RESUMEN
TNF-α, a potent proinflammatory cytokine, is generated in a precursor form called transmembrane (m)TNF-α that is expressed as a type II polypeptide on the surface of certain cells. mTNF-α was shown to act both as a ligand by binding to TNF-α receptors, as well as a receptor that transmits outside-to-inside (reverse) signals back into the mTNF-α-bearing cells. In this study, we show that nonactivated macrophages express basal levels of mTNF-α and respond to anti-TNF-α Abs by triggering the MAPK kinase 4 signaling pathway. The pathway induces TGF-ß. Based on inhibitory experiments, the production of TGF-ß1 is regulated via Jun kinases, whereas that of other TGF-ßs is regulated via p38 MAPKs. Exposure to LPS further induced the expression of mTNF-α, and triggering of mTNF-α strongly suppressed the LPS-induced proinflammatory response. Neutralizing TGF-ß by Abs prevented the mTNF-α-mediated suppression of LPS-induced proinflammatory cytokine formation, indicating that the immune-suppressive effect of mTNF-α is mediated via TGF-ß. Although apoptotic cells are also known to suppress LPS-induced proinflammatory cytokine formation in macrophages by upregulating TGF-ß, we show that they do not use the mTNF-α signaling pathway. Because TGF-ß possesses a wide range of immune-suppressive effects, our data indicate that upregulation of TGF-ß synthesis by those TNF-α-targeting molecules, which are able to trigger mTNF-α, might contribute to their therapeutic effect in the treatment of certain inflammatory diseases such as Crohn's disease, Wegener's granulomatosis, or sarcoidosis. Additionally, none of the TNF-α-targeting molecules is expected to interfere with the immune-silencing effects of apoptotic cells.
Asunto(s)
Citocinas/biosíntesis , Macrófagos/inmunología , Transducción de Señal/inmunología , Factor de Crecimiento Transformador beta/inmunología , Factor de Necrosis Tumoral alfa/inmunología , Animales , Antiinflamatorios/farmacología , Apoptosis/inmunología , Western Blotting , Citocinas/inmunología , Técnica del Anticuerpo Fluorescente , Humanos , Inmunohistoquímica , Lipopolisacáridos/inmunología , Lipopolisacáridos/farmacología , Macrófagos/efectos de los fármacos , Masculino , Ratones , Microscopía Confocal , Reacción en Cadena de la Polimerasa , Transducción de Señal/efectos de los fármacos , Factor de Crecimiento Transformador beta/biosíntesisRESUMEN
Transglutaminase 2 (TG2) has been known for a long time to be associated with the in vivo apoptosis program of various cell types, including T cells. Though the expression of the enzyme is strongly induced in mouse thymocytes following apoptosis induction in vivo, no significant induction of TG2 can be detected, when thymocytes are induced to die by the same stimuli in vitro indicating that signals arriving from the tissue environment are required for the proper in vivo induction of the enzyme. Previous studies from our laboratory have demonstrated that two of these signals, transforming growth factor-ß (TGF-ß) and retinoids, are produced by macrophages engulfing apoptotic cells. However, in addition to TGF-ß and retinoids, engulfing macrophages produce adenosine as well. Here, we show that in vitro adenosine, adenosine, and retinoic acid or adenosine, TGF-ß and retinoic acids together can significantly enhance the TG2 mRNA expression in dying thymocytes. The effect of adenosine is mediated via adenosine A2A receptors (A2ARs) and the A2AR-triggered adenylate cyclase signaling pathway. In accordance, loss of A2ARs in A2AR null mice significantly attenuates the in vivo induction of TG2 following apoptosis induction in the thymus indicating that adenosine indeed contributes in vivo to the apoptosis-related appearance of the enzyme. We also demonstrate that adenosine is produced extracellularly during engulfment of apoptotic thymocytes, partly from adenine nucleotides released via thymocyte pannexin-1 channels. Our data reveal a novel crosstalk between macrophages and apoptotic cells, in which apoptotic cell uptake-related adenosine production contributes to the appearance of TG2 in the dying thymocytes.
Asunto(s)
Adenosina/farmacología , Proteínas de Unión al GTP/genética , Macrófagos/efectos de los fármacos , Receptor de Adenosina A2A/genética , Timocitos/efectos de los fármacos , Transglutaminasas/genética , Adenosina/metabolismo , Adenosina Trifosfato/metabolismo , Animales , Apoptosis , Comunicación Celular , Técnicas de Cocultivo , Proteínas de Unión al GTP/agonistas , Proteínas de Unión al GTP/metabolismo , Expresión Génica , Macrófagos/citología , Macrófagos/inmunología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Fagocitosis , Proteína Glutamina Gamma Glutamiltransferasa 2 , ARN Mensajero/genética , ARN Mensajero/metabolismo , Receptor de Adenosina A2A/metabolismo , Transducción de Señal , Timocitos/inmunología , Timocitos/patología , Factor de Crecimiento Transformador beta/metabolismo , Factor de Crecimiento Transformador beta/farmacología , Transglutaminasas/metabolismo , Tretinoina/metabolismo , Tretinoina/farmacologíaRESUMEN
Tumor necrosis factor (TNF)-α is a potent pro-inflammatory cytokine exerting pleiotropic effects on various cell types. It is synthesized in a precursor form called transmembrane TNF-α (mTNF-α) which, after being processed by metalloproteinases, is released in a soluble form to mediate its biological activities through Type 1 and 2 TNF receptors in TNF receptor expressing cells. In addition to acting in soluble form, TNF-α also acts in the transmembrane form both as a ligand by activating TNF receptors, as well as a receptor that transmits outside-to-inside (reverse) signals back into mTNF-α bearing cells. Since the discovery that TNF-α plays a determining role in the pathogenesis of several chronic inflammatory diseases, anti-TNF agents are increasingly being used in the treatment of a rapidly expanding number of rheumatic and systemic autoimmune diseases, such as rheumatoid arthritis, Crohn's disease, psoriasis, psoriatic arthritis, ankyloting spondylitis, Wegener granulomatosis and sarcoidosis. There are 5 TNF antagonists currently available: etanercept, a soluble TNF receptor construct; infliximab, a chimeric monoclonal antibody; adalimumab and golimumab, fully human antibodies; and certolizumab pegol, an Fab' fragment of a humanized anti-TNF-α antibody. Though each compound can efficiently neutralize TNF-α, increasing evidence suggests that they show different efficacy in the treatment of these diseases. These observations indicate that in addition to neutralizing TNF-α, other biological effects induced by TNF-α targeting molecules dictate the success of the therapy. Recently, we found that mTNF-α reverse signaling leads to transforming growth factor (TGF)-ß production in macrophages and anti-TNF agents selectively trigger this pathway. In this review we will focus on the potential contribution of the activation of the mTNF-α signaling pathway to the success of the anti-TNF therapy.
Asunto(s)
Antiinflamatorios/farmacología , Antiinflamatorios/uso terapéutico , Proteínas de la Membrana/metabolismo , Transducción de Señal/fisiología , Factor de Crecimiento Transformador beta/metabolismo , Factor de Necrosis Tumoral alfa/metabolismo , Animales , Humanos , Inflamación/tratamiento farmacológico , Inflamación/metabolismo , Macrófagos/efectos de los fármacos , Transducción de Señal/efectos de los fármacosRESUMEN
Efficient execution of apoptotic cell death followed by efficient clearance mediated by professional macrophages is a key mechanism in maintaining tissue homeostasis. Removal of apoptotic cells usually involves three central elements: 1) attraction of phagocytes via soluble "find me" signals, 2) recognition and phagocytosis via cell surface-presenting "eat me" signals, and 3) suppression or initiation of inflammatory responses depending on additional innate immune stimuli. Suppression of inflammation involves both direct inhibition of proinflammatory cytokine production and release of anti-inflammatory factors, which all contribute to the resolution of inflammation. In the current study, using wild-type and adenosine A(2A) receptor (A2AR) null mice, we investigated whether A2ARs, known to mediate anti-inflammatory signals in macrophages, participate in the apoptotic cell-mediated immunosuppression. We found that macrophages engulfing apoptotic cells release adenosine in sufficient amount to trigger A2ARs, and simultaneously increase the expression of A2ARs, as a result of possible activation of liver X receptor and peroxisome proliferators activated receptor δ. In macrophages engulfing apoptotic cells, stimulation of A2ARs suppresses the NO-dependent formation of neutrophil migration factors, such as macrophage inflammatory protein-2, using the adenylate cyclase/protein kinase A pathway. As a result, loss of A2ARs results in elevated chemoattractant secretion. This was evident as pronounced neutrophil migration upon exposure of macrophages to apoptotic cells in an in vivo peritonitis model. Altogether, our data indicate that adenosine is one of the soluble mediators released by macrophages that mediate engulfment-dependent apoptotic cell suppression of inflammation.
Asunto(s)
Adenosina/inmunología , Apoptosis/inmunología , Inflamación/inmunología , Fagocitosis/inmunología , Receptor de Adenosina A2A/inmunología , Animales , Inflamación/patología , Macrófagos/inmunología , Ratones , Ratones Noqueados , Peritonitis/inmunologíaRESUMEN
INTRODUCTION: Transglutaminase 2 (TG2), a protein crosslinking enzyme with multiple biochemical functions, has been connected to various inflammatory processes. In this study, the involvement of TG2 in monosodium urate (MSU) crystal-induced inflammation was studied. METHODS: Immunohistochemistry, reverse transcription-polymerase chain reaction (RT-PCR) were performed to detect TG2 expression in synovial fluid mononuclear cells (SFMCs) and synovial tissue from patients with gouty arthritis. MSU crystal-exposed RAW264.7 mouse macrophages were analyzed for interleukin-1ß (IL-1ß), tumor necrosis factor-α (TNF-α), transforming growth factor ß1 (TGF-ß1) and TG2 expression by RT-PCR and enzyme-linked immunosorbent assay (ELISA). TG2 small interfering (si)-RNA-mediated silencing and overexpression in RAW264.7 cells were used to evaluate the involvement of TG2 in resolving MSU crystal-induced inflammation. The role of metastatic tumor antigen 1 (MTA1), a master chromatin modifier, was investigated by MTA1 si-RNA-mediated knockdown. In addition, the inflammatory responses were followed in wild type and TG2 null mice after being challenged with MSU crystals in an in vivo peritonitis model. RESULTS: TG2 expression was up-regulated in the synovium tissue and SFMCs from patients with gouty arthritis. The levels of MTA1, TG2, TGF-ß1, IL-1ß and TNF-α mRNAs were consistently increased in MSU crystal-stimulated RAW264.7 cells. si-MTA1 impaired the basal, as well as the MSU crystal-induced expression of TG2 and TGF-ß1, but increased that of IL-1ß and TNF-α. TG2 overexpression dramatically suppressed MSU crystal-induced IL-1ß and TNF-α, but significantly enhanced the TGF-ß1 production. Neutralizing TGF-ß antibodies or inhibition of the crosslinking activity of TG2 attenuated these effects. On the contrary, loss of TG2 resulted in a reduced TGF-ß, but in an increased IL-1ß and TNF-α production in MSU crystal-stimulated RAW264.7 cells and mouse embryonic fibroblasts (MEFs). MSU crystal-stimulated IL-1ß production was Janus kinase 2 (JAK2)-signaling dependent and TG2-induced TGF-ß suppressed the activity of it. Finally, TG2-deficient mice exhibited hyper inflammatory responses after being challenged with MSU crystals in an in vivo peritonitis model. CONCLUSIONS: These findings reveal an inherent regulatory role of the MTA1-TG2 pathway in the self-limitation of MSU crystal-induced inflammation via positively regulating the levels of active TGF-ß1 in macrophages that opposes the MSU crystal-induced JAK2-dependent pro-inflammatory cytokine formation.
Asunto(s)
Artritis Gotosa/metabolismo , Proteínas de Unión al GTP/biosíntesis , Histona Desacetilasas/biosíntesis , Proteínas Represoras/biosíntesis , Factor de Crecimiento Transformador beta1/biosíntesis , Transglutaminasas/biosíntesis , Regulación hacia Arriba/fisiología , Ácido Úrico/toxicidad , Animales , Artritis Gotosa/patología , Línea Celular , Humanos , Inflamación/metabolismo , Inflamación/patología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Proteína Glutamina Gamma Glutamiltransferasa 2 , Transactivadores , Regulación hacia Arriba/efectos de los fármacosRESUMEN
Timed initiation of apoptotic cell death followed by efficient removal mediated by professional macrophages is a key mechanism in maintaining tissue homeostasis. Besides phagocytosis, clearance of apoptotic cells also involves suppression of inflammatory responses by apoptotic cells mediated by both direct inhibition of pro-inflammatory cytokine production and release of soluble anti-inflammatory factors, which act in a paracrine or autocrine fashion to amplify or sustain the anti-inflammatory response. Previous work has demonstrated that during engulfment of apoptotic cells adenosine is produced in sufficient amounts to trigger both adenosine A2A receptors (A2ARs) and A3 receptors (A3Rs). Adenosine bound to A2ARs of macrophages activated the adenylate cyclase pathway to suppress the apoptotic-cell induced, NO-dependent formation of neutrophil migration factors. Here we show by using A3R null engulfing macrophages that the adenosine produced triggers the A3Rs as well, which attenuate the A2AR signaling by inhibiting adenylate cyclase. As a result, the balance in the activation of A2ARs and A3Rs determines the amounts of NO and consequently the levels of neutrophil chemoattractants formed. Since during phagocytosis of apoptotic cells the expression of A2ARs increases, while that of A3Rs decreases, on long term adenosine suppresses the proinflammatory responses in engulfing macrophages.
Asunto(s)
Adenosina/inmunología , Apoptosis/inmunología , Macrófagos Peritoneales/inmunología , Fagocitosis/fisiología , Receptor de Adenosina A3/inmunología , Adenosina/genética , Adenilil Ciclasas/genética , Adenilil Ciclasas/inmunología , Animales , Apoptosis/genética , Comunicación Autocrina/fisiología , Macrófagos Peritoneales/citología , Ratones , Ratones Noqueados , Neutrófilos/citología , Neutrófilos/inmunología , Comunicación Paracrina/fisiología , Receptor de Adenosina A2A/genética , Receptor de Adenosina A2A/inmunología , Receptor de Adenosina A3/genéticaRESUMEN
Transglutaminase 2 (TG2) is a protein crosslinking enzyme with several additional biochemical functions. Loss of TG2 in vivo results in impaired phagocytosis of apoptotic cells and altered proinflammatory cytokine production by macrophages engulfing apoptotic cells leading to autoimmunity. It has been proposed that TG2 acts as an integrin ß(3) coreceptor in the engulfment process, while altered proinflammatory cytokine production is related to the lack of latent TGFß activation by TG2 null macrophages. Here we report that TG2 null macrophages respond to lipopolysaccharide treatment by elevated IL-6 and TNFα production. Though TGFß has been proposed to act as a feed back regulator of proinflammatory cytokine production in LPS-stimulated macrophages, this phenomenon is not related to the lack of active TGFß production. Instead, in the absence of TG2 integrin ß(3) maintains an elevated basal Src family kinase activity in macrophages, which leads to enhanced phosphorylation and degradation of the IκBα. Low basal levels of IκBα explain the enhanced sensitivity of TG2 null macrophages to signals that regulate NF-κB. Our data suggest that TG2 null macrophages bear a proinflammatory phenotype, which might contribute to the enhanced susceptibility of these mice to develop autoimmunity and atherosclerosis.