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1.
J Allergy Clin Immunol ; 131(6): 1635-43, 2013 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-23006543

RESUMEN

BACKGROUND: The exact pathogenesis of the pediatric disorder periodic fever, aphthous stomatitis, pharyngitis, cervical adenitis (PFAPA) syndrome is unknown. OBJECTIVES: We hypothesized that PFAPA might be due to dysregulated monocyte IL-1ß production linked to genetic variants in proinflammatory genes. METHODS: Fifteen patients with PFAPA syndrome were studied during and outside a febrile episode. Hematologic profile, inflammatory markers, and cytokine levels were measured in the blood. The capacity of LPS-stimulated PBMCs and monocytes to secrete IL-1ß was assessed by using ELISA, and active IL-1ß secretion was visualized by means of Western blotting. Real-time quantitative PCR was performed to assess cytokine gene expression. DNA was screened for variants of the MEFV, TNFRSF1A, MVK, and NLRP3 genes in a total of 57 patients with PFAPA syndrome. RESULTS: During a febrile attack, patients with PFAPA syndrome revealed significantly increased neutrophil counts, erythrocyte sedimentation rates, and C-reactive protein, serum amyloid A, myeloid-related protein 8/14, and S100A12 levels compared with those seen outside attacks. Stimulated PBMCs secreted significantly more IL-1ß during an attack (during a febrile episode, 575 ± 88 pg/mL; outside a febrile episode, 235 ± 56 pg/mL; P < .001), and this was in the mature active p17 form. IL-1ß secretion was inhibited by ZYVAD, a caspase inhibitor. Similar results were found for stimulated monocytes (during a febrile episode, 743 ± 183 pg/mL; outside a febrile episode, 227 ± 92 pg/mL; P < .05). Genotyping identified variants in 15 of 57 patients, with 12 NLRP3 variants, 1 TNFRSF1A variant, 4 MEFV variants, and 1 MVK variant. CONCLUSION: Our data strongly suggest that IL-1ß monocyte production is dysregulated in patients with PFAPA syndrome. Approximately 20% of them were found to have NLRP3 variants, suggesting that inflammasome-related genes might be involved in this autoinflammatory syndrome.


Asunto(s)
Fiebre/metabolismo , Interleucina-1beta/biosíntesis , Linfadenitis/metabolismo , Monocitos/metabolismo , Faringitis/metabolismo , Estomatitis Aftosa/metabolismo , Adolescente , Adulto , Anciano , Niño , Femenino , Fiebre/genética , Fiebre/inmunología , Variación Genética , Humanos , Inflamación/inmunología , Inflamación/metabolismo , Mediadores de Inflamación/sangre , Proteína Antagonista del Receptor de Interleucina 1/metabolismo , Recuento de Leucocitos , Leucocitos Mononucleares/inmunología , Leucocitos Mononucleares/metabolismo , Lipopolisacáridos/inmunología , Linfadenitis/genética , Linfadenitis/inmunología , Masculino , Persona de Mediana Edad , Monocitos/inmunología , Neutrófilos , Faringitis/genética , Faringitis/inmunología , Estomatitis Aftosa/genética , Estomatitis Aftosa/inmunología , Síndrome , Adulto Joven
2.
J Immunol ; 186(4): 2495-502, 2011 Feb 15.
Artículo en Inglés | MEDLINE | ID: mdl-21239716

RESUMEN

Basic calcium phosphate (BCP) crystals are associated with severe osteoarthritis and acute periarticular inflammation. Three main forms of BCP crystals have been identified from pathological tissues: octacalcium phosphate, carbonate-substituted apatite, and hydroxyapatite. We investigated the proinflammatory effects of these BCP crystals in vitro with special regard to the involvement of the NLRP3-inflammasome in THP-1 cells, primary human monocytes and macrophages, and mouse bone marrow-derived macrophages (BMDM). THP-1 cells stimulated with BCP crystals produced IL-1ß in a dose-dependent manner. Similarly, primary human cells and BMDM from wild-type mice also produced high concentrations of IL-1ß after crystal stimulation. THP-1 cells transfected with short hairpin RNA against the components of the NLRP3 inflammasome and mouse BMDM from mice deficient for NLRP3, apoptosis-associated speck-like protein, or caspase-1 did not produce IL-1ß after BCP crystal stimulation. BCP crystals induced macrophage apoptosis/necrosis as demonstrated by MTT and flow cytometric analysis. Collectively, these results demonstrate that BCP crystals induce IL-1ß secretion through activating the NLRP3 inflammasome. Furthermore, we speculate that IL-1 blockade could be a novel strategy to inhibit BCP-induced inflammation in human disease.


Asunto(s)
Fosfatos de Calcio/farmacología , Proteínas Portadoras/fisiología , Inflamasomas/fisiología , Interleucina-1beta/metabolismo , Macrófagos/inmunología , Monocitos/inmunología , Animales , Células de la Médula Ósea/inmunología , Células de la Médula Ósea/metabolismo , Células de la Médula Ósea/patología , Pirofosfato de Calcio/farmacología , Caspasa 1/deficiencia , Caspasa 1/fisiología , Línea Celular Tumoral , Células Cultivadas , Cristalización , Humanos , Inflamasomas/metabolismo , Macrófagos/metabolismo , Macrófagos/patología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Monocitos/metabolismo , Monocitos/patología , Proteína con Dominio Pirina 3 de la Familia NLR , Ácido Úrico/farmacología
3.
Arthritis Rheum ; 63(2): 422-33, 2011 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-21279999

RESUMEN

OBJECTIVE: To determine the mechanisms involved in inflammatory responses to octacalcium phosphate (OCP) crystals in vivo. METHODS: OCP crystal-induced inflammation was monitored using a peritoneal model of inflammation in mice with different deficiencies affecting interleukin-1 (IL-1) secretion (IL-1α(-/-) , IL-1ß(-/-) , ASC(-/-) , and NLRP3(-/-) mice) or in mice pretreated with IL-1 inhibitors (anakinra [recombinant IL-1 receptor antagonist] and anti-IL-1ß). The production of IL-1α, IL-1ß, and myeloid-related protein 8 (MRP-8)-MRP-14 complex was determined by enzyme-linked immunosorbent assay. Peritoneal neutrophil recruitment and cell viability were determined by flow cytometry. Depletion of mast cells or resident macrophages was performed by pretreatment with compound 48/80 or clodronate liposomes, respectively. RESULTS: OCP crystals induced peritoneal inflammation, as demonstrated by neutrophil recruitment and up-modulation of IL-1α, IL-1ß, and MRP-8-MRP-14 complex, to levels comparable with those induced by monosodium urate monohydrate crystals. This OCP crystal-induced inflammation was both IL-1α- and IL-1ß-dependent, as shown by the inhibitory effects of anakinra and anti-IL-1ß antibody treatment. Accordingly, OCP crystal stimulation resulted in milder inflammation in IL-1α(-/-) and IL-1ß(-/-) mice. Interestingly, ASC(-/-) and NLRP3(-/-) mice did not show any alteration in their inflammation status in response to OCP crystals. Depletion of the resident macrophage population resulted in a significant decrease in crystal-induced neutrophil infiltration and proinflammatory cytokine production in vivo, whereas mast cell depletion had no effect. Finally, OCP crystals induced apoptosis/necrosis of peritoneal cells in vivo. CONCLUSION: These data indicate that macrophages, rather than mast cells, are important for initiating and driving OCP crystal-induced inflammation. Additionally, OCP crystals induce IL-1-dependent peritoneal inflammation without requiring the NLRP3 inflammasome.


Asunto(s)
Sustitutos de Huesos/toxicidad , Fosfatos de Calcio/toxicidad , Proteínas Portadoras/metabolismo , Interleucina-1/metabolismo , Peritonitis/inducido químicamente , Animales , Conservadores de la Densidad Ósea/farmacología , Supervivencia Celular/efectos de los fármacos , Ácido Clodrónico/farmacología , Cristalización , Modelos Animales de Enfermedad , Femenino , Inyecciones Intraperitoneales , Macrófagos Peritoneales/efectos de los fármacos , Macrófagos Peritoneales/patología , Masculino , Mastocitos/efectos de los fármacos , Mastocitos/patología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Proteína con Dominio Pirina 3 de la Familia NLR , Neutrófilos/efectos de los fármacos , Neutrófilos/patología , Peritoneo/efectos de los fármacos , Peritoneo/patología , Peritonitis/metabolismo , Peritonitis/patología , p-Metoxi-N-metilfenetilamina/farmacología
4.
Immunology ; 134(1): 33-40, 2011 Sep.
Artículo en Inglés | MEDLINE | ID: mdl-21718313

RESUMEN

Apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC) is an important component of the inflammasome, functioning as an adaptor protein that facilitates the recruitment and activation of procaspases that in turn promote the maturation of interleukin-1ß (IL-1ß) and IL-18. Despite initial focus on the inflammatory properties of ASC there is emerging evidence that highlights the importance of ASC in facilitating adaptive immune responses. However, the cellular and molecular basis for the involvement of ASC in adaptive immunity remains largely unexplored. We have previously demonstrated that activated ASC-deficient T cells have dampened proliferative responses. We have therefore explored the underlying cellular mechanism(s) by which ASC regulates T-cell proliferation. We show that under activating conditions (anti-CD3/CD28 stimulation) in bulk T-cell cultures the presence of ASC(-/-) CD4(+) T cells is sufficient to suppress the proliferative responses of neighbouring T cells. Furthermore, ASC(-/-) CD4(+) T cells upon activation exhibit a suppressive cytokine profile, with elevated production of IL-10 and reduced secretion of T helper type 1 cytokines, interferon-γ and IL-2. This increase in IL-10 secretion within the activated ASC(-/-) CD4(+) T-cell compartment was not associated with a proportional increase in conventional Foxp3(+) regulatory T (Treg) cells. Interestingly, when equal numbers of fluorescence-activated cell sorted ASC(+/+) and ASC(-/-) Treg cells (CD4(+) CD44(intermediate/high) CD25(+)) were activated in vitro, the ASC(-/-) fraction produced significantly more IL-10 than their wild-type counterparts, suggesting that ASC(-/-) Treg cells have greater suppressive capacity. Collectively, these results imply that the ASC may influence the development and functioning of Treg cells.


Asunto(s)
Efecto Espectador/inmunología , Proteínas del Citoesqueleto/fisiología , Tolerancia Inmunológica/fisiología , Interleucina-10/metabolismo , Activación de Linfocitos/inmunología , Linfocitos T Reguladores/metabolismo , Linfocitos T/inmunología , Animales , Anticuerpos Monoclonales/inmunología , Anticuerpos Monoclonales/farmacología , Antígenos CD/metabolismo , Proteínas Reguladoras de la Apoptosis , Artritis/inmunología , Proteínas Adaptadoras de Señalización CARD , Antígenos CD28/inmunología , Complejo CD3/inmunología , Linfocitos T CD4-Positivos/efectos de los fármacos , Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD4-Positivos/metabolismo , Linfocitos T CD8-positivos/efectos de los fármacos , Linfocitos T CD8-positivos/inmunología , Linfocitos T CD8-positivos/metabolismo , Proteínas Portadoras/genética , Recuento de Células , Proliferación Celular/efectos de los fármacos , Técnicas de Cocultivo , Medios de Cultivo Condicionados/farmacología , Citocinas/metabolismo , Proteínas del Citoesqueleto/deficiencia , Proteínas del Citoesqueleto/genética , Interferón gamma/metabolismo , Interleucina-10/inmunología , Interleucina-10/farmacología , Interleucinas/metabolismo , Ganglios Linfáticos/citología , Ganglios Linfáticos/inmunología , Activación de Linfocitos/efectos de los fármacos , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Proteína con Dominio Pirina 3 de la Familia NLR , Bazo/citología , Bazo/inmunología , Linfocitos T/efectos de los fármacos , Linfocitos T/metabolismo , Linfocitos T Reguladores/efectos de los fármacos , Linfocitos T Reguladores/inmunología , Factor de Necrosis Tumoral alfa/metabolismo
5.
J Immunol ; 183(6): 4003-12, 2009 Sep 15.
Artículo en Inglés | MEDLINE | ID: mdl-19717512

RESUMEN

Because IL-1beta plays an important role in inflammation in human and murine arthritis, we investigated the contribution of the inflammasome components ASC, NALP-3, IPAF, and caspase-1 to inflammatory arthritis. We first studied the phenotype of ASC-deficient and wild-type mice during Ag-induced arthritis (AIA). ASC(-/-) mice showed reduced severity of AIA, decreased levels of synovial IL-1beta, and diminished serum amyloid A levels. In contrast, mice deficient in NALP-3, IPAF, or caspase-1 did not show any alteration of joint inflammation, thus indicating that ASC associated effects on AIA are independent of the classical NALP-3 or IPAF inflammasomes. Because ASC is a ubiquitous cytoplasmic protein that has been implicated in multiple cellular processes, we explored other pathways through which ASC may modulate inflammation. Ag-specific proliferation of lymph node and spleen cells from ASC-deficient mice was significantly decreased in vitro, as was the production of IFN-gamma, whereas IL-10 production was enhanced. TCR ligation by anti-CD3 Abs in the presence or absence of anti-CD28 Abs induced a reduction in T cell proliferation in ASC(-/-) T cells compared with wild-type ones. In vivo lymph node cell proliferation was also significantly decreased in ASC(-/-) mice, but no effects on apoptosis were observed either in vitro or in vivo in these mice. In conclusion, these results strongly suggest that ASC modulates joint inflammation in AIA through its effects on cell-mediated immune responses but not via its implication in inflammasome formation.


Asunto(s)
Proteínas Reguladoras de la Apoptosis/deficiencia , Artritis Experimental/etiología , Proteínas de Unión al Calcio/deficiencia , Proteínas Portadoras/genética , Caspasa 1/deficiencia , Proteínas del Citoesqueleto/fisiología , Inflamación/etiología , Animales , Antígenos/toxicidad , Artritis Experimental/patología , Proteínas Adaptadoras de Señalización CARD , Proliferación Celular , Artropatías/patología , Ganglios Linfáticos/patología , Ratones , Ratones Noqueados , Complejos Multiproteicos/inmunología , Proteína con Dominio Pirina 3 de la Familia NLR , Bazo/patología
6.
Immunology ; 129(2): 178-85, 2010 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-19824913

RESUMEN

The NACHT, LRR and PYD domains containing protein (NALP3) inflammasome is a key regulator of interleukin-1 beta (IL-1 beta) secretion. As there is strong evidence for a pro-inflammatory role of IL-1 beta in rheumatoid arthritis (RA) and in murine models of arthritis, we explored the expression of the different components of the NALP3 inflammasome as well as other nucleotide oligomerization domain (NOD)-like receptors (NLRs) in synovium obtained from patients with RA. The expression of NLRs was also studied in fibroblast lines derived from joint tissue. By immunohistology, NALP3 and apoptosis-associated speck-like protein containing a CARD domain (ASC) were expressed in myeloid and endothelial cells and B cells. T cells expressed ASC but lacked NALP3. In synovial fibroblast lines, NALP3 expression was not detected at the RNA and protein levels and stimulation with known NALP3 agonists failed to induce IL-1 beta secretion. Interestingly, we were unable to distinguish RA from osteoarthritis synovial samples on the basis of their basal level of RNA expression of known NLR proteins, though RA samples contained higher levels of caspase-1 assayed by enzyme-linked immunosorbent assay. These results indicate that myeloid and endothelial cells are the principal sources of inflammasome-mediated IL-1 beta production in the synovium, and that synovial fibroblasts are unable to activate caspase-1 because they lack NALP3. The NALP3 inflammasome activity does not account for the difference in level of inflammation between RA and osteoarthritis.


Asunto(s)
Artritis Reumatoide/inmunología , Proteínas Portadoras/metabolismo , Proteínas Adaptadoras de Señalización NOD/metabolismo , Osteoartritis/inmunología , Membrana Sinovial/metabolismo , Anciano , Anciano de 80 o más Años , Proteínas Reguladoras de la Apoptosis/genética , Proteínas Reguladoras de la Apoptosis/inmunología , Proteínas Reguladoras de la Apoptosis/metabolismo , Artritis Reumatoide/genética , Artritis Reumatoide/metabolismo , Linfocitos B/inmunología , Linfocitos B/metabolismo , Linfocitos B/patología , Proteínas Portadoras/genética , Proteínas Portadoras/inmunología , Caspasa 1/metabolismo , Línea Celular , Células Endoteliales/inmunología , Células Endoteliales/metabolismo , Células Endoteliales/patología , Femenino , Fibroblastos/inmunología , Fibroblastos/metabolismo , Fibroblastos/patología , Humanos , Interleucina-1beta/metabolismo , Masculino , Persona de Mediana Edad , Células Mieloides/inmunología , Células Mieloides/metabolismo , Células Mieloides/patología , Proteína con Dominio Pirina 3 de la Familia NLR , Proteínas Adaptadoras de Señalización NOD/genética , Proteínas Adaptadoras de Señalización NOD/inmunología , Osteoartritis/genética , Osteoartritis/metabolismo , Ribonucleoproteínas/genética , Ribonucleoproteínas/inmunología , Ribonucleoproteínas/metabolismo , Membrana Sinovial/patología , Linfocitos T/inmunología , Linfocitos T/metabolismo , Linfocitos T/patología
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