RESUMEN
BACKGROUND: Asthma is a chronic respiratory condition, with airway hyperresponsiveness (AHR) and inflammation as hallmarks. The hypothesis that the substantially increased expression of arginase 1 in activated macrophages limits the availability of L-arginine for nitric oxide synthesis, and thus increases AHR in lungs of mice with experimentally induced allergic asthma was recently refuted by several studies. In the present study, we tested the hypothesis that, instead, a low circulating concentration of arginine aggravates AHR in the same murine asthma model. Female FVB F/A2 tg/tg transgenic mice, which overexpress rat arginase 1 in their enterocytes, exhibit a ~ 50% decrease of their plasma L-arginine concentration. METHODS: Adult female F/A2 tg/tg mice and their wild-type littermates (F/A2 wt/wt ) were sensitized and challenged with ovalbumin (OVA/OVA). Lung function was assessed with the flexiVent™ system. Adaptive changes in the expression of arginine-metabolizing or -transporting enzymes, chemokines and cytokines, and lung histology were quantified with qPCR, ELISA, and immunohistochemistry, respectively. RESULTS: Reduction of circulating L-arginine concentration significantly increased AHR in OVA/OVA-treated mice and, to a lesser extent, even in PBS/OVA-treated mice. The pulmonary inflammatory response in OVA/OVA-treated F/A2 tg/tg and F/A2 wt/wt mice was comparable. OVA/OVA-treated F/A2 tg/tg mice differed from similarly treated female mice, in which arginase 1 expression in lung macrophages was eliminated, by a complete absence of an adaptive increase in the expression of arginine-metabolizing or -transporting enzymes. CONCLUSION: A reduction of the circulating L-arginine concentration rather than the macrophage-mediated increase of arginine catabolism worsens AHR.
Asunto(s)
Arginina/sangre , Asma/sangre , Pulmón/metabolismo , Hipersensibilidad Respiratoria/sangre , Animales , Arginasa/biosíntesis , Arginina/deficiencia , Asma/patología , Hiperreactividad Bronquial/sangre , Hiperreactividad Bronquial/patología , Femenino , Pulmón/patología , Ratones , Ratones Transgénicos , Hipersensibilidad Respiratoria/patologíaRESUMEN
BACKGROUND: (Over-)expression of arginase may limit local availability of arginine for nitric oxide synthesis. We investigated the significance of arginase1 (ARG1) for the development of airway hyperresponsiveness (AHR) and lung inflammation in female mice with ovalbumin (OVA)-induced allergic asthma. METHODS: Arg1 was ablated in the lung by crossing Arg1 fl/fl and Tie2Cre tg/- mice. OVA sensitization and challenge were conducted, and AHR to methacholine was determined using the Flexivent system. Changes in gene expression, chemokine and cytokine secretion, plasma IgE, and lung histology were quantified using RT-qPCR, ELISA, and immunohistochemistry, respectively. RESULTS: Arg1 ablation had no influence on the development of OVA-induced AHR, but attenuated OVA-induced increases in expression of Arg2 and Nos2, Slc7a1, Slc7a2, and Slc7a7 (arginine transporters), Il4, Il5 and Il13 (TH2-type cytokines), Ccl2 and Ccl11 (chemokines), Ifng (TH1-type cytokine), Clca3 and Muc5ac (goblet cell markers), and OVA-specific IgE. Pulmonary IL-10 protein content increased, but IL-4, IL-5, IL-13, TNFα and IFNγ content, and lung histopathology, were not affected. Arg1 elimination also decreased number and tightness of correlations between adaptive changes in lung function and inflammatory parameters in OVA/OVA-treated female mice. OVA/OVA-treated female mice mounted a higher OVA-IgE response than males, but the correlation between lung function and inflammation was lower. Arg1-deficient OVA/OVA-treated females differed from males in a more pronounced decline of arginine-metabolizing and -transporting genes, higher plasma arginine levels, a smaller OVA-specific IgE response, and no improvement of peripheral lung function. CONCLUSION: Complete ablation of Arg1 in the lung affects mRNA abundance of arginine-transporting and -metabolizing genes, and pro-inflammatory genes, but not methacholine responsiveness or accumulation of inflammatory cells.
Asunto(s)
Arginasa/genética , Asma/genética , Asma/metabolismo , Citocinas/genética , ARN Mensajero/metabolismo , Resistencia de las Vías Respiratorias/genética , Sistema de Transporte de Aminoácidos y+/genética , Sistema de Transporte de Aminoácidos y+L , Sistemas de Transporte de Aminoácidos Básicos/genética , Animales , Arginasa/metabolismo , Arginina/sangre , Asma/inducido químicamente , Asma/fisiopatología , Transportador de Aminoácidos Catiónicos 1/genética , Citocinas/metabolismo , Femenino , Expresión Génica , Inmunoglobulina E/sangre , Macrófagos/metabolismo , Ratones , Ratones Noqueados , Células Mieloides , Óxido Nítrico Sintasa de Tipo II/genética , Ovalbúmina , Neumonía/genética , Neumonía/patología , Mecánica Respiratoria/genéticaRESUMEN
Variegate porphyria (VP) and acute intermittent porphyria (AIP), the two most common types of acute porphyrias (AHPs), result from a partial deficiency of protoporphyrinogen oxidase (PPOX) and hydroxymethylbilane synthase (HMBS), respectively. A rare but serious complication in the AHPs is hepatocellular carcinoma (HCC). However, the underlying pathomechanisms are yet unknown. We performed DNA sequence analysis in cancerous and non-cancerous liver tissue of a VP and an AIP patient, both with HCC. In samples of both cancerous and non-cancerous liver tissues from the patients, we identified the underlying PPOX and HMBS germline mutations, c.1082dupC and p.G111R, respectively. Additionally, we detected a second somatic mutation, only in the cancer tissue i.e., p.L416X in the PPOX gene of the VP patient and p.L220X in the HMBS gene of the AIP patient, both located in trans to the respective germline mutations. Both somatic mutations were not detected in 10 non-porphyria-associated HCCs. Our data demonstrate that in the hepatic cancer tissue of AHP patients, somatic second-hit mutations result in nearly complete inactivation of the enzymes catalyzing major steps in the heme biosynthetic pathway. Both PPOX and HMBS, which might act as tumor suppressors, play a crucial role in the development of HCC in these individuals.
Asunto(s)
Carcinoma Hepatocelular/etiología , Carcinoma Hepatocelular/genética , Flavoproteínas/genética , Hidroximetilbilano Sintasa/genética , Neoplasias Hepáticas/etiología , Neoplasias Hepáticas/genética , Proteínas Mitocondriales/deficiencia , Proteínas Mitocondriales/genética , Mutación , Porfiria Intermitente Aguda/complicaciones , Porfiria Intermitente Aguda/genética , Porfiria Variegata/complicaciones , Porfiria Variegata/genética , Protoporfirinógeno-Oxidasa/genética , Anciano , Anciano de 80 o más Años , Carcinoma Hepatocelular/enzimología , Femenino , Mutación de Línea Germinal , Humanos , Neoplasias Hepáticas/enzimología , Porfiria Intermitente Aguda/enzimología , Porfiria Variegata/enzimología , Proteínas Supresoras de Tumor/deficiencia , Proteínas Supresoras de Tumor/genéticaRESUMEN
Asthma is a chronic inflammatory disease of the small airways, with airway hyperresponsiveness (AHR) and inflammation as hallmarks. Recent studies suggest a role for arginase in asthma pathogenesis, possibly because arginine is the substrate for both arginase and NO synthase and because NO modulates bronchial tone and inflammation. Our objective was to investigate the importance of increased pulmonary arginase 1 expression on methacholine-induced AHR and lung inflammation in a mouse model of allergic asthma. Arginase 1 expression in the lung was ablated by crossing Arg1(fl/fl) with Tie2Cre(tg/-) mice. Mice were sensitized and then challenged with ovalbumin. Lung function was measured with the Flexivent. Adaptive changes in gene expression, chemokine and cytokine secretion, and lung histology were quantified with quantitative PCR, ELISA, and immunohistochemistry. Arg1 deficiency did not affect the allergic response in lungs and large-airway resistance, but it improved peripheral lung function (tissue elastance and resistance) and attenuated adaptive increases in mRNA expression of arginine-catabolizing enzymes Arg2 and Nos2, arginine transporters Slc7a1 and Slc7a7, chemokines Ccl2 and Ccl11, cytokines Tnfa and Ifng, mucus-associated epithelial markers Clca3 and Muc5ac, and lung content of IL-13 and CCL11. However, expression of Il4, Il5, Il10, and Il13 mRNA; lung content of IL-4, IL-5, IL-10, TNF-α, and IFN-γ protein; and lung pathology were not affected. Correlation analysis showed that Arg1 ablation disturbed the coordinated pulmonary response to ovalbumin challenges, suggesting arginine (metabolite) dependence of this response. Arg1 ablation in the lung improved peripheral lung function and affected arginine metabolism but had little effect on airway inflammation.
Asunto(s)
Arginasa/fisiología , Asma/fisiopatología , Hiperreactividad Bronquial/patología , Hipersensibilidad/patología , Pulmón/fisiología , Neumonía/patología , Sistema Respiratorio/patología , Resistencia de las Vías Respiratorias/fisiología , Animales , Western Blotting , Hiperreactividad Bronquial/inducido químicamente , Hiperreactividad Bronquial/metabolismo , Broncoconstrictores/toxicidad , Quimiocinas/metabolismo , Citocinas/metabolismo , Células Dendríticas/citología , Células Dendríticas/metabolismo , Femenino , Perfilación de la Expresión Génica , Hipersensibilidad/metabolismo , Técnicas para Inmunoenzimas , Pulmón/citología , Macrófagos/citología , Macrófagos/metabolismo , Masculino , Cloruro de Metacolina/toxicidad , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Células Mieloides/citología , Células Mieloides/metabolismo , Ovalbúmina/fisiología , Neumonía/inducido químicamente , Neumonía/metabolismo , ARN Mensajero/genética , Reacción en Cadena en Tiempo Real de la Polimerasa , Sistema Respiratorio/efectos de los fármacos , Sistema Respiratorio/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
RATIONALE: Chronic obstructive pulmonary disease (COPD) is associated with increased numbers of CD8(+) cytotoxic T lymphocytes (CTLs) in the lung, but the functional activity of CTLs remains unknown. Granzyme A (GrA) and B (GrB) are serine proteases considered to be important effector molecules of CTLs and natural killer cells. OBJECTIVE: To investigate protein and mRNA expression of GrA and GrB in peripheral lung tissue from patients with COPD and control subjects with normal lung function. METHODS: Paraffin-embedded sections of surgical lung specimens from 22 patients with COPD (FEV(1), 22% predicted; GOLD stage 4) and 15 control subjects (FEV(1), 108% predicted) were immunostained for GrA and GrB, and semiquantified on a 3-point scale. Messenger RNA expression in total lung, specific cell types enriched for by laser capture microdissection, and freshly isolated primary cells were determined by reverse transcriptase-polymerase chain reaction. MEASUREMENTS AND MAIN RESULTS: GrA and GrB immunoreactivity was observed in CD8(+) CTLs and CD57(+) natural killer cells, but also in type II pneumocytes and alveolar macrophages in both groups. Bronchiolar epithelium stained positive for GrA, but negative for GrB. These observations were confirmed by reverse transcriptase-polymerase chain reaction on total lung, laser capture microdissection-enriched specific cell types and freshly isolated primary type II pneumocytes. The scores of GrA-expressing type II pneumocytes were significantly higher in patients with COPD versus control subjects. CONCLUSIONS: GrA and GrB mRNA and protein are detectable in human lung tissue. GrA expression is increased in type II pneumocytes of patients with very severe COPD. These results indicate that GrA may be important in the development of COPD.
Asunto(s)
Bronquios/enzimología , Expresión Génica , Granzimas/genética , Alveolos Pulmonares/enzimología , Enfermedad Pulmonar Obstructiva Crónica , ARN Mensajero/genética , Bronquios/patología , Femenino , Estudios de Seguimiento , Granzimas/biosíntesis , Humanos , Masculino , Persona de Mediana Edad , Reacción en Cadena de la Polimerasa , Pronóstico , Alveolos Pulmonares/patología , Enfermedad Pulmonar Obstructiva Crónica/enzimología , Enfermedad Pulmonar Obstructiva Crónica/genética , Enfermedad Pulmonar Obstructiva Crónica/patología , Índice de Severidad de la EnfermedadRESUMEN
This study investigated local and systemic innate immune responses in lipopolysaccharide (LPS)-induced lung inflammation in mice. Intratracheal LPS exposure resulted in increased pulmonary mRNA expression for acute-phase reactants (APRs) alpha(1)-antitrypsin (alpha(1)-AT), alpha(1)-acid glycoprotein (AGP), and LPS-binding protein (LBP) from 4 hours post exposure. Although pulmonary serum amyloid P component (SAP) mRNA was not increased, systemic levels of SAP, AGP, and LBP were elevated from 24 hours post exposure. Systemic APRs increase was associated with hepatic mRNA expression. As in vivo neutralization of interleukin (IL)-6, but not tumor necrosis factor (TNF)-alpha, fully ablated hepatic APR mRNA expression, IL-6 may act as signaling molecule between lung and liver. In conclusion, pulmonary LPS exposure induced rapid APR expression in lung, which precedes IL-6-mediated systemic elevation of APRs associated with hepatic APRs expression.