 or 2 ng/kg) was insufficient to increase plasma IL-18 in five healthy adults measured 3, 12, and 24 hr following challenge. In contrast, in the first 96 hr of admission to the surgical intensive care unit, mean maximal serum IL-18 was elevated (1,122 +/- 259 pg/ml) in nine septic patients compared to six healthy adults (191 +/- 42 pg/ml), P < 0.01). Serum IL-18 concentrations in septic patients did not correlate with other measured inflammatory mediators: tumor necrosis factor, IL-6, IL-10, or secretory leukocyte protease inhibitor. Therefore, IL-18 circulates in healthy adults and is a component of the human systemic inflammatory response. Further, stimuli other than LPS may induce IL-18 production in vivo in human sepsis.

Asunto(s)

Interleucina-18/sangre , Sepsis/inmunología , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Estudios de Casos y Controles , Femenino , Humanos , Mediadores de Inflamación/sangre , Lipopolisacáridos/toxicidad , Masculino , Persona de Mediana Edad

RESUMEN

OBJECTIVES: To document changes in serum secretory leukocyte protease inhibitor (SLPI) in human sepsis and in experimental endotoxemia in vivo. To compare changes in serum SLPI in human sepsis with changes in interleukin (IL)-6, IL-10, and tumor necrosis factor (TNF)-alpha. To determine whether or not changes in SLPI correlate with the severity of multiple organ dysfunction syndrome as measured by the maximal multiple organ dysfunction score. Finally, because neutrophils have been implicated in tissue injury associated with organ dysfunction, to determine whether recombinant human SLPI blocks activation of isolated human neutrophils. DESIGN: Case-control study and ex-vivo cellular assay. SETTING: Surgical intensive care unit and clinical research center of university hospitals; laboratory of a medical school. INTERVENTIONS: None. MEASUREMENTS AND MAIN RESULTS: There was a significant dose-dependent elevation (50.2+/-4.0 ng/mL, p = .01) in plasma SLPI 12 hrs after administration of lipopolysaccharide to seven healthy adults (36.4+/-2.3 ng/mL). Further, serum concentrations of SLPI (132+/-15 ng/mL) were elevated in septic surgical patients compared with healthy controls (43+/-2 ng/mL, p < .01) and nonseptic surgical controls (69+/-10 ng/mL, p = .01). Serum SLPI concentrations correlated (r2 = .71, p < .01) better with organ dysfunction as measured by maximal multiple organ dysfunction score than did serum IL-6 (r2 = .49, p < .01), IL-10 (r2 = .05, p = .22), or TNF-alpha (r2 = .02, p = .44). We found that recombinant human SLPI in vitro inhibits TNF-alpha-induced hydrogen peroxide production by human neutrophils (ID50 = 1-2 microg/mL). CONCLUSIONS: Serum SLPI is elevated in human sepsis and experimental endotoxemia. Maximal concentrations of serum SLPI correlate significantly with maximal multiple organ dysfunction scores in patients with sepsis. Secretory leukocyte protease inhibitor may function to limit ongoing neutrophil-mediated tissue injury associated with organ dysfunction.

Asunto(s)

Endotoxemia/sangre , Activación Neutrófila/inmunología , Proteínas/metabolismo , Choque Séptico/inmunología , Síndrome de Respuesta Inflamatoria Sistémica/inmunología , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Estudios de Casos y Controles , Citocinas/sangre , Escherichia coli/inmunología , Femenino , Humanos , Unidades de Cuidados Intensivos , Lipopolisacáridos/inmunología , Masculino , Persona de Mediana Edad , Proteínas Inhibidoras de Proteinasas Secretoras , Estallido Respiratorio/inmunología , Inhibidor Secretorio de Peptidasas Leucocitarias

RESUMEN

Over the past decade, reactive nitrogen intermediates joined reactive oxygen intermediates as a biochemically parallel and functionally non-redundant pathway for mammalian host resistance to many microbial pathogens. The past year has brought a new appreciation that these two pathways are partially redundant, such that each can compensate in part for the absence of the other. In combination, their importance to defense of the murine host is greater than previously appreciated. In addition to direct microbicidal actions, reactive nitrogen intermediates have immunoregulatory effects relevant to the control of infection. Genes have been characterized in Mycobacterium tuberculosis and Salmonella typhimurium that may regulate the ability of pathogens to resist reactive nitrogen and oxygen intermediates produced by activated macrophages.

Asunto(s)

Mycobacterium tuberculosis/patogenicidad , Óxido Nítrico/metabolismo , Salmonella typhimurium/patogenicidad , Animales , Predisposición Genética a la Enfermedad , Humanos , Macrófagos/inmunología , Macrófagos/metabolismo , Ratones , Mycobacterium tuberculosis/genética , Mycobacterium tuberculosis/inmunología , Especies Reactivas de Oxígeno/metabolismo , Infecciones por Salmonella/inmunología , Infecciones por Salmonella/microbiología , Salmonella typhimurium/genética , Salmonella typhimurium/inmunología , Tuberculosis/inmunología , Tuberculosis/microbiología

RESUMEN

BACKGROUND: Septic shock is a leading cause of mortality in intensive care units. No new interventions in the last 20 years have made a substantial impact on the outcome of patients with septic shock. Identification of inhibitable pathways that mediate death in shock is an important goal. MATERIALS AND METHODS: Two novel caspase inhibitors, (2-indolyl)-carbonyl-Ala-Asp-fluoromethylketone (IDN 1529) and (1-methyl-3-methyl-2-indolyl)-carbonyl-Val-Asp-fluoromethylketone (IDN 1965), were studied in a murine model of endotoxic shock. RESULTS: IDN 1529 prolonged survival when given before or up to 3 hr after high-dose LPS (p < 0.01) and increased by 2.2-fold the number of animals surviving longterm after a lower dose of LPS (p < 0.01). Despite its similar chemical structure, IDN 1965 lacked these protective effects. Both compounds inhibited caspases 1, 2, 3, 6, 8, and 9, and both afforded comparable reduction in Fas- and LPS-induced caspase 3-like activity and apoptosis. Paradoxically, administration of IDN 1529 but not IDN 1965 led to an increase in the LPS-induced elevation of serum cytokines related directly (IL-1beta, IL-18) or indirectly (IL-1alpha, IL-1Ra) to the action of caspase 1. CONCLUSIONS: A process that appears to be distinct from both apoptosis and the release of inflammatory cytokines is a late-acting requirement for lethality in endotoxic shock. Inhibition of this process can rescue mice even when therapy is initiated after LPS has made the mice severely ill.

Asunto(s)

Inhibidores de Cisteína Proteinasa/farmacología , Indoles/farmacología , Oligopéptidos/farmacología , Choque Séptico/tratamiento farmacológico , Animales , Anticuerpos Monoclonales/farmacología , Apoptosis/efectos de los fármacos , Caspasa 1/efectos de los fármacos , Caspasa 1/genética , Caspasa 1/metabolismo , Caspasa 3 , Inhibidores de Caspasas , Caspasas/metabolismo , Citocinas/sangre , Citocinas/efectos de los fármacos , Femenino , Interleucina-1/metabolismo , Lipopolisacáridos , Hígado/efectos de los fármacos , Hígado/patología , Macrófagos/efectos de los fármacos , Macrófagos/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Proteínas Recombinantes/efectos de los fármacos , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Choque Séptico/mortalidad , Tasa de Supervivencia , Receptor fas/inmunología

RESUMEN

Evidence implicating inducible nitric oxide synthase (iNOS) in the alterations of cardiac function characteristic of septic shock has come mostly from studies on anesthetized animals, isolated hearts, cultured myocytes, or hosts treated with pharmacologic inhibitors that lack complete specificity for iNOS. Platelet-activating factor (PAF) can participate in the induction of iNOS and has also been implicated in cardiac dysfunction in sepsis. The present studies assessed cardiac function in a model of sepsis in awake mice in which the gene for iNOS was either normal or selectively disrupted. Mice of each genotype were treated with parenteral fluids or with a highly specific antagonist of PAF. Endotoxic shock was induced by challenge with bacterial lipopolysaccharide (LPS) after priming with heat-killed Propionobacterium acnes. Wild-type mice increased stroke volume and cardiac output in response to LPS. These changes were absent in iNOS-deficient mice. When treated with parenteral fluids, LPS-challenged wild-type and iNOS-deficient mice both had a marked reduction in cardiac output. Antagonism of PAF had no effect on echocardiographic indices in wild-type mice, but selectively overcame the bradycardia and reduced cardiac output elicited by fluid administration in LPS-shocked, iNOS-deficient mice. Thus, there are major cardiovascular effects of PAF that are shared by rather than mediated by iNOS. Neither complete iNOS deficiency nor antagonism of PAF improved survival, whether tested as single or combined intervention. On the contrary, complete deficiency of iNOS was detrimental to survival. Finally, we tested the hypothesis that iNOS deficiency might improve survival if the deficiency were specific but partial. For this, we used mice with one normal and one disrupted gene for iNOS. No survival advantage was evident for these iNOS heterozygotes. Thus, partial or complete inhibition of iNOS, with or without antagonism of PAF, afforded no evident benefit beyond the previously demonstrated reduction in hypotension. Finally, these studies demonstrate that echocardiography preceded by acclimatization is feasible in unanesthetized mice, a finding which should expand the value of genetically manipulated animals for analysis of cardiac function.

Asunto(s)

Óxido Nítrico Sintasa/deficiencia , Factor de Activación Plaquetaria/antagonistas & inhibidores , Choque Séptico/diagnóstico por imagen , Choque Séptico/genética , Animales , Ecocardiografía , Lipopolisacáridos , Ratones , Ratones Noqueados/genética , Óxido Nítrico Sintasa/genética , Óxido Nítrico Sintasa de Tipo II , Vehículos Farmacéuticos/farmacología , Propionibacterium acnes , Choque Séptico/inducido químicamente , Choque Séptico/microbiología , Análisis de Supervivencia

RESUMEN

Taxol, a plant-derived antitumor agent, stabilizes microtubules. Taxol also elicits cell signals in a manner indistinguishable from bacterial lipopolysaccharide (LPS). LPS-like actions of Taxol are controlled by the lps gene and are independent of binding to the known Taxol target, beta-tubulin. Using biotin-labeled Taxol, avidin-agarose affinity chromatography, and peptide mass fingerprinting, we identified two Taxol targets from mouse macrophages and brain as heat shock proteins (Hsps) of the 70- and 90-kDa families. Geldanamycin, a specific inhibitor of the Hsp 90 family, blocked the nuclear translocation of NF-kappaB and expression of tumor necrosis factor in macrophages treated with Taxol or with LPS. Geldanamycin did not block microtubule bundling by Taxol or macrophage activation by tumor necrosis factor. Thus, Taxol binds Hsps, and Hsp 90 helps mediate the activation of macrophages by Taxol and by LPS.

Asunto(s)

Proteínas HSP90 de Choque Térmico/metabolismo , Lipopolisacáridos/farmacología , Activación de Macrófagos , Macrófagos/efectos de los fármacos , Paclitaxel/farmacología , Animales , Antineoplásicos/farmacología , Benzoquinonas , Biotinilación , Encéfalo/metabolismo , Proteínas de Unión al ADN/análisis , Proteínas HSP90 de Choque Térmico/antagonistas & inhibidores , Inmunohistoquímica , Lactamas Macrocíclicas , Ratones , Microtúbulos/efectos de los fármacos , FN-kappa B/antagonistas & inhibidores , FN-kappa B/metabolismo , Proteínas Nucleares/análisis , Quinonas/farmacología , Factor de Necrosis Tumoral alfa/farmacología

RESUMEN

Two classes of oxidants are thought to play a critical role in tissue damage in septic shock: reactive oxygen intermediates (ROI) and reactive nitrogen intermediates (RNI). Particular importance has been ascribed to peroxynitrite, a product arising from the reaction of nitric oxide with superoxide. A major source of ROI is the respiratory burst oxidase of neutrophils, eosinophils, monocytes, and macrophages. A major source of RNI is inducible nitric oxide synthase (iNOS), an enzyme expressed in leukocytes, hepatocytes, vascular smooth muscle cells, endothelium, and cardiac myocytes during inflammation. In previous studies using various mouse models of endotoxic shock, genetic deficiency of iNOS as a sole intervention did not consistently alter survival. Here, using Salmonella typhimurium endotoxic bacterial lipopolysaccharide (LPS) as a sole challenge, genetic deficiency of iNOS was associated with no protection or a reduction in survival, depending on the dose of LPS. Further, no protection from lethality was observed when LPS was injected into mice genetically deficient in the 91 kDa subunit of the respiratory burst oxidase (gp91phox) nor in mice genetically deficient in both gp91phox and iNOS (gp91phox-/-/NOS2-/- mice). For the latter experiments, mice were challenged either with S. typhimurium LPS alone or with inactivated bacille Calmette-Guerin (BCG) followed by Escherichia coli LPS. Deficiency of gp91phox impaired the inflammatory response to inactivated Propionobacterium acnes, rendering survival studies following priming with P. acnes difficult to interpret. Thus, in two models of endotoxic shock, major reductions in the ability to form nitric oxide or superoxide, alone or in combination, failed to improve survival.

Asunto(s)

NADH NADPH Oxidorreductasas/genética , NADPH Oxidasas , Óxido Nítrico Sintasa/genética , Choque Séptico/genética , Animales , Modelos Animales de Enfermedad , Susceptibilidad a Enfermedades/fisiopatología , Endotoxinas/toxicidad , Escherichia coli/patogenicidad , Lipopolisacáridos , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos , Ratones Mutantes , NADH NADPH Oxidorreductasas/deficiencia , Óxido Nítrico Sintasa/deficiencia , Óxido Nítrico Sintasa de Tipo II , Salmonella typhimurium/patogenicidad , Tasa de Supervivencia

RESUMEN

C3H/HeJ mice carry a mutant allele (Lpsd) of a recently identified gene whose normal allele (Lpsn) confers responsiveness to bacterial LPS in C3H/HeN and most other mouse strains. Recently we reported differential display analysis of matched macrophage-derived cell lines from C3H/HeJ and C3H/HeN mice under LPS-free conditions. Of the approximately 12,000 transcripts evaluated, 4 were differentially expressed. One transcript represented secretory leukocyte protease inhibitor. In this study, we report another differentially expressed transcript, mouse matrix metalloprotease-9 (MMP-9). Like secretory leukocyte protease inhibitor, MMP-9 was expressed constitutively in the Lpsd macrophage cell line and not in the Lpsn cell line. Similarly, two additional macrophage cell lines that respond readily to LPS by producing nitric oxide and TNF expressed no MMP-9 under LPS-free conditions. However, in all four cell lines, LPS induced MMP-9 or augmented its expression. In primary macrophages, concentrations of LPS in the ng/ml range augmented the expression of MMP-9 mRNA. Paradoxically, macrophages from Lpsd mice expressed more MMP-9 transcripts than macrophages from Lpsn mice. In contrast, the induction of TNF in response to LPS was much more pronounced in Lpsn macrophages. The present findings with MMP-9 suggest that homozygosity at Lpsd does not so much prevent a response to LPS as dysregulate it, resulting in the suppression of some LPS signaling pathways and the preservation of others.

Asunto(s)

Colagenasas/biosíntesis , Lipopolisacáridos/farmacología , Macrófagos/enzimología , Alelos , Animales , Línea Celular , Colagenasas/genética , Relación Dosis-Respuesta Inmunológica , Inducción Enzimática/genética , Inducción Enzimática/inmunología , Femenino , Regulación de la Expresión Génica/inmunología , Inyecciones Intraperitoneales , Macrófagos/inmunología , Macrófagos Peritoneales/efectos de los fármacos , Macrófagos Peritoneales/enzimología , Macrófagos Peritoneales/inmunología , Metaloproteinasa 9 de la Matriz , Ratones , Ratones Endogámicos C3H , Tioglicolatos/administración & dosificación , Transcripción Genética/inmunología

RESUMEN

The new synthetic oleanane triterpenoid 2-cyano-3,12-dioxoolean-1,9-dien-28-oic acid (CDDO) is a potent, multifunctional molecule. It induces monocytic differentiation of human myeloid leukemia cells and adipogenic differentiation of mouse 3T3-L1 fibroblasts and enhances the neuronal differentiation of rat PC12 pheochromocytoma cells caused by nerve growth factor. CDDO inhibits proliferation of many human tumor cell lines, including those derived from estrogen receptor-positive and -negative breast carcinomas, myeloid leukemias, and several carcinomas bearing a Smad4 mutation. Furthermore, it suppresses the abilities of various inflammatory cytokines, such as IFN-gamma, interleukin-1, and tumor necrosis factor-alpha, to induce de novo formation of the enzymes inducible nitric oxide synthase (iNos) and inducible cyclooxygenase (COX-2) in mouse peritoneal macrophages, rat brain microglia, and human colon fibroblasts. CDDO will also protect rat brain hippocampal neurons from cell death induced by beta-amyloid. The above activities have been found at concentrations ranging from 10(-6) to 10(-9) M in cell culture, and these results suggest that CDDO needs further study in vivo, for either chemoprevention or chemotherapy of malignancy as well as for neuroprotection.

Asunto(s)

Antiinflamatorios no Esteroideos/farmacología , Antineoplásicos/farmacología , Ácido Oleanólico/análogos & derivados , Células 3T3 , Animales , Diferenciación Celular/efectos de los fármacos , Ciclooxigenasa 2 , Relación Dosis-Respuesta a Droga , Humanos , Isoenzimas/efectos de los fármacos , Proteínas de la Membrana , Ratones , Óxido Nítrico Sintasa/antagonistas & inhibidores , Óxido Nítrico Sintasa de Tipo II , Ácido Oleanólico/farmacología , Prostaglandina-Endoperóxido Sintasas/efectos de los fármacos , Ratas

RESUMEN

To determine the relative contributions of respiratory burst-derived reactive oxygen intermediates (ROI) versus reactive nitrogen intermediates (RNI) to macrophage-mediated intracellular host defense, mice genetically deficient in these mechanisms were challenged with Leishmania donovani, a protozoan that selectively parasitizes visceral tissue macrophages. During the early stage of liver infection at wk 2, both respiratory burst-deficient gp91(phox)-/- (X-linked chronic granulomatous disease [X-CGD]) mice and inducible nitric oxide synthase (iNOS) knockout (KO) mice displayed comparably increased susceptibility. Thereafter, infection was unrestrained in mice lacking iNOS but was fully controlled in X-CGD mice. Mononuclear cell influx into infected liver foci in X-CGD and iNOS KO mice was also overtly impaired at wk 2. However, granuloma assembly in parasitized tissue eventually developed in both hosts but with divergent effects: mature granulomas were functionally active (leishmanicidal) in X-CGD mice but inert in iNOS-deficient animals. These results suggest that (a) ROI and RNI probably act together in the early stage of intracellular infection to regulate both tissue recruitment of mononuclear inflammatory cells and the initial extent of microbial replication, (b) RNI alone are necessary and sufficient for eventual control of visceral infection, and (c) although mature granulomas have traditionally been associated with control of such infections, these structures fail to limit intracellular parasite replication in the absence of iNOS.

Asunto(s)

Enfermedad Granulomatosa Crónica/inmunología , Leishmania donovani/inmunología , Leishmaniasis Visceral/inmunología , Activación de Macrófagos/fisiología , Macrófagos/fisiología , Glicoproteínas de Membrana/fisiología , NADPH Oxidasas/fisiología , Óxido Nítrico Sintasa/fisiología , Óxido Nítrico/inmunología , Especies Reactivas de Oxígeno/inmunología , Animales , Predisposición Genética a la Enfermedad , Granuloma/etiología , Granuloma/inmunología , Granuloma/parasitología , Granuloma/patología , Enfermedad Granulomatosa Crónica/complicaciones , Enfermedad Granulomatosa Crónica/genética , Leishmaniasis Visceral/complicaciones , Hígado/parasitología , Macrófagos/parasitología , Glicoproteínas de Membrana/deficiencia , Glicoproteínas de Membrana/genética , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , NADPH Oxidasa 2 , NADPH Oxidasas/deficiencia , NADPH Oxidasas/genética , Óxido Nítrico Sintasa/deficiencia , Óxido Nítrico Sintasa/genética , Óxido Nítrico Sintasa de Tipo II

RESUMEN

Viral infection often activates the interferon (IFN)-gamma-inducible gene, nitric oxide synthase 2 (NOS2). Expression of NOS2 can limit viral growth but may also suppress the immune system and damage tissue. This study assessed each of these effects in genetically deficient NOS2(-/-) mice after infection with influenza A, a virus against which IFN-gamma has no known activity. At inocula sufficient to cause consolidating pneumonitis and death in wild-type control mice, NOS2(-/-) hosts survived with little histopathologic evidence of pneumonitis. Moreover, they cleared influenza A virus from their lungs by an IFN-gamma-dependent mechanism that was not evident in wild-type mice. Even when the IFN-gamma-mediated antiviral activity was blocked in NOS2(-/-) mice with anti-IFN-gamma mAb, such mice failed to succumb to disease. Further evidence that this protection was independent of viral load was provided by treating NOS2(+/+) mice with the NOS inhibitor, Nomega-methyl-L-arginine (L-NMA). L-NMA prevented mortality without affecting viral growth. Thus, host NOS2 seems to contribute more significantly to the development of influenza pneumonitis in mice than the cytopathic effects of viral replication. Although NOS2 mediates some antiviral effects of IFN-gamma, during influenza infection it can suppress another IFN-gamma-dependent antiviral mechanism. This mechanism was observed only in the complete absence of NOS2 activity and appeared sufficient to control influenza A virus growth in the absence of changes in cytotoxic T lymphocyte activity.

Asunto(s)

Virus de la Influenza A/inmunología , Interferón gamma/fisiología , Óxido Nítrico Sintasa/fisiología , Infecciones por Orthomyxoviridae/inmunología , Animales , Pulmón/patología , Pulmón/virología , Ratones , Ratones Endogámicos C57BL , Óxido Nítrico/biosíntesis , Óxido Nítrico Sintasa/deficiencia , Óxido Nítrico Sintasa de Tipo II , Neumonía Viral/prevención & control , Replicación Viral

RESUMEN

To assess whether nitric oxide synthase 2 (NOS2) fulfills the criteria of an innate resistance locus against an acute viral infection, we inoculated genetically deficient NOS2-/- mice with virulent ectromelia virus (EV), the causative agent of mousepox. NOS2-/- mice proved highly susceptible to EV yet showed no diminution in other well-characterized anti-EV immune responses, i.e. , gamma interferon secretion and NK cell and EV-specific cytotoxic T lymphocyte activities. Thus, the NOS2 locus can be considered a critical monogenic determinant of EV resistance that contributes to host survival.

Asunto(s)

Virus de la Ectromelia/fisiología , Óxido Nítrico Sintasa/fisiología , Animales , Línea Celular , Chlorocebus aethiops , Virus de la Ectromelia/inmunología , Femenino , Eliminación de Gen , Inmunidad Innata , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Óxido Nítrico Sintasa/genética , Óxido Nítrico Sintasa de Tipo II

RESUMEN

Mouse secretory leukocyte protease inhibitor (SLPI) was recently characterized as a lipopolysaccharide (LPS)-induced product of macrophages that antagonizes their LPS-induced activation of NF-kappaB and production of NO and tumor necrosis factor (TNF) (F. Y. Jin, C. Nathan, D. Radzioch, and A. Ding, Cell 88:417-426, 1997). To better understand the role of SLPI in innate immune and inflammatory responses, we examined the kinetics of SLPI expression in response to LPS, LPS-induced cytokines, and LPS-mimetic compounds. SLPI mRNA was detectable in macrophages by Northern blot analysis within 30 min of exposure to LPS but levels peaked only at 24 to 36 h and remained elevated at 72 h. Despite the slowly mounting and prolonged response, early expression of SLPI mRNA was cycloheximide resistant. Two LPS-induced proteins-interleukin-10 (IL-10) and IL-6-also induced SLPI, while TNF and IL-1beta did not. The slow attainment of maximal induction of SLPI by LPS in vitro was mimicked by infection with Pseudomonas aeruginosa in vivo, where SLPI expression in the lung peaked at 3 days. Two LPS-mimetic molecules-taxol from yew bark and lipoteichoic acid (LTA) from gram-positive bacterial cell walls-also induced SLPI. Transfection of macrophages with SLPI inhibited their LTA-induced NO production. An anti-inflammatory role for macrophage-derived SLPI seems likely based on SLPI's slowly mounting production in response to constituents of gram-negative and gram-positive bacteria, its induction both as a direct response to LPS and as a response to anti-inflammatory cytokines induced by LPS, and its ability to suppress the production of proinflammatory products by macrophages stimulated with constituents of both gram-positive and gram-negative bacteria.

Asunto(s)

Lipopolisacáridos/inmunología , Macrófagos/inmunología , Biosíntesis de Proteínas , Animales , Inducción Enzimática , Escherichia coli/inmunología , Femenino , Genes Inmediatos-Precoces , Interleucina-10/inmunología , Interleucina-6/inmunología , Enfermedades Pulmonares/inmunología , Ratones , Ratones Endogámicos C3H , Ratones Endogámicos C57BL , Paclitaxel/farmacología , Proteínas Inhibidoras de Proteinasas Secretoras , Proteínas/genética , Infecciones por Pseudomonas/inmunología , Inhibidor Secretorio de Peptidasas Leucocitarias , Staphylococcus aureus/inmunología

RESUMEN

Resuscitation from hemorrhagic shock induces profound changes in the physiologic processes of many tissues and activates inflammatory cascades that include the activation of stress transcriptional factors and upregulation of cytokine synthesis. This process is accompanied by acute organ damage (e.g., lungs and liver). We have previously demonstrated that the inducible nitric oxide synthase (iNOS) is expressed during hemorrhagic shock. We postulated that nitric oxide production from iNOS would participate in proinflammatory signaling. Using the iNOS inhibitor N6-(iminoethyl)-L-lysine or iNOS knockout mice we found that the activation of the transcriptional factors nuclear factor kappaB and signal transducer and activator of transcription 3 and increases in IL-6 and G-CSF messenger RNA levels in the lungs and livers measured 4 h after resuscitation from hemorrhagic shock were iNOS dependent. Furthermore, iNOS inhibition resulted in a marked reduction of lung and liver injury produced by hemorrhagic shock. Thus, induced nitric oxide is essential for the upregulation of the inflammatory response in resuscitated hemorrhagic shock and participates in end organ damage under these conditions.

Asunto(s)

Inflamación/etiología , Óxido Nítrico/fisiología , Choque Hemorrágico/inmunología , Animales , Proteínas de Unión al ADN/metabolismo , Factor Estimulante de Colonias de Granulocitos/genética , Interleucina-6/genética , Pulmón/patología , Masculino , Ratones , Ratones Noqueados , FN-kappa B/metabolismo , Óxido Nítrico Sintasa/antagonistas & inhibidores , Óxido Nítrico Sintasa/fisiología , Óxido Nítrico Sintasa de Tipo II , Ratas , Ratas Sprague-Dawley , Factor de Transcripción STAT3 , Transactivadores/metabolismo

RESUMEN

Mutagenesis of the host immune system has helped identify response pathways necessary to combat tuberculosis. Several such pathways may function as activators of a common protective gene: inducible nitric oxide synthase (NOS2). Here we provide direct evidence for this gene controlling primary Mycobacterium tuberculosis infection using mice homozygous for a disrupted NOS2 allele. NOS2(-/-) mice proved highly susceptible, resembling wild-type littermates immunosuppressed by high-dose glucocorticoids, and allowed Mycobacterium tuberculosis to replicate faster in the lungs than reported for other gene-deficient hosts. Susceptibility appeared to be independent of the only known naturally inherited antimicrobial locus, NRAMP1. Progression of chronic tuberculosis in wild-type mice was accelerated by specifically inhibiting NOS2 via administration of N6-(1-iminoethyl)-L-lysine. Together these findings identify NOS2 as a critical host gene for tuberculostasis.

Asunto(s)

Proteínas Portadoras/genética , Proteínas de Transporte de Catión , Inmunidad Innata/genética , Proteínas de la Membrana/genética , Mycobacterium tuberculosis/fisiología , Óxido Nítrico Sintasa/deficiencia , Óxido Nítrico Sintasa/genética , Tuberculosis/genética , Tuberculosis/inmunología , Alelos , Animales , Proteínas Portadoras/biosíntesis , Cruzamientos Genéticos , Susceptibilidad a Enfermedades , Exones , Femenino , Genotipo , Glucocorticoides/farmacología , Haplotipos , Heterocigoto , Homocigoto , Terapia de Inmunosupresión , Isoenzimas/biosíntesis , Isoenzimas/deficiencia , Isoenzimas/genética , Pulmón/microbiología , Pulmón/patología , Masculino , Proteínas de la Membrana/biosíntesis , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos , Ratones Noqueados , Mycobacterium tuberculosis/aislamiento & purificación , Óxido Nítrico Sintasa/biosíntesis , Reacción en Cadena de la Polimerasa , Polimorfismo Genético , Tuberculosis/patología

RESUMEN

The identification of human inflammatory cells that express inducible nitric oxide synthase and the clarification of the role of inducible nitric oxide synthase in human infectious or inflammatory processes have been elusive. In neutrophil-enriched fractions from urine, we demonstrate a 43-fold increase in nitric oxide synthase activity in patients with urinary tract infections compared with that in neutrophil-enriched fractions from noninfected controls. Partially purified inducible nitric oxide synthase is primarily membrane associated, calcium independent, and inhibited by arginine analogues with a rank order consistent with that of purified human inducible nitric oxide synthase. Molecular, biochemical, and immunocytochemical evidence unequivocally identifies inducible nitric oxide synthase as the major nitric oxide synthase isoform found in neutrophils isolated from urine during urinary tract infections. Elevated inducible nitric oxide synthase activity and elevated nitric oxide synthase protein measured in patients with urinary tract infections and treated with antibiotics does not decrease until 6-10 d of antibiotic treatment. The extended elevation of neutrophil inducible nitric oxide synthase during urinary tract infections may have both antimicrobial and proinflammatory functions.

Asunto(s)

Infecciones Bacterianas/enzimología , Neutrófilos/enzimología , Óxido Nítrico Sintasa/biosíntesis , Infecciones Urinarias/enzimología , Adulto , Anciano , Antibacterianos/farmacología , Arginina/farmacología , Infecciones Bacterianas/orina , Western Blotting , Canavanina/farmacología , Membrana Celular/enzimología , Citrulina/análogos & derivados , Citrulina/farmacología , Femenino , Guanidinas/farmacología , Humanos , Inmunohistoquímica , Isoenzimas/biosíntesis , Isoenzimas/aislamiento & purificación , Isoenzimas/metabolismo , Antígenos Comunes de Leucocito/inmunología , Masculino , Persona de Mediana Edad , NG-Nitroarginina Metil Éster/farmacología , Óxido Nítrico Sintasa/efectos de los fármacos , Óxido Nítrico Sintasa/aislamiento & purificación , Nitroarginina/farmacología , Ornitina/análogos & derivados , Ornitina/farmacología , Reacción en Cadena de la Polimerasa , ARN Mensajero/análisis , Sulfonamidas/farmacología , Tiourea/análogos & derivados , Tiourea/farmacología , Trifluoperazina/farmacología , Infecciones Urinarias/orina , omega-N-Metilarginina/farmacología

RESUMEN

IFN-gamma and TNF-alpha, potent inhibitors of hematopoiesis, induce nitric oxide synthase (NOS) in various cell types. When normal human bone marrow (BM) or CD34+ cells were exposed to NO, inhibition of colony formation was dose dependent and direct. NO induced apoptosis in BM progenitors, as shown by electrophoretic detection of DNA degradation and deoxynucleotidyl transferase assay. Using PCR and immunoprecipitation, we found inducible NOS (iNOS) mRNA and iNOS protein in BM after stimulation with IFN-gamma or TNF-alpha. iNOS mRNA was also detected by PCR in highly purified CD34+ cells; TNF-alpha or IFN-gamma increased iNOS expression. The presence of iNOS in CD34+ cells was confirmed in single cells by immunochemical staining. NG-Monomethyl-L-arginine (MM-Arg), an NOS inhibitor, partially reversed the effects of TNF-alpha and, to a lesser extent, IFN-gamma in methylcellulose culture of total BM and CD34+ cells, and inhibited apoptosis of BM cells induced by these cytokines. When the effects of competitive iNOS inhibition were tested on more immature progenitors, MM-Arg increased the number of long-term BM culture-initiating cells in control cultures but failed to protect these cells from the inhibitory action of IFN-gamma and TNF-alpha. Our results suggest that NO may be one mediator of cytokine-induced hematopoietic suppression.

Asunto(s)

Aminoácido Oxidorreductasas/metabolismo , Hematopoyesis/efectos de los fármacos , Células Madre Hematopoyéticas/efectos de los fármacos , Interferón gamma/farmacología , Óxido Nítrico/farmacología , Factor de Necrosis Tumoral alfa/farmacología , Apoptosis/efectos de los fármacos , Arginina/análogos & derivados , Arginina/farmacología , Secuencia de Bases , Benzamidas/farmacología , Células de la Médula Ósea , Células Cultivadas , Ensayo de Unidades Formadoras de Colonias , Inducción Enzimática/efectos de los fármacos , Humanos , Datos de Secuencia Molecular , Óxido Nítrico Sintasa , Reacción en Cadena de la Polimerasa , ARN Mensajero/biosíntesis , ARN Mensajero/genética , omega-N-Metilarginina

RESUMEN

Hemorrhagic tumor necrosis is an inflammatory event that leads to selective destruction of malignant tissues, with both potentially toxic and beneficial consequences. A pilot clinical trial was undertaken combining tumor necrosis factor-alpha (TNF-alpha) with the monoclonal antibody R24 (MoAb R24) against GD3 ganglioside in patients with metastatic melanoma. Patients received MoAb R24 to recruit leukocytes to the tumor followed by low doses of recombinant TNF-alpha to activate leukocytes. Eight patients were treated and seven patients had mild toxicity. One patient with extensive metastatic melanoma developed tumor lysis syndrome within hours after treatment with almost complete necrosis of bulky tumors in multiple visceral sites. To our knowledge, this is the first documented case of hemorrhagic tumor necrosis in a patient with metastatic cancer in multiple visceral sites.

Asunto(s)

Anticuerpos Monoclonales/uso terapéutico , Protocolos de Quimioterapia Combinada Antineoplásica/efectos adversos , Gangliósidos/inmunología , Melanoma/terapia , Síndrome de Lisis Tumoral/etiología , Factor de Necrosis Tumoral alfa/efectos adversos , Adulto , Anciano , Anticuerpos Monoclonales/administración & dosificación , Femenino , Humanos , Masculino , Melanoma/patología , Melanoma/secundario , Persona de Mediana Edad , N-Formilmetionina Leucil-Fenilalanina/farmacología , Necrosis , Neutrófilos/efectos de los fármacos , Neutrófilos/metabolismo , Proteínas Recombinantes/efectos adversos , Acetato de Tetradecanoilforbol/farmacología , Factor de Necrosis Tumoral alfa/administración & dosificación

RESUMEN

Microtubule-associated proteins may mediate the activation of macrophages by bacterial LPS. Three lines of evidence support this hypothesis: a) Taxol, a microtubule-binding diterpene, mimics the ability of LPS to induce cytokines and down-regulate receptors for TNF-alpha. In recombinant inbred mouse strains differing at the Lps gene, responsiveness to these effects of Taxol co-segregates with responsiveness to LPS. b) In vitro, LPS binds specifically to MT and preferentially to beta-tubulin. c) LPS activates microtubule-associated protein-2 kinase (MAPK). The present studies bring together and extend these lines of evidence. a) Taxol, like LPS, rapidly induces tyrosine phosphorylation of MAPK in mouse macrophages, and triggers MAPK to phosphorylate an exogenous substrate. b) Tyrosine phosphorylation of MAPK is an extremely rapid cellular response both to taxol and LPS. c) Macrophages from C3H/HeJ mice, which carry a defective Lps gene, fail to activate MAPK in response to taxol or LPS, although they activate MAPK in response to insulin or IFN-gamma. These results suggest that tyrosine phosphorylation of MAPK is among the earliest known response of macrophages to LPS. Taxol mimics LPS with respect to immediate MAPK activation, later transcriptional events, and the genetic control of both sets of responses. LPS and taxol thus appear to share an early step in a functionally important signal transduction pathway that may involve MT.

Asunto(s)

Proteínas Quinasas Dependientes de Calcio-Calmodulina/metabolismo , Lipopolisacáridos/farmacología , Paclitaxel/farmacología , Tirosina/metabolismo , Animales , Activación Enzimática/efectos de los fármacos , Femenino , Insulina/farmacología , Interferón gamma/farmacología , Macrófagos/metabolismo , Ratones , Ratones Endogámicos C3H , Fosforilación

RESUMEN

The formation of nitric oxide by human phagocytes as measured by nitrite production is controversial. We report here that nitrite production by phorbol myristate acetate (PMA)-stimulated human polymorphonuclear leukocytes (PMN) is considerably increased by the addition of azide and a further increase occurs when catalase also is added. Nitrite production by the PMN-PMA-azide-catalase system is unaffected by superoxide dismutase or monomethylarginine but is markedly reduced by the substitution of chronic granulomatous disease for normal neutrophils. The stimulated neutrophils could be replaced by the H2O2-generating enzyme system glucose-glucose oxidase. These findings suggest that nitrite production does not, in this instance, reflect nitric oxide synthase activity by human neutrophils but rather the catalase-catalyzed conversion of azide to nitrite in the presence of H2O2 generated by the stimulated PMN.

Asunto(s)

Azidas/metabolismo , Catalasa/metabolismo , Neutrófilos/metabolismo , Nitritos/metabolismo , Glucosa/metabolismo , Humanos , Peróxido de Hidrógeno/metabolismo , Neutrófilos/efectos de los fármacos , Oxidación-Reducción , Acetato de Tetradecanoilforbol/farmacología