RESUMEN
Regulation of neutrophil activation is critical for disease control. Neutrophil extracellular traps (NETs), which are web-like structures composed of DNA and neutrophil-derived proteins, are formed following pro-inflammatory signals; however, if this process is uncontrolled, NETs contribute to disease pathogenesis, exacerbating inflammation and host tissue damage1,2. Here we show that myeloid inhibitory C-type lectin-like (MICL), an inhibitory C-type lectin receptor, directly recognizes DNA in NETs; this interaction is vital to regulate neutrophil activation. Loss or inhibition of MICL functionality leads to uncontrolled NET formation through the ROS-PAD4 pathway and the development of an auto-inflammatory feedback loop. We show that in the context of rheumatoid arthritis, such dysregulation leads to exacerbated pathology in both mouse models and in human patients, where autoantibodies to MICL inhibit key functions of this receptor. Of note, we also detect similarly inhibitory anti-MICL autoantibodies in patients with other diseases linked to aberrant NET formation, including lupus and severe COVID-19. By contrast, dysregulation of NET release is protective during systemic infection with the fungal pathogen Aspergillus fumigatus. Together, we show that the recognition of NETs by MICL represents a fundamental autoregulatory pathway that controls neutrophil activity and NET formation.
RESUMEN
Neutrophil extracellular traps play a key role in defense against extracellular pathogens. The release of these chromatin structures, that contain a combination of cytoplasmic and granule proteins, is known as NETosis, a regulated cell death modality typical of neutrophils. NETosis is induced by pathogens as well as other stimuli such as activated platelets. Our understanding of the molecular events underlying this phenomenon remains incomplete. The currently used experimental approaches to study NETs are semi-quantitative, subjective in nature, and low throughput, rendering it difficult to compare results between laboratories. This is highlighted in two articles published in this issue of the European Journal of Immunology which present what appear to be contradicting results on NET formation. Considering the extensive research on NETosis and the importance of this phenomenon in the immune response, we find it timely to briefly review the lacunae in the most commonly used methods to investigate NETosis. The impact these technical difficulties have on the advancement of our knowledge in this field as well as potential solutions are also discussed.
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Trampas Extracelulares/inmunología , Trampas Extracelulares/metabolismo , Proteínas Quinasas/inmunología , Proteínas Quinasas/metabolismo , Proteína Serina-Treonina Quinasas de Interacción con Receptores/inmunología , Proteína Serina-Treonina Quinasas de Interacción con Receptores/metabolismo , Transducción de Señal/inmunología , Animales , Femenino , HumanosRESUMEN
BACKGROUND: Myeloid inhibitory C-type lectin-like receptor (MICL, Clec12A) is a C-type lectin receptor (CLR) expressed predominantly by myeloid cells. Previous studies have suggested that MICL is involved in controlling inflammation. OBJECTIVE: To determine the role of this CLR in inflammatory pathology using Clec12A(-/-) mice. METHODS: Clec12A(-/-) mice were generated commercially and primarily characterised using the collagen antibody-induced arthritis (CAIA) model. Mechanisms and progress of disease were characterised by clinical scoring, histology, flow cytometry, irradiation bone-marrow chimera generation, administration of blocking antibodies and in vivo imaging. Characterisation of MICL in patients with rheumatoid arthritis (RA) was determined by immunohistochemistry and single nucleotide polymorphism analysis. Anti-MICL antibodies were detected in patient serum by ELISA and dot-blot analysis. RESULTS: MICL-deficient animals did not present with pan-immune dysfunction, but exhibited markedly exacerbated inflammation during CAIA, owing to the inappropriate activation of myeloid cells. Polymorphisms of MICL were not associated with disease in patients with RA, but this CLR was the target of autoantibodies in a subset of patients with RA. In wild-type mice the administration of such antibodies recapitulated the Clec12A(-/-) phenotype. CONCLUSIONS: MICL plays an essential role in regulating inflammation during arthritis and is an autoantigen in a subset of patients with RA. These data suggest an entirely new mechanism underlying RA pathogenesis, whereby the threshold of myeloid cell activation can be modulated by autoantibodies that bind to cell membrane-expressed inhibitory receptors.
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Artritis Experimental/genética , Artritis Reumatoide/genética , Lectinas Tipo C/fisiología , Receptores Mitogénicos/fisiología , Animales , Artritis Reumatoide/sangre , Artritis Reumatoide/etiología , Artritis Reumatoide/patología , Autoanticuerpos/metabolismo , Ensayo de Inmunoadsorción Enzimática , Humanos , Lectinas Tipo C/deficiencia , Lectinas Tipo C/inmunología , Ratones , Células Mieloides/metabolismo , Polimorfismo Genético , Receptores Mitogénicos/deficiencia , Receptores Mitogénicos/inmunología , Membrana Sinovial/patologíaRESUMEN
Many cationic drugs are concentrated in acidic cell compartments due to low retro-diffusion of the protonated molecule (ion trapping), with an ensuing vacuolar and autophagic cytopathology. In solid tissues, there is evidence that phagocytic cells, e.g., histiocytes, preferentially concentrate cationic drugs. We hypothesized that peripheral blood leukocytes could differentially take up a fluorescent model cation, quinacrine, depending on their phagocytic competence. Quinacrine transport parameters were determined in purified or total leukocyte suspensions at 37 °C. Purified polymorphonuclear leukocytes (PMNLs, essentially neutrophils) exhibited a quinacrine uptake velocity inferior to that of lymphocytes, but a consistently higher affinity (apparent KM 1.1 vs. 6.3 µM, respectively). However, the vacuolar (V)-ATPase inhibitor bafilomycin A1 prevented quinacrine transport or initiated its release in either cell type. PMNLs capture most of the quinacrine added at low concentrations to fresh peripheral blood leukocytes compared with lymphocytes and monocytes (cytofluorometry). Accumulation of the autophagy marker LC3-II occurred rapidly and at low drug concentrations in quinacrine-treated PMNLs (significant at ≥2.5 µM, ≥2 h). Lymphocytes contained more LAMP1 than PMNLs, suggesting that the mass of lysosomes and late endosomes is a determinant of quinacrine uptake Vmax. PMNLs, however, exhibited the highest capacity for pinocytosis (uptake of fluorescent dextran into endosomes). The selectivity of quinacrine distribution in peripheral blood leukocytes may be determined by the collaboration of a non-concentrating plasma membrane transport mechanism, tentatively identified as pinocytosis in PMNLs, with V-ATPase-mediated concentration. Intracellular reservoirs of cationic drugs are a potential source of toxicity (e.g., loss of lysosomal function in phagocytes).
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Leucocitos/metabolismo , Neutrófilos/metabolismo , Quinacrina/sangre , ATPasas de Translocación de Protón Vacuolares/sangre , Autofagia/efectos de los fármacos , Inhibidores Enzimáticos/sangre , Inhibidores Enzimáticos/farmacocinética , Inhibidores Enzimáticos/farmacología , Humanos , Immunoblotting , Cinética , Leucocitos/enzimología , Macrólidos/sangre , Macrólidos/farmacología , Microscopía Fluorescente , Neutrófilos/enzimología , Pinocitosis/fisiología , Análisis de Regresión , ATPasas de Translocación de Protón Vacuolares/antagonistas & inhibidoresRESUMEN
We previously described a non-classical mechanism that arrests FcγRIIa signaling in human neutrophils once engaged by immune complexes or opsonized pathogens. The engagement of FcγRIIa leads to its ubiquitination by the ubiquitin ligase c-Cbl and degradation by the proteasome. Herein, we further examined some of the events regulating this novel pathway. The adaptor protein CIN85 was described in other systems to be involved in the regulation of the c-Cbl-dependent pathway. We found that CIN85 is expressed in human neutrophils and that it translocates like c-Cbl from the cytosol to the plasma membrane following receptor cross-linking. CIN85 was also recruited to the same subset of high density detergent-resistant membrane fractions in which stimulated FcγRIIa partitioned with c-Cbl. The integrity of these microdomains is essential to the FcγRIIa degradation process because the cholesterol-depleting agent methyl-ß-cyclodextrin inhibits this event. Silencing the expression of CIN85 by siRNA in dibutyryl cyclic AMP-differentiated PLB 985 cells prevented FcγRIIa degradation and increased IgG-mediated phagocytosis. Confocal microscopy revealed that the presence of CIN85 is essential to the proper sorting of FcγRIIa during endocytosis. We also provide direct evidence that CIN85 is a substrate of serine/threonine kinase PKCs. Classical PKCs positively regulate FcγRIIa ubiquitination and degradation because these events were inhibited by Gö6976, a classical PKC inhibitor. We conclude that the ubiquitination and degradation of stimulated FcγRIIa mediated by c-Cbl are positively regulated by the adaptor protein CIN85 in a PKC-dependent manner and that these events contribute to the termination of FcγRIIa signaling.
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Proteínas Adaptadoras Transductoras de Señales/fisiología , Neutrófilos/metabolismo , Proteína Quinasa C/metabolismo , Proteínas Proto-Oncogénicas c-cbl/fisiología , Receptores de IgG/biosíntesis , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Regulación hacia Abajo/genética , Humanos , Estabilidad Proteica , Transporte de Proteínas , Transducción de Señal/inmunología , UbiquitinaciónRESUMEN
Angiostatin is a potent inhibitor of angiogenesis. One mechanism through which angiostatin inhibits angiogenesis is by binding to the cell surface protein p80-angiomotin. The p80-angiomotin protein promotes angiogenesis, in part, by conferring a hypermigratory phenotype to endothelial cells. Although p80-angiomotin is extensively characterized, less is known about the related protein angiomotin-like 1. We report that angiomotin-like 1 forms part of a protein complex containing p80-angiomotin. Structure-function studies revealed that angiomotin-like 1 associates with this p80-angiomotin-containing complex via its coiled-coil domain. Since p80-angiomotin plays a role in cell migration, a process that involves the remodeling of the actin cytoskeleton, we then addressed the hypothesis that angiomotin-like 1 may interact with the cytoskeleton. Immunofluorescence studies reveal that angiomotin-like 1 not only co-localizes with filamentous actin but also significantly modifies the architecture of the actin cytoskeleton. Regarding migration, angiomotin-like 1 increases the velocity of migration and decreases the persistence of migration directionality. Together these observations strongly suggest that angiomotin-like 1 is involved in actin-cytoskeleton-based processes, in part, via its interaction with a p80-angiomotin-containing complex and the actin cytoskeleton. These findings have important implications for angiogenesis-driven disease since angiomotin and angiomotin-like 1 are both expressed in capillaries.
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Actinas/metabolismo , Citoesqueleto/metabolismo , Péptidos y Proteínas de Señalización Intercelular/metabolismo , Proteínas de la Membrana/metabolismo , Neovascularización Fisiológica , Angiomotinas , Movimiento Celular/fisiología , Células HeLa , Humanos , Proteínas de la Membrana/genética , Proteínas de Microfilamentos , Estructura Terciaria de Proteína/genética , Estructura Terciaria de Proteína/fisiologíaRESUMEN
Flow cytometry-based methods have been developed to measure most neutrophil responses. The assessment of the mobilization of calcium, however, is routinely performed on neutrophils isolated from whole blood. This report describes a flow cytometry-based assay to measure the mobilization of calcium in neutrophils directly in whole blood. This method requires minimal sample manipulation, small volumes of blood and is performed in a short period of time. Both clinical and research laboratories will be able to assess neutrophil function and the quality of granulocyte preparations using a more time and cost effective calcium mobilization test.
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Células Sanguíneas/metabolismo , Calcio/análisis , Calcio/metabolismo , Citometría de Flujo/métodos , Humanos , Cinética , N-Formilmetionina Leucil-Fenilalanina , Factores de Tiempo , ZimosanRESUMEN
CD16b is unique in that it is the only Fc receptor linked to the plasma membrane by a GPI (glycosylphosphatidylinositol) anchor. GPI-anchored proteins often preferentially localize to DRMs (detergent-resistant membranes) that are rich in sphingolipids and cholesterol and play an important role in signal transduction. Even though the responses to CD16b engagement have been intensively investigated, the importance of DRM integrity for CD16b signalling has not been characterized in human neutrophils. We provide direct evidence that CD16b constitutively partitions with both low- and high-density DRMs. Moreover, upon CD16b engagement, a significant increase in the amount of the receptor is observed in high-density DRMs. Similarly to CD16b, CD11b also resides in low- and high-density DRMs. In contrast with CD16b, the partitioning of CD11b in DRMs does not change in response to CD16b engagement. We also provide evidence for the implication of Syk in CD16b signalling and its partitioning to DRMs in resting and activated PMNs (polymorphonuclear neutrophils). Additionally, DRM-disrupting agents, such as nystatin and methyl-beta-cyclodextrin, alter cellular responses to CD16b receptor ligation. Notably, a significant increase in the mobilization of intracellular Ca2+ and in tyrosine phosphorylation of intracellular substrates after CD16b engagement is observed. Altogether, the results of this study provide evidence that high-density DRMs play a role in CD16b signalling in human neutrophils.
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Antígenos CD/metabolismo , Membrana Celular/química , Membrana Celular/metabolismo , Neutrófilos/citología , Neutrófilos/metabolismo , Receptores de IgG/metabolismo , Calcio/metabolismo , Proteínas Ligadas a GPI , Humanos , Antígeno de Macrófago-1/metabolismo , Nistatina/farmacología , Unión Proteica , Transducción de Señal , beta-Ciclodextrinas/farmacologíaRESUMEN
Tec kinases belong to the second largest family of nonreceptor tyrosine kinases. Although these kinases are expressed in myeloid cells, little is known about their implication in neutrophil function. We recently reported the participation of Tec kinases in the responses of human neutrophils to the bacterial peptide N-formyl-l-methionyl-l-leucyl-l-phenylalanine via G-coupled protein receptors. In this study, we extended our investigations of Tec kinases to the signaling of the glycosylphosphatidylinositol-linked receptor CD16b, which is highly and specifically expressed in neutrophils. The results obtained indicate that Tec is translocated to the plasma membrane, phosphorylated, and activated upon CD16b cross-linking and that the activation of Tec is inhibited by Src-specific inhibitors as well as by the phosphatidylinositol-3 kinase inhibitor, wortmannin. As no specific inhibitor of Tec exists, the role of Tec kinases was further investigated using a-Cyano-b-hydroxy-b-methyl-N-(2,5-dibromophenyl)propenamide (LFM-A13), a compound known to inhibit Bruton's tyrosine kinase. We show that this compound also inhibits the kinase activity of Tec and provide evidence that the mobilization of intracellular calcium and the tyrosine phosphorylation of phospholipase Cgamma2 (PLCgamma2) induced upon CD16b engagement are inhibited by LFM-A13. We also show that Tec kinases are important for CD16b-dependent degranulation of neutrophils. In summary, we provide direct evidence for the implication of Tec in CD16b signaling and suggest that Tec kinases are involved in the phosphorylation and activation of PLCgamma2 and subsequently, in the mobilization of calcium in human neutrophils.
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Antígenos CD/metabolismo , Señalización del Calcio/fisiología , Neutrófilos/metabolismo , Proteínas Tirosina Quinasas/metabolismo , Receptores de IgG/metabolismo , Amidas/farmacología , Androstadienos/farmacología , Señalización del Calcio/efectos de los fármacos , Degranulación de la Célula/efectos de los fármacos , Degranulación de la Célula/fisiología , Membrana Celular/metabolismo , Células Cultivadas , Proteínas Ligadas a GPI , Humanos , N-Formilmetionina Leucil-Fenilalanina/farmacología , Nitrilos/farmacología , Fosfolipasa C gamma , Inhibidores de Proteínas Quinasas/farmacología , Transporte de Proteínas/efectos de los fármacos , Transporte de Proteínas/fisiología , Proteínas Tirosina Quinasas/antagonistas & inhibidores , Fosfolipasas de Tipo C/metabolismo , Wortmanina , Familia-src Quinasas/antagonistas & inhibidores , Familia-src Quinasas/metabolismoRESUMEN
The motin family of proteins is comprised of three polypeptides, angiomotin, angiomotin-like 1, and angiomotin-like 2. Angiomotin is an angiostatin-binding protein that promotes endothelial cell motility and is involved in angiogenesis. The function of human angiomotin-like-1 and angiomotin-like-2, however, remains unknown. In this report, we investigated the potential for molecular diversity within the motin family of proteins through the identification and characterization of alternatively spliced transcripts in endothelial cells, human tissues and a variety of cell lines. We report that the motins display variability at the mRNA level suggesting an intricate regulatory system at the transcriptional and potentially protein level. Some alternative transcripts are expressed in a tissue-specific manner and others give rise to novel protein isoforms. The alternative splicing that occurs within this protein family may have important implications in the regulation of the expression and function of the motins.
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Empalme Alternativo , Proteínas Portadoras/genética , Péptidos y Proteínas de Señalización Intercelular/genética , Precursores del ARN/genética , Angiomotinas , Northern Blotting , Proteínas Portadoras/metabolismo , Línea Celular , Línea Celular Tumoral , Exones , Femenino , Perfilación de la Expresión Génica , Genes/genética , Células HT29 , Células HeLa , Humanos , Péptidos y Proteínas de Señalización Intercelular/metabolismo , Intrones , Células K562 , Proteínas de la Membrana , Proteínas de Microfilamentos , Microscopía Fluorescente , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Eliminación de Secuencia , Transfección , Células U937RESUMEN
In human peripheral blood neutrophils or in myeloid PLB-985 cells differentiated towards a neutrophil-like phenotype, the peptide N-formyl-L-norleucyl-L-leucyl-L-phenylalanyl-L-norleucyl-L-tyrosyl-L-leucyl-fluorescein isothiocyanate (f-Nle-Leu-Phe-Nle-Tyr-Lys-FITC) binds to and activates formyl peptide receptor1 (FPR1) and is submitted to receptor-mediated endocytosis (microscopy, cytofluorometry). This peptide may be considered a C-terminally extended version of f-Met-Leu-Phe which carries a fluorescent cargo into cells. By analogy to other peptide hormones for which we have evaluated epitope-tagged agonists as carriers of antibody cargoes, we have designed and evaluated f-Nle-Leu-Phe-Nle-Tyr-Lys-myc, C-terminally extended with the 10-residue myc tag. This peptide is as potent as f-Met-Leu-Phe to compete for f-Nle-Leu-Phe-Nle-Tyr-Lys-FITC uptake by PLB-985 cells, but did not mediate (10-1000nM) the internalization of the fluorescent anti-myc monoclonal antibody 4A6 added to the extracellular fluid at ~7nM (microscopy). The nonfluorescent version of the antibody (28nM) acts as a pre-receptor antagonist of f-Nle-Leu-Phe-Nle-Tyr-Lys-myc, but not of f-Met-Leu-Phe (superoxide release assay in differentiated PLB-985 cells). A further prolonged analog, f-Nle-Leu-Phe-Nle-Tyr-Lys-(Asn-Gly)5-myc, designed to decrease the possible steric hindrance between FPR1 and the bound anti-myc antibody, has little affinity for the receptor, precluding a direct assessment of this issue. Thus, the relatively low-affinity anti-myc antibody used at a high concentration functionally behaves as a selective pre-receptor antagonist of the agonist f-Nle-Leu-Phe-Nle-Tyr-Lys-myc.
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Epítopos/metabolismo , Células Mieloides/inmunología , N-Formilmetionina Leucil-Fenilalanina/metabolismo , Neutrófilos/inmunología , Proteínas Proto-Oncogénicas c-myc/metabolismo , Receptores de Formil Péptido/agonistas , Anticuerpos Monoclonales/metabolismo , Diferenciación Celular , Línea Celular , Endocitosis/genética , Epítopos/genética , Epítopos/inmunología , Humanos , Ligandos , N-Formilmetionina Leucil-Fenilalanina/análogos & derivados , Unión Proteica , Proteínas Proto-Oncogénicas c-myc/genética , Proteínas Proto-Oncogénicas c-myc/inmunología , Proteínas Recombinantes de Fusión/genética , EstereoisomerismoRESUMEN
INTRODUCTION: Monosodium urate crystals (MSU), the etiological agent of gout, are one of the most potent proinflammatory stimuli for neutrophils. The modulation of MSU-induced neutrophil activation by inhibitory receptors remains poorly characterized. The expression of the myeloid inhibitory C-type lectin-like receptor (MICL) in neutrophils is downregulated by several proinflammatory stimuli, suggestive of a role for this receptor in neutrophil function. We thus investigated the potential role of MICL in MSU-induced neutrophil activation. METHODS: The expression of MICL was monitored in human neutrophils by flow cytometry and Western blot analysis after stimulation with MSU. Protein tyrosine phosphorylation was also assessed by Western blot analysis and the production of IL-1 and IL-8 by enzyme-linked immunosorbent assay. Changes in the concentration of cytoplasmic free calcium were monitored with the Fura-2-acetoxymethyl ester calcium indicator. MICL expression was modulated with an anti-MICL antibody in neutrophils and siRNA in the PLB-985 neutrophil-like cell line. RESULTS: MSU induced the downregulation of MICL expression in neutrophils. A diminution in the expression of MICL induced by antibody cross-linking or siRNA enhanced the MSU-dependent increase in cytoplasmic calcium levels, protein tyrosine phosphorylation and IL-8 but not IL-1 production. Pretreatment of neutrophils with colchicine inhibited the MSU-induced downregulation of MICL expression. CONCLUSIONS: Our findings strongly suggest that MICL acts as an inhibitory receptor in human neutrophils since the downregulation of MICL expression enhances MSU-induced neutrophil activation. Since MSU downregulates the expression of MICL, MICL may play a pathogenic role in gout by enhancing neutrophil effector functions. In support of this notion, colchicine counteracts the MSU-induced loss of MICL expression. Our findings thus also provide further insight into the potential molecular mechanisms behind the anti-inflammatory properties of this drug.
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Gota/metabolismo , Lectinas Tipo C/metabolismo , Activación Neutrófila/fisiología , Neutrófilos/metabolismo , Receptores Mitogénicos/metabolismo , Ácido Úrico/metabolismo , Western Blotting , Células Cultivadas , Ensayo de Inmunoadsorción Enzimática , Citometría de Flujo , Humanos , Inmunohistoquímica , Interleucina-1/biosíntesis , Interleucina-8/biosíntesisRESUMEN
OBJECTIVE: The CLEC12A gene codes for an immune inhibitory receptor that maps to 12p13.2. Since an increase in CLEC12A mRNA correlates with rheumatoid factor values greater than 40 IU/ml in rheumatoid fibroblast-like synovial cells, this study assessed the potential of an association between CLEC12A and rheumatoid arthritis (RA) using a phenotype-based approach. METHODS: A discovery cohort of Western European ethnicity was genotyped for eight tag single nucleotide polymorphisms. Statistical analyses relied on the transmission disequilibrium test, relative risk and 95% confidence interval (CI) calculations. Observed haplotype frequencies were compared to expected frequencies using a family-based association test. Statistically significant associations were further tested in a second cohort of unrelated West-European RA patients. RESULTS: An overtransmission of the C allele of the rs1323461 tag single nucleotide polymorphism was observed (56.6% of allele C transmission, P = 0.046) in the discovery cohort. The relative risk of the AC and CC genotypes when compared to the AA genotype was high (relative risk = 4.08; 95% CI: 1.52-10.95, uncorrected P = 2.1 × 10(-3)), particularly in the subgroup of erosive RA (relative risk = 5.27; 95% CI: 1.53-18.19, uncorrected P = 2.1 × 10(-3)), both remaining statistically significant after conservative Bonferroni's correction. The CGAGCCGA haplotype was observed more frequently than expected (P = 0.013). In the second cohort, the C allele had a tendency to be more frequent in RA patients (82.4%) than controls (79.2%) (P = 0.069). CONCLUSION: We report a potential genetic association of CLEC12A with RA. Since CLEC12A encodes for the myeloid inhibitory C-type lectin-like receptor that modulates cytokine synthesis, this receptor may contribute to the pathogenesis of RA.
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Artritis Reumatoide/etnología , Artritis Reumatoide/genética , Lectinas Tipo C/genética , Polimorfismo de Nucleótido Simple/genética , Receptores Mitogénicos/genética , Adulto , Anciano , Anciano de 80 o más Años , Artritis Reumatoide/epidemiología , Estudios de Casos y Controles , Estudios de Cohortes , Europa (Continente)/epidemiología , Femenino , Frecuencia de los Genes/genética , Genotipo , Haplotipos/genética , Humanos , Desequilibrio de Ligamiento/genética , Masculino , Persona de Mediana Edad , FenotipoRESUMEN
LATS2 kinase functions as part of the Hippo pathway to promote contact inhibition of growth and tumor suppression by phosphorylating and inhibiting the transcriptional coactivator YAP. LATS2 is activated by the MST2 kinase. How LATS2 is activated by MST2 in response to changes in cell density is unknown. Here we identify the angiomotin-family tight junction protein AMOTL2 as a novel activator of LATS2. Like AMOTL2, the other angiomotin-family proteins AMOT and AMOTL1 also activate LATS2 through a novel conserved domain that binds and activates LATS2. AMOTL2 binds MST2, LATS2, and YAP, suggesting that AMOTL2 might serve as a scaffold protein. We show that LATS2, AMOTL2, and YAP all localize to tight junctions, raising the possibility that clustering of Hippo pathway components at tight junctions might function to trigger LATS2 activation and growth inhibition in response to increased cell density.
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Proteínas Adaptadoras Transductoras de Señales/metabolismo , Proteínas Portadoras/genética , Proteínas Portadoras/metabolismo , Péptidos y Proteínas de Señalización Intercelular/metabolismo , Proteínas de la Membrana/metabolismo , Fosfoproteínas/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Supresoras de Tumor/metabolismo , Proteínas Adaptadoras Transductoras de Señales/genética , Angiomotinas , Línea Celular Tumoral , Inhibición de Contacto/genética , Células HEK293 , Humanos , Péptidos y Proteínas de Señalización Intercelular/genética , Proteínas de la Membrana/genética , Proteínas de Microfilamentos , Fosfoproteínas/genética , Fosforilación , Unión Proteica/genética , Proteínas Serina-Treonina Quinasas/genética , Estructura Terciaria de Proteína , ARN Interferente Pequeño/genética , Serina-Treonina Quinasa 3 , Transducción de Señal , Uniones Estrechas/metabolismo , Factores de Transcripción , Proteínas Supresoras de Tumor/genética , Proteínas Señalizadoras YAPRESUMEN
BACKGROUND: Microfracture and drilling elicit a cartilage repair whose quality depends on subchondral bone repair. Alternatively activated (AA) macrophages express arginase-1, release angiogenic factors, and could be potential mediators of trabecular bone repair. HYPOTHESIS: Chitosan-glycerol phosphate (GP)/blood implants elicit arginase-1+ macrophages in vivo through neutrophil-dependent mechanisms and improve trabecular bone repair of drilled defects compared with drilling alone. STUDY DESIGN: Controlled laboratory study. METHODS: Bilateral trochlear cartilage defects were created in 15 rabbits, microdrilled, and treated or not with chitosan-GP/blood implant to analyze AA macrophages, CD-31+ blood vessels, bone, and cartilage repair after 1, 2, or 8 weeks. Neutrophil and macrophage chemotaxis to rabbit subcutaneous implants of autologous blood and chitosan-GP (+/-blood) was quantified at 1 or 7 days. In vitro, sera from human chitosan-GP/blood and whole blood clots cultured at 37 degrees C were analyzed by proteomics and neutrophil chemotaxis assays. RESULTS: Chitosan-GP/blood clots and whole blood clots released a similar profile of chemotactic factors (PDGF-BB, IL-8/CXCL8, MCP-1/CCL2, and no IL-1beta or IL-6), although chitosan clot sera attracted more neutrophils in vitro. Subcutaneous chitosan-GP (+/-blood) implants attracted more neutrophils (P < .001) and AA macrophages than whole blood clots in vivo. In repairing subchondral drill holes, chitosan-GP/blood implant attracted more AA macrophages at 1 and 2 weeks and more blood vessels at 2 weeks compared with drilled controls. Treatment elicited a more complete woven bone repair at 8 weeks than controls (P = .0011) with a more uniform, integrated collagen type II+ cartilage repair tissue. CONCLUSION AND CLINICAL RELEVANCE: AA macrophages may play a role in the regeneration of subchondral bone, and chitosan-GP can attract and transiently accumulate these cells in the repair tissue. The resulting improved subchondral repair could be advantageous toward enhancing integration of a restored chondral surface to the subchondral bone.
Asunto(s)
Artroplastia Subcondral , Enfermedades de los Cartílagos/tratamiento farmacológico , Enfermedades de los Cartílagos/cirugía , Cartílago Articular/fisiología , Factores Quimiotácticos/biosíntesis , Quimiotaxis de Leucocito/fisiología , Condrogénesis/efectos de los fármacos , Regeneración Tisular Dirigida , Macrófagos/fisiología , Neutrófilos/fisiología , Adulto , Inductores de la Angiogénesis/metabolismo , Animales , Arginasa/metabolismo , Coagulación Sanguínea/efectos de los fármacos , Coagulación Sanguínea/fisiología , Quimiocina CCL2/biosíntesis , Quimiocinas/biosíntesis , Quitosano/farmacología , Quitosano/uso terapéutico , Coagulantes/farmacología , Coagulantes/uso terapéutico , Femenino , Glicerol/farmacología , Glicerol/uso terapéutico , Humanos , Interleucina-8/biosíntesis , Macrófagos/enzimología , Masculino , Persona de Mediana Edad , Modelos Animales , Fosfatos/farmacología , Fosfatos/uso terapéutico , Conejos , Andamios del Tejido , Adulto JovenRESUMEN
INTRODUCTION: Osteoarthritis is characterized by the progressive destruction of cartilage in the articular joints. Novel therapies that promote resurfacing of exposed bone in focal areas are of interest in osteoarthritis because they may delay the progression of this disabling disease in patients who develop focal lesions. Recently, the addition of 80% deacetylated chitosan to cartilage microfractures was shown to promote the regeneration of hyaline cartilage. The molecular mechanisms by which chitosan promotes cartilage regeneration remain unknown. Because neutrophils are transiently recruited to the microfracture site, the effect of 80% deacetylated chitosan on the function of neutrophils was investigated. Most studies on neutrophils use preparations of chitosan with an uncertain degree of deacetylation. For therapeutic purposes, it is of interest to determine whether the degree of deacetylation influences the response of neutrophils to chitosan. The effect of 95% deacetylated chitosan on the function of neutrophils was therefore also investigated and compared with that of 80% deacetylated chitosan. METHODS: Human blood neutrophils from healthy donors were isolated by centrifugation on Ficoll-Paque. Chemotaxis was performed using the chemoTX system. Production of superoxide anions was evaluated using the cytochrome c reduction assay. Degranulation was determined by evaluating the release of myeloperoxidase and lactoferrin. The internalization of fluorescently labelled 80% deacetylated chitosan by neutrophils was studied by confocal microscopy. RESULTS: Neutrophils were dose dependently attracted to 80% deacetylated chitosan. In contrast, 95% deacetylated chitosan was not chemotactic for neutrophils. Moreover, the majority of the chemotactic effect of 80% deacetylated chitosan was mediated by phospholipase-A2-derived bioactive lipids. Contrary to the induction of chemotaxis, neither 80% nor 95% deacetylated chitosan activated the release of granule enzymes or the generation of active oxygen species. Despite the distinct response of neutrophils toward 80% and 95% deacetylated chitosan, both chitosans were internalized by neutrophils. CONCLUSIONS: Eighty per cent deacetylated chitosan induces a phenotype in neutrophils that is distinct from the classical phenotype induced by pro-inflammatory agents. Our observations also indicate that the degree of deacetylation is an important factor to consider in the use of chitosan as an accelerator of repair because neutrophils do not respond to 95% deacetylated chitosan.
Asunto(s)
Cartílago Articular/fisiología , Quitosano/química , Quitosano/farmacología , Neutrófilos/fisiología , Fenotipo , Cicatrización de Heridas/fisiología , Acetilación , Adulto , Cartílago Articular/efectos de los fármacos , Cartílago Articular/metabolismo , Quimiotaxis de Leucocito/efectos de los fármacos , Quimiotaxis de Leucocito/fisiología , Quitosano/metabolismo , Humanos , Neutrófilos/efectos de los fármacos , Neutrófilos/metabolismo , Cicatrización de Heridas/efectos de los fármacosRESUMEN
The effects of cholesterol-perturbing agents on the mobilization of calcium induced upon the stimulation of human neutrophils by chemotactic factors were tested. Methyl-beta-cyclodextrin and filipin did not alter the initial peak of calcium mobilization but shortened the duration of the calcium spike that followed the addition of fMet-Leu-Phe. These agents also inhibited the influx of Mn(2+) induced by fMet-Leu-Phe or thapsigargin. Methyl-beta-cyclodextrin and filipin completely abrogated the mobilization of calcium induced by 10(-10) m platelet-activating factor, which at this concentration depends to a major extent on an influx of calcium as well as the influx of calcium induced by 10(-7) m platelet-activating factor. On the other hand, methyl-beta-cyclodextrin and filipin enhanced the mobilization of calcium induced by ligation of FcgammaRIIA, an agonist that did not induce a detectable influx of calcium. Finally, methyl-beta-cyclodextrin and filipin enhanced the stimulation of the profile of tyrosine phosphorylation, the activity of phospholipase D (PLD), and the production of superoxide anions induced by fMet-Leu-Phe. These results suggest that the calcium channels utilized by chemotactic factors in human neutrophils are either located in cholesterol-rich regions of the plasma membrane, or that the mechanisms that lead to their opening depend on the integrity of these microdomains.
Asunto(s)
Calcio/metabolismo , Colesterol/metabolismo , Neutrófilos/efectos de los fármacos , Neutrófilos/metabolismo , beta-Ciclodextrinas , Membrana Celular/metabolismo , Ciclodextrinas/farmacología , Citoplasma/metabolismo , Humanos , N-Formilmetionina Leucil-Fenilalanina/farmacología , Fosfolipasa D/metabolismo , Fosforilación , Factor de Activación Plaquetaria/farmacología , Estructura Terciaria de Proteína , Superóxidos , Factores de Tiempo , Tirosina/metabolismoRESUMEN
The signal transduction mechanisms associated with the ligation of FcgammaRIIA in human neutrophils are as yet only incompletely characterized. In the present study, we have investigated the distribution and fate of FcgammaRIIA following its cross-linking. The results obtained indicate that cross-linking of FcgammaRIIA led, within a few seconds, to its translocation into a nonionic detergent-insoluble fraction. This was followed, within a couple of minutes, by a substantial loss of immunoreactive FcgammaRIIA in the cells. The stimulated degradation of FcgammaRIIA was blocked by the Src kinase inhibitor PP1 but not by wortmannin, ST-638, piceatannol, or cytochalasin B. Cross-linked FcgammaRIIA could be solubilized by saponin (in the presence of Nonidet P-40) and by beta-octylglucoside. Sucrose gradient analysis of the distribution of FcgammaRIIA revealed that its cross-linking led to its translocation into the pellets and not the light buoyant density fractions classically associated with lipid rafts. Disruption of cholesterol-containing membrane microdomains with filipin prevented the degradation of FcgammaRIIA but did not inhibit the stimulation of the pattern of tyrosine phosphorylation or the mobilization of calcium that followed FcgammaRIIA cross-linking. These data suggest that both cholesterol-rich domains and Src kinases are required for the degradation of the activated FcgammaRIIA and provide new insights into the early events following FcgammaRIIA cross-linking.
Asunto(s)
Antígenos CD/metabolismo , Detergentes/farmacología , Neutrófilos/inmunología , Neutrófilos/metabolismo , Receptores de IgG/metabolismo , Adulto , Antígenos CD/inmunología , Antígenos CD/fisiología , Reactivos de Enlaces Cruzados/metabolismo , Inhibidores Enzimáticos/farmacología , Filipina/farmacología , Glucósidos/farmacología , Humanos , Microdominios de Membrana/efectos de los fármacos , Microdominios de Membrana/inmunología , Microdominios de Membrana/metabolismo , Activación Neutrófila/inmunología , Neutrófilos/efectos de los fármacos , Neutrófilos/enzimología , Fosforilación/efectos de los fármacos , Estructura Terciaria de Proteína/efectos de los fármacos , Transporte de Proteínas/efectos de los fármacos , Transporte de Proteínas/inmunología , Pirazoles/farmacología , Pirimidinas/farmacología , Receptores de IgG/antagonistas & inhibidores , Receptores de IgG/inmunología , Receptores de IgG/fisiología , Saponinas/farmacología , Transducción de Señal/inmunología , Solubilidad , Tirosina/metabolismo , Familia-src Quinasas/antagonistas & inhibidoresRESUMEN
A novel mammalian plasma membrane bound nucleoside triphosphate diphosphohydrolase (NTPDase), named NTPDase8, has been cloned and characterized. Analysis of cDNA reveals an open reading frame of 1491 base pairs encoding a protein of 497 amino acid residues with an estimated molecular mass of 54650 Da and a predicted isoelectric point of 5.94. In a mouse, the genomic sequence is located on chromosome 2A3 and is comprised of 10 exons. The deduced amino acid sequence reveals eight putative N-glycosylation sites, two transmembrane domains, five apyrase-conserved regions, and 20-50% amino acid identity with other mammalian NTPDases. mRNA expression was detected in liver, jejunum, and kidney. Both intact cells and crude cell lysates from COS-7 cells expressing NTPDase8 hydrolyzed P2 receptor agonists, namely, ATP, ADP, UTP, and UDP, but did not hydrolyze AMP. There was an absolute requirement for divalent cations for the catalytic activity (Ca(2+) > Mg(2+)) with an optimal pH between 5.5 and 8.0 for ATP and 6.4 for ADP hydrolysis. Kinetic parameters derived from analysis of crude cell lysates showed that the enzyme had lower apparent K(m) values for adenine nucleotides and for triphosphonucleosides (K(m,app) of 13 microM for ATP, 41 microM for ADP, 47 microM for UTP, and 171 microM for UDP). Hydrolysis of triphosphonucleosides resulted in a transient accumulation of the corresponding diphosphonucleoside, as expected from the apparent K(m) values. Enzymatic properties of NTPDase8 differ from those of other NTPDases suggesting an alternative way to modulate nucleotide levels and consequently P2 receptor activation.