RESUMEN
We used an animal model of salt-sensitive hypertension (SSH) in which ovariectomized (oVx) rats developed hypertension with high salt (HS) intake. Hypertension is accompanied by changes in the percentage of CD4+ T lymphocytes, immune CD45+ cell infiltration into renal tissue, and changes in Na+, K+- ATPase (NKA) expression in both renal tissue and peripheral blood mononuclear cells (PBMCs). To determine whether the observed changes resulted from HS intake, high blood pressure, or both, hydralazine (HDZ) was used to lower blood pressure. The oVx HS rats received two HDZ schedules either to prevent or to treat hypertension. NKA was overexpressed in the kidneys of all oVx groups and in PBMCs of oVx HS rats. This pattern was not altered with HDZ treatment. Changes in CD4+ T lymphocytes and renal infiltration of CD45+ cells were not reversed either. High salt, but not high blood pressure, induces immune cell activation and renal infiltration. Overexpressed NKA is the primary event, and HS is the perturbation to the system in this model of SSH, which resembles the postmenopausal state.
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Hipertensión , Riñón , Ovariectomía , Ratas Wistar , Animales , Femenino , Ratas , Riñón/patología , Riñón/metabolismo , Riñón/inmunología , Hipertensión/inmunología , Hipertensión/patología , Hipertensión/metabolismo , ATPasa Intercambiadora de Sodio-Potasio/metabolismo , Cloruro de Sodio Dietético/efectos adversos , Presión Sanguínea/efectos de los fármacos , Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD4-Positivos/metabolismo , Hidralazina/farmacologíaRESUMEN
SummarySystemic lupus erythematosus (SLE) is an autoimmune condition developing thrombocytopenia in about 10-15% of cases, however, mechanisms leading to low platelet count were not deeply investigated in this illness. Here we studied possible causes of thrombocytopenia, including different mechanisms of platelet clearance and impairment in platelet production. Twenty-five SLE patients with and without thrombocytopenia were included. Platelet apoptosis, assessed by measurement of loss of mitochondrial membrane potential, active caspase 3 and phosphatidylserine exposure, was found to increase in thrombocytopenic patients. Plasma from 67% SLE patients (thrombocytopenic and non-thrombocytopenic) induced loss of sialic acid (Ricinus communis agglutinin I and/or Peanut agglutinin binding) from normal platelet glycoproteins. Concerning platelet production, SLE plasma increased megakaryopoiesis (evaluated using normal human cord blood CD34+ hematopoietic progenitors), but inhibited thrombopoiesis (proplatelet count). Anti-platelet autoantibody depletion from SLE plasma reverted this inhibition. Overall, abnormalities were more frequently observed in thrombocytopenic than non-thrombocytopenic SLE patients and in those with active disease (SLEDAI≥5). In conclusion, platelet clearance due to apoptosis and desialylation, and impaired platelet production mainly due to inhibition of thrombopoiesis, could be relevant mechanisms leading to thrombocytopenia in SLE. These findings could provide a rational basis for the choice of proper therapies to correct platelet counts in these patients.[Figure: see text].
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Lupus Eritematoso Sistémico , Púrpura Trombocitopénica Idiopática , Trombocitopenia , Autoanticuerpos , Plaquetas , Humanos , Lupus Eritematoso Sistémico/complicaciones , Recuento de Plaquetas , Trombocitopenia/complicaciones , TrombopoyesisRESUMEN
NEW FINDINGS: What is the central question of this study? In a model of salt-sensitive hypertension in ovariectomized (oVx) adult Wistar rats, what is the expression of proteins related to sodium transport in peripheral blood mononuclear cells (PBMCs), and how does the response of proteins to high sodium intake compare with changes in blood pressure in intact female rats? What is the main finding and its importance? Sodium transport proteins in PBMCs react to high sodium and blood pressure markedly differently in oVx versus intact female rats. Protein expression shows sodium and pressure sensitivity. Renal immune cells increase in oVx under high salt. ABSTRACT: Hypertension is a worldwide public health problem. High sodium consumption is associated with hypertension, and hypertensive mechanisms involve immunity cells. Peripheral blood mononuclear cells (PBMCs) are endowed with proteins related to sodium transport. We studied their abundance in PBMCs from intact (IF) or ovariectomized (oVx) adult Wistar rats under normal (NS) or high (HS) salt intake. Ovariectomy was performed at 60 days of life. At 145 days, one group of IF and oVx rats received NS or HS intake for 5 days. Another group of IF HS and oVx HS rats received hydralazine (HDZ) to reduce blood pressure (BP). Sodium balance and BP were recorded. Expression of Na+ ,K+ -ATPase (NKA), Na+ -K+ -2Cl- cotransporter 1 (NKCC1), serum/glucocorticoid-regulated kinase 1 (SGK1), dopamine D1 like receptor (D1DR), CD4+ and CD8+ were determined in PBMCs and CD45+ leukocytes in renal tissue. IF HS rats showed increased natriuresis and normal BP. NKA and CD4+ expression diminished in IF HS. Instead, oVx HS rats had sodium retention and high BP and increased the expression of NKA, NKCC1, D1DR, CD4+ and CD8+ in PBMCs. Renal CD45+ leukocytes increased in oVx HS rats. HDZ decreased BP in all rats. Upon HDZ treatment, NKA did not change, NKCC1 decreased in oVx HS rats, while SGK1 increased in both IF HS and oVx HS rats. Hormonal background determines BP response and the expression of proteins related to sodium transport in PBMCs and renal immune cells at HS intake. The analysis of NKA, NKCC1 and SGK1 expression in PBMCs differentiated salt-sensitivity from BP variations.
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Hipertensión , Cloruro de Sodio Dietético , Animales , Presión Sanguínea/fisiología , Proteínas Portadoras , Femenino , Humanos , Leucocitos Mononucleares/metabolismo , Ovariectomía , Ratas , Ratas Wistar , Sodio/metabolismo , Cloruro de Sodio Dietético/metabolismo , ATPasa Intercambiadora de Sodio-PotasioRESUMEN
Germline RUNX1 mutations lead to thrombocytopenia and platelet dysfunction in familial platelet disorder with predisposition to acute myelogenous leukemia (AML). Multiple aspects of platelet function are impaired in these patients, associated with altered expression of genes regulated by RUNX1 We aimed to identify RUNX1-targets involved in platelet function by combining transcriptome analysis of patient and shRUNX1-transduced megakaryocytes (MK). Down-regulated genes included TREM-like transcript (TLT)-1 (TREML1) and the integrin subunit alpha (α)-2 (ITGA2) of collagen receptor α2-beta (ß)-1, which are involved in platelet aggregation and adhesion, respectively. RUNX1 binding to regions enriched for H3K27Ac marks was demonstrated for both genes using chromatin immunoprecipitation. Cloning of these regions upstream of the respective promoters in lentivirus allowing mCherry reporter expression showed that RUNX1 positively regulates TREML1 and ITGA2, and this regulation was abrogated after deletion of RUNX1 sites. TLT-1 content was reduced in patient MK and platelets. A blocking anti-TLT-1 antibody was able to block aggregation of normal but not patient platelets, whereas recombinant soluble TLT-1 potentiated fibrinogen binding to patient platelets, pointing to a role for TLT-1 deficiency in the platelet function defect. Low levels of α2 integrin subunit were demonstrated in patient platelets and MK, coupled with reduced platelet and MK adhesion to collagen, both under static and flow conditions. In conclusion, we show that gene expression profiling of RUNX1 knock-down or mutated MK provides a suitable approach to identify novel RUNX1 targets, among which downregulation of TREML1 and ITGA2 clearly contribute to the platelet phenotype of familial platelet disorder with predisposition to AML.
Asunto(s)
Trastornos de las Plaquetas Sanguíneas/genética , Trastornos de las Plaquetas Sanguíneas/metabolismo , Subunidad alfa 2 del Factor de Unión al Sitio Principal/metabolismo , Regulación de la Expresión Génica , Integrina alfa2/genética , Leucemia Mieloide Aguda/etiología , Receptores Inmunológicos/genética , Trastornos de las Plaquetas Sanguíneas/sangre , Plaquetas/metabolismo , Susceptibilidad a Enfermedades , Perfilación de la Expresión Génica , Humanos , Leucemia Mieloide Aguda/diagnóstico , Megacariocitos/metabolismo , Mutación , Agregación Plaquetaria , Pruebas de Función Plaquetaria , Unión ProteicaRESUMEN
The SDF-1-CXCR4 axis plays an essential role in the regulation of platelet production, by directing megakaryocyte (MK) migration toward the vascular niche, thus allowing terminal maturation and proplatelet formation, and also regulates platelet function in an autocrine manner. Inherited thrombocytopenias (IT) comprise a spectrum of diverse clinical conditions caused by mutations in genes involved in platelet production and function. We assessed CXCR4 expression and SDF-1 levels in a panel of well-characterized forms of IT. Decreased surface CXCR4 levels were found in 8 of 27 (29.6%) IT patients by flow cytometry, including 4 of 6 patients with ANKRD26-RT, 3 of 3 patients with GPS and 1 of 6 patients with FPD/AML. Low CXCR4 levels were associated with impaired SDF-1-triggered platelet aggregation, indicating that this decrease is functionally relevant, whereas a normal platelet response was shown in patients harbouring preserved membrane CXCR4. Reduced CXCR4 was not due to decreased gene expression, as platelet RNA levels were normal or increased, suggesting a post-transcriptional defect. Increased ligand-induced receptor internalization was ruled out, as circulating SDF-1 levels were similar to controls. MK CXCR4 expression was normal, indicating that the defect in CXCR4 arises after the step of platelet biogenesis. In conclusion, the finding of defective CXCR4 expression specifically associated with certain IT disorders highlights the fact that abnormalities in several megakaryocytic regulators underlie IT pathogenesis and further reveal the heterogeneous nature of these conditions.
Asunto(s)
Plaquetas/metabolismo , Quimiocina CXCL12/biosíntesis , Regulación de la Expresión Génica , Enfermedades Genéticas Congénitas/sangre , Megacariocitos/metabolismo , Receptores CXCR4/biosíntesis , Trombocitopenia/sangre , Adolescente , Adulto , Anciano , Plaquetas/patología , Niño , Femenino , Enfermedades Genéticas Congénitas/genética , Enfermedades Genéticas Congénitas/patología , Humanos , Péptidos y Proteínas de Señalización Intercelular , Masculino , Megacariocitos/patología , Persona de Mediana Edad , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Agregación Plaquetaria/genética , Trombocitopenia/genética , Trombocitopenia/patologíaRESUMEN
BACKGROUND: Anagrelide represents a treatment option for essential thrombocythemia, although its place in therapy remains controversial. AIM: To assess the impact of mutational status in response rates and development of adverse events during long-term use of anagrelide. METHODS: We retrospectively evaluated 67 patients with essential thrombocythemia treated with anagrelide during 68 (4-176) months. RESULTS: Mutational frequencies were 46.3%, 28.3%, and 1.5% for JAK2V617F, CALR and MPL mutations. Anagrelide yielded a high rate of hematologic responses, which were complete in 49.25% and partial in 46.25%, without differences among molecular subsets. The rate of thrombosis during treatment was one per 100 patient-years, without excess bleeding. Anemia was the major adverse event, 30.3% at 5-yr follow-up, being more frequent in CALR(+) (P < 0.05). Myelofibrotic transformation developed in 14.9% (12.9%, 21%, and 12.5% in JAK2V617F(+), CALR(+), and triple-negative patients, respectively, P = NS) and those treated >60 months were at higher risk, OR (95% CI) 9.32 (1.1-78.5), P < 0.01, indicating the need for bone marrow monitoring during prolonged treatment. CONCLUSION: Although CALR(+) patients were at higher risk of developing anemia, anagrelide proved effective among all molecular subsets, indicating that mutational status does not seem to represent a major determinant of choice of cytoreductive treatment among essential thrombocythemia therapies.
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Calreticulina/genética , Janus Quinasa 2/genética , Inhibidores de Agregación Plaquetaria/administración & dosificación , Quinazolinas/administración & dosificación , Receptores de Trombopoyetina/genética , Trombocitemia Esencial/tratamiento farmacológico , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Anemia/etiología , Anemia/patología , Calreticulina/inmunología , Niño , Femenino , Estudios de Seguimiento , Expresión Génica , Humanos , Janus Quinasa 2/inmunología , Masculino , Persona de Mediana Edad , Mutación , Inhibidores de Agregación Plaquetaria/efectos adversos , Mielofibrosis Primaria/etiología , Mielofibrosis Primaria/patología , Quinazolinas/efectos adversos , Receptores de Trombopoyetina/inmunología , Estudios Retrospectivos , Trombocitemia Esencial/genética , Trombocitemia Esencial/inmunología , Trombocitemia Esencial/patologíaRESUMEN
The pathophysiological mechanisms contributing to the decreased platelet count in immune thrombocytopenia (ITP) are not entirely understood. Here, we investigated the key step of proplatelet formation (PPF) by studying the effect of ITP plasma in thrombopoiesis. Normal cord blood-derived mature megakaryocytes were cultured in the presence of recalcified plasma from ITP patients, and PPF was evaluated by microscopic analysis. Patient samples induced a dose-dependent inhibition in PPF, as well as decreased complexity of proplatelet architecture. Although slightly increased, plasma-induced megakaryocyte apoptosis was not related to PPF impairment. Purified IgG reproduced the inhibitory effect, while platelet-adsorbed plasma induced its reversion, suggesting the involvement of auto-antibodies in the inhibition of thrombopoiesis. Impaired PPF, induced by ITP plasmas bearing anti-GPIIb-IIIa antibodies, was related to their ability to interfere with the normal function of this integrin, as assessed by megakaryocyte PAC-1 binding and ß3 integrin phosphorylation while the presence of anti-glycoprotein Ia-IIa auto-antibodies was associated with loss of normal inhibition of PPF induced by type I collagen. In conclusion, abnormal thrombopoiesis comprising decreased PPF and morphological changes in proplatelet structure are induced by patient samples, unveiling new mechanisms contributing to decreased platelet count in ITP.
Asunto(s)
Recuento de Plaquetas , Púrpura Trombocitopénica Idiopática/sangre , Púrpura Trombocitopénica Idiopática/inmunología , Trombopoyesis , Adulto , Anciano , Anciano de 80 o más Años , Especificidad de Anticuerpos/inmunología , Apoptosis/inmunología , Autoanticuerpos/inmunología , Plaquetas/citología , Humanos , Integrinas/inmunología , Megacariocitos/citología , Megacariocitos/inmunología , Persona de Mediana Edad , Complejo GPIIb-IIIa de Glicoproteína Plaquetaria/inmunología , Glicoproteínas de Membrana Plaquetaria/inmunología , Trombopoyesis/inmunología , Adulto JovenRESUMEN
BACKGROUND: Multiple blood cell abnormalities participate in the development of inflammation in systemic lupus erythematosus (SLE). Although platelets have been suggested as one of these contributors through the release of their content during activation, there are limited specific data about their role as immune players in SLE. MATERIALS AND METHODS: Thirteen SLE patients were included. Flow cytometry was used to measure Toll-like receptors (TLR) 2, 4, and 9 in resting platelets, platelet-activation markers (PAC-1 binding, P-selectin, CD63, and CD40 ligand -L) and platelet-leukocyte aggregates before and after specific TLR stimulation. Soluble CD40L and von Willebrand factor (vWf) release from stimulated platelets was measured using ELISA. RESULTS: In resting conditions, SLE platelets showed normal expression levels of TLR 2, 4 and 9. Platelet surface activation markers, soluble CD40L, and vWf release were normal at baseline and after TLR stimulation. Platelet-monocyte aggregates were elevated in resting conditions in SLE samples and showed only a marginal increase after TLR stimulation, while baseline and stimulated platelet-neutrophil and platelet-lymphocyte aggregates were normal. C-reactive protein levels positively correlated with platelet-monocyte aggregates both at baseline and after stimulation with the TLR-2 agonist PAM3CSK4, suggesting these complexes could reflect the inflammatory activity in SLE. In our cohort, 12 of 13 patients received treatment with hydroxychloroquine (HCQ), a known inhibitor of endosomal activity and a potential inhibitor of platelet activation. The fact that SLE platelets showed an adequate response to TLR agonists suggests that, despite this treatment, they retain the ability to respond to the increased levels of damage-associated molecular patterns (DAMPs), which represent known TLR ligands, present in the circulation of SLE patients. Interestingly, elevated plasma levels of high mobility group box 1 (HMGB1), a classical DAMP, correlated with vWf release from TLR-stimulated platelets, suggesting that HMGB1 may also be released by platelets, thereby creating a positive feedback loop for platelet activation that contributes to inflammation. CONCLUSION: Our study demonstrates normal platelet TLR expression and function together with increased circulating platelet-monocyte aggregates. In addition, a direct correlation was observed between plasma HMGB1 levels and platelet vWf release following TLR2 stimulation. This platelet behavior in a group of patients undergoing HCQ treatment suggests that platelets could play a role in the inflammatory state of SLE.
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Proteína HMGB1 , Lupus Eritematoso Sistémico , Humanos , Proteína HMGB1/metabolismo , Ligando de CD40 , Factor de von Willebrand/metabolismo , Receptores Toll-Like/metabolismo , Plaquetas/metabolismo , Inflamación/metabolismo , Receptor Toll-Like 9Asunto(s)
Autoanticuerpos/inmunología , Células de la Médula Ósea/inmunología , Proteínas de la Matriz Extracelular/inmunología , Megacariocitos/inmunología , Púrpura Trombocitopénica Idiopática/inmunología , Adulto , Anciano , Anciano de 80 o más Años , Estudios de Casos y Controles , Adhesión Celular/inmunología , Células Cultivadas , Femenino , Humanos , Masculino , Megacariocitos/fisiología , Persona de Mediana Edad , Trombopoyesis/inmunología , Adulto JovenRESUMEN
Myelofibrosis (MF) is a clonal hematopoietic stem cell disorder classified among chronic myeloproliferative neoplasms, characterized by exacerbated myeloid and megakaryocytic proliferation and bone marrow fibrosis. It is induced by driver (JAK2/CALR/MPL) and high molecular risk mutations coupled to a sustained inflammatory state that contributes to disease pathogenesis. Patient outcome is determined by stratification into risk groups and refinement of current prognostic systems may help individualize treatment decisions. Circulating cell-free (cf)DNA comprises short fragments of double-stranded DNA, which promotes inflammation by stimulating several pathways, including inflammasome activation, which is responsible for IL-1ß and IL-18 maturation and release. In this work, we assessed the contribution of cfDNA as a marker of disease progression and mediator of inflammation in MF. cfDNA was increased in MF patients and higher levels were associated with adverse clinical outcome, a high-risk molecular profile, advanced disease stages and inferior overall survival, indicating its potential value as a prognostic marker. Cell-free DNA levels correlated with tumor burden parameters and markers of systemic inflammation. To mimic the effects of cfDNA, monocytes were stimulated with poly(dA:dT), a synthetic double-stranded DNA. Following stimulation, patient monocytes released higher amounts of inflammasome-processed cytokine, IL-18 to the culture supernatant, reflecting enhanced inflammasome function. Despite overexpression of cytosolic DNA inflammasome sensor AIM2, IL-18 release from MF monocytes was shown to rely mainly on the NLRP3 inflammasome, as it was prevented by NLRP3-specific inhibitor MCC950. Circulating IL-18 levels were increased in MF plasma, reflecting in vivo inflammasome activation, and highlighting the previously unrecognized involvement of this cytokine in MF cytokine network. Monocyte counts were higher in patients and showed a trend towards correlation with IL-18 levels, suggesting monocytes represent a source of circulating IL-18. The close correlation shown between IL-18 and cfDNA levels, together with the finding of enhanced DNA-triggered IL-18 release from monocytes, suggest that cfDNA promotes inflammation, at least in part, through inflammasome activation. This work highlights cfDNA, the inflammasome and IL-18 as additional players in the complex inflammatory circuit that fosters MF progression, potentially providing new therapeutic targets.
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Ácidos Nucleicos Libres de Células , Mielofibrosis Primaria , Humanos , Inflamasomas/metabolismo , Citocinas/metabolismo , Interleucina-18/metabolismo , Proteína con Dominio Pirina 3 de la Familia NLR/metabolismo , Mielofibrosis Primaria/genética , Inflamación/inducido químicamente , ADN , Progresión de la EnfermedadRESUMEN
Bone marrow stromal cells provide a proper environment for the development of hematologic lineages. The incorporation of different stromal cells into in vitro culture systems would be an attractive model to study megakaryopoiesis and thrombopoiesis. Our objective was to evaluate the participation of different types of stromal cells in in vitro megakaryopoiesis, thrombopoiesis, and megakaryocyte (MK) survival. CD34-positive progenitors from umbilical cord blood were differentiated into MK precursors and then cocultured with umbilical vein endothelial cells (HUVECs), bone marrow mesenchymal stem cells (MSCs), skin fibroblasts (SFs) (all human), or the mouse fibroblast cell line L929. The number of MKs (CD61-positive cells) was increased in the presence of HUVECs and SFs, whereas L929 cells decreased total and mature MK counts. With respect to thrombopoiesis, HUVECs increased proplatelet (PP)-producing MKs, while MSCs, L929 cells, and SFs had the opposite effect (immunofluorescence staining and microscopic analysis). MK survival was enhanced in MSC and SF co-cultures, as assessed by evaluation of pyknotic nuclei. However, HUVECs and L929 did not prevent apoptosis of MKs. Reciprocally, the cloning efficiency of MSCs was decreased in the presence of MKs, while the ability of stromal cells (either MSCs or SFs) to produce the extracellular matrix proteins type III collagen, fibronectin, dermatan sulfate, heparan sulfate, and prolyl 4-hydroxylase subunit ß was preserved. These data indicate that each stromal cell type performs distinctive functions that differentially modulate MK growth and platelet production and, at the same time, that MKs also modify stromal cell behavior. Overall, our results highlight the relevance of considering the influence of stromal cells in MK research as well as the close interplay of different cell types within the bone marrow milieu.
Asunto(s)
Plaquetas , Megacariocitos , Proliferación Celular , Células del EstromaRESUMEN
An amendment to this paper has been published and can be accessed via a link at the top of the paper.
RESUMEN
Essential thrombocythemia (ET) is comprised among chronic myeloproliferative neoplasms (MPN) and is caused by driver mutations in JAK2, CALR, and MPL, which lead to megakaryocyte proliferation and prominent thrombocytosis. Thrombosis remains the main cause of morbidity in ET and is driven by the interplay between blood cells, the endothelium, the clotting cascade, and host-derived inflammatory mediators. Platelet activation plays a key role in the thrombotic predisposition, although the underlying mechanisms remain poorly defined. In addition to their role in hemostasis, platelets participate in innate immunity and inflammation owing to the expression of toll-like receptors (TLR), which recognize inflammatory signals, triggering platelet functional responses. Considering the impact of inflammation on ET procoagulant state, we assessed the contribution of TLR2 and TLR4 to platelet hemostatic and inflammatory properties in ET patients, by using Pam3CSK4 and lipopolysaccharide (LPS) as specific TLR2 and TLR4 ligands, respectively. TLR2 ligation induced increased surface translocation of α-granule-derived P-selectin and CD40L, which mediate platelet interaction with leukocytes and endothelial cells, respectively, and higher levels of dense granule-derived CD63 in patients, whereas PAC-1 binding was not increased and LPS had no effect on these platelet responses. Platelet-neutrophil aggregate formation was elevated in ET at baseline and after stimulation of both TLR2 and TLR4. In addition, ET patients displayed higher TLR2- and TLR4-triggered platelet secretion of the chemokine RANTES (CCL5), whereas von Willebrand factor release was not enhanced, revealing a differential releasate pattern for α-granule-stored inflammatory molecules. TLR-mediated hyperresponsiveness contrasted with impaired or preserved responses to classic platelet hemostatic agonists, such as TRAP-6 and thrombin. TLR2 and TLR4 expression on the platelet surface was normal, whereas phosphorylation of downstream effector ERK1/2 was higher in patients at baseline and after incubation with Pam3CSK4, which may partly explain the enhanced TLR2 response. In conclusion, exacerbated response to TLR stimulation may promote platelet activation in ET, boosting platelet/leukocyte/endothelial interactions and secretion of inflammatory mediators, overall reinforcing the thromboinflammatory state. These findings highlight the role of platelets as inflammatory sentinels in MPN prothrombotic scenario and provide additional evidence for the close intertwining between thrombosis and inflammation in this setting.
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Plaquetas/fisiología , Inflamación/etiología , Trombocitemia Esencial/complicaciones , Trombosis/etiología , Receptores Toll-Like/fisiología , Adulto , Anciano , Quimiocina CCL5/fisiología , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Femenino , Humanos , Masculino , Persona de Mediana Edad , Neutrófilos/fisiología , Fosforilación , Activación Plaquetaria , Trombocitemia Esencial/inmunologíaRESUMEN
This study investigated the involvement of chemokines including stromal derived factor 1 (SDF-1), interleukin 8 (IL-8), growth-related oncogene alpha (GRO-alpha) and their receptors, CXCR4, CXCR2 and CXCR1 in essential thrombocythemia (ET), a chronic myeloproliferative disease characterized by megakaryocytic hyperplasia and high platelet count. Fifty-three ET patients were studied. Plasma levels of SDF-1, IL-8 and GRO-alpha, evaluated by enzyme-linked immunosorbent assay, and flow cytometric analysis of CXCR1 and CXCR2 on the platelet membrane, were found to be normal in ET patients. CXCR4 expression on platelet surface as well as platelet CXCR4 mRNA detected by real-time reverse transcription polymerase chain reaction, were decreased. Platelet CXCR4 internalization rate was normal while SDF-1-induced platelet aggregation was delayed, decreased or absent. Immunohistochemical staining revealed that megakaryocytes were also affected. CXCR4 decrease was not observed either in peripheral white blood cells or in circulating CD34(+) precursors. These results show that CXCR4 is decreased in the megakaryocytic lineage in ET, mainly due to a reduced CXCR4 production, and an abnormal platelet response to SDF-1. This report is the first to describe platelet and megakaryocytic CXCR4 deficiency in a human disease and the presence of this abnormality in a megakaryocytic-related illness highlights the important role of SDF-1/CXCR4 axis in platelet development.
Asunto(s)
Quimiocina CXCL12/metabolismo , Megacariocitos/metabolismo , Receptores CXCR4/metabolismo , Trombocitemia Esencial/metabolismo , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Plaquetas/química , Plaquetas/metabolismo , Estudios de Casos y Controles , Quimiocina CXCL1/sangre , Quimiocina CXCL12/sangre , Niño , Femenino , Citometría de Flujo , Humanos , Inmunohistoquímica , Interleucina-8/sangre , Masculino , Megacariocitos/química , Persona de Mediana Edad , Agregación Plaquetaria , ARN Mensajero/análisis , Receptores CXCR4/análisis , Receptores CXCR4/genética , Receptores de Interleucina-8A/análisis , Receptores de Interleucina-8B/análisis , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa/métodos , Trombocitemia Esencial/sangreRESUMEN
Mechanisms leading to low platelet count in immune thrombocytopenia (ITP) involves both decreased production and increased destruction of platelet. However, the contribution of these pathologic mechanisms to clinical outcome of individual patients is uncertain. Here we evaluated different pathogenic mechanisms including in vitro megakaryopoiesis, platelet/megakaryocyte (MK) desialylation and MK apoptosis, and compared these effects with thrombopoyesis and platelet apoptosis in the same cohort of ITP patients. Normal umbilical cord blood-CD34+ cells, mature MK derived cells or platelets were incubated with plasma from ITP patients. Despite inhibition of thrombopoiesis previously observed, megakaryopoiesis was normal or even increased. Plasma from ITP patients affected the sialylation pattern of control platelets and this effect occurred concomitantly with apoptosis in 35% ITP samples. However, none of these abnormalities were observed in control MKs incubated with ITP plasma. Addition of mononuclear cells as immune effectors did not lead to phosphatidylserine exposure in MK, ruling out an antibody-mediated cytotoxic effect. These results suggest that both desialylation and apoptosis may be relevant mechanisms leading to platelet destruction although, they do not interfere with MK function. Analysis of these thrombocytopenic factors in individual patients showed no specific distribution pattern. However, the presence of circulating antiplatelet autoantibodies was associated with higher incidence of abnormalities. In conclusion, the causes of thrombocytopenia are multifactorial and may occur together, providing a rational basis for the use of combination therapies targeting concomitant ITP mechanisms in patients with refractory disease.
RESUMEN
Megakaryopoiesis and platelet production are driven by transcription factors and cytokines present in bone marrow environment. Essential thrombocythemia (ET) is a chronic myeloproliferative disorder characterized by high platelet count and megakaryocytic hyperplasia. In the present work we evaluated plasmatic levels of cytokines involved in megakaryocytic development in a group of patients with ET that were not on treatment, as well as thrombopoietin (TPO) levels before and during anagrelide treatment. The assays were carried out using ELISA techniques. Among the cytokines mainly involved in proliferation of megakaryocytic progenitors, interleukin 3 (IL-3) levels were found increased in patients compared to normal controls (p = 0.0383). Granulocyte-macrophage colony stimulating factor and stem cell factor levels were normal. Interleukin 6, as well as interleukin 11 and erythropoietin (EPO), cytokines mainly related to megakaryocytic maturation, were normal. Plasma TPO levels before treatment were within the normal range and increased during treatment but the difference was not statistically significant. Patients who displayed spontaneous platelet aggregation had higher plasma TPO levels compared to those who did not (p = 0.049). We did not find any relationship between cytokine levels and clinical or laboratory parameters. The high IL-3 levels seen in some patients with ET could contribute to megakaryocytic proliferation. The simultaneous occurrence of higher TPO levels and elevated platelet count could be a predisposing factor for the development of spontaneous platelet aggregation in ET patients.
Asunto(s)
Megacariocitos/fisiología , Trombocitemia Esencial/sangre , Trombopoyesis/fisiología , Trombopoyetina/sangre , Ensayo de Inmunoadsorción Enzimática , Factor Estimulante de Colonias de Granulocitos y Macrófagos/sangre , Factor Estimulante de Colonias de Granulocitos y Macrófagos/efectos de los fármacos , Humanos , Interleucina-3/sangre , Megacariocitos/efectos de los fármacos , Agregación Plaquetaria/efectos de los fármacos , Agregación Plaquetaria/fisiología , Inhibidores de Agregación Plaquetaria/uso terapéutico , Quinazolinas/uso terapéutico , Estudios Retrospectivos , Estadísticas no Paramétricas , Factor de Células Madre/sangre , Factor de Células Madre/efectos de los fármacos , Trombocitemia Esencial/tratamiento farmacológico , Trombocitosis/inducido químicamente , Trombopoyesis/efectos de los fármacos , Trombopoyetina/efectos de los fármacosRESUMEN
Mechanisms leading to decreased platelet count in immune thrombocytopenia (ITP) are heterogeneous. This study describes increased platelet apoptosis involving loss of mitochondrial membrane potential (ΔΨm), caspase 3 activation (aCasp3) and phosphatidylserine (PS) externalization in a cohort of adult ITP patients. Apoptosis was not related to platelet activation, as PAC-1 binding, P-selectin exposure and GPIb-IX internalization were not increased. Besides, ITP platelets were more sensitive to apoptotic stimulus in terms of aCasp3. Incubation of normal platelets with ITP plasma induced loss of ΔΨm, while PS exposure and aCasp3 remained unaltered. The increase in PS exposure observed in ITP platelets could be reproduced in normal platelets incubated with ITP plasma by adding normal CD3+ lymphocytes to the system as effector cells. Addition of leupeptin -a cathepsin B inhibitor- to this system protected platelets from apoptosis. Increased PS exposure was also observed when normal platelets and CD3+ lymphocytes were incubated with purified IgG from ITP patients and was absent when ITP plasma was depleted of auto-antibodies, pointing to the latter as responsible for platelet damage. Apoptosis was present in platelets from all patients carrying anti-GPIIb-IIIa and anti-GPIb auto-antibodies but was absent in the patient with anti-GPIa-IIa auto-antibodies. Platelet damage inversely correlated with platelet count and decreased during treatment with a thrombopoietin receptor agonist. These results point to a key role for auto-antibodies in platelet apoptosis and suggest that antibody-dependent cell cytotoxicity is the mechanism underlying this phenomenon.
Asunto(s)
Autoanticuerpos/inmunología , Plaquetas/patología , Púrpura Trombocitopénica Idiopática/inmunología , Adulto , Anciano , Anciano de 80 o más Años , Plaquetas/efectos de los fármacos , Plaquetas/inmunología , Complejo CD3/metabolismo , Calcimicina/farmacología , Caspasa 3/metabolismo , Humanos , Linfocitos/inmunología , Linfocitos/metabolismo , Persona de Mediana Edad , Fosfatidilserinas/metabolismo , Plasma , Activación Plaquetaria , Púrpura Trombocitopénica Idiopática/sangre , Adulto JovenRESUMEN
OBJECTIVE: In a previous study, we found increased plasma soluble receptor for interleukin-6 (sIL-6R) levels in patients with essential thrombocythemia (ET) that could promote megakaryopoiesis through IL-6 binding and further interaction with the signal transducer gp130. Here we have searched for the cell source of sIL-6R within mononuclear cells in these patients and the underlying abnormalities involved in its overproduction. MATERIALS AND METHODS: Thirty patients with the diagnosis of ET were studied. sIL-6R levels were measured by enzyme-linked immunosorbent assay technique in the supernatants of peripheral monocyte and lymphocyte cultures. Expression of membrane-anchored IL-6R was determined by flow cytometry. In order to study the mechanism of sIL-6R production, tumor necrosis factor-α protease inhibitor was added to specifically block IL-6R shedding. Gene expression of sIL-6R levels were evaluated by reverse transcription polymerase chain reaction. RESULTS: Monocytes were the main source of sIL-6R. Besides, in ET patients, monocyte sIL-6R release was higher than that of controls (p = 0.0014). Lymphocytes enhanced monocyte sIL-6R production by cell-mediated contact in normal controls, but this cooperation could not be seen in patients. Membrane expression of IL-6R was increased after monocyte adhesion in ET. sIL-6R synthesis was upregulated in most patients, while messenger RNA was normal. CONCLUSIONS: Our results indicate that ET monocytes are responsible for sIL-6R overproduction within mononuclear cells through synthesis upregulation. In addition, the lack of cooperation of lymphocytes in monocyte sIL-6R production in ET could be due to a monocyte abnormality. The agonistic effect of sIL-6R on IL-6 action could contribute to the exacerbated megakaryocytic growth in ET.
Asunto(s)
Linfocitos/metabolismo , Monocitos/metabolismo , Receptores de Interleucina-6/sangre , Trombocitemia Esencial/sangre , Adolescente , Adulto , Anciano , Membrana Celular/metabolismo , Células Cultivadas , Ensayo de Inmunoadsorción Enzimática , Femenino , Citometría de Flujo , Regulación de la Expresión Génica , Humanos , Janus Quinasa 2/genética , Masculino , Persona de Mediana Edad , Mutación , Receptores de Interleucina-6/genética , Receptores de Interleucina-6/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Solubilidad , Trombocitemia Esencial/genética , Trombocitemia Esencial/metabolismo , Adulto JovenRESUMEN
OBJECTIVE: JAK2V617F mutation rate in granulocytes from essential thrombocythemia (ET) patients ranges from 12% to 57%. Our aim was to evaluate the frequency of this mutation in the megakaryocyte/platelet lineage, and to analyze its clinical associations in ET. In addition, we determined whether this mutation leads to constitutive phosphorylation of STAT5 in platelets. MATERIALS AND METHODS: Consecutive patients with ET were included and clinical features were retrospectively reviewed. Mutation detection was performed by allele specific RT-PCR (AS-RT-PCR) and Restriction fragment length polymorphism (RFLP) analysis of platelet RNA. Constitutive phosphorylation of STAT5 in platelets was studied by Western blot. RESULTS: Fifty patients were included, 24 (48%) were JAK2V617F-positive by both AS-RT-PCR and RFLP. Patients with the mutation were older, had significantly higher hemoglobin levels, and lower platelet counts. Besides, higher frequency of thrombotic events was found in JAK2V617F-positive patients younger than 60, 53% vs. 4%, P = 0.0008. In addition, constitutive STAT5 phosphorylation was not detected in platelets from 12 patients. CONCLUSIONS: The frequency of the JAK2V617F mutation in platelets was similar to that reported in granulocytes in the literature, suggesting this mutation does not occur as an isolated event in the megakaryocyte lineage. If confirmed in a larger study, the observed higher frequency of thrombosis in patients younger than 60 might be a useful predictive marker for thrombosis in this subset of patients. Even though this mutation has been predicted to constitutively activate the JAK2 kinase, spontaneous phosphorylation of STAT5 does not seem to be a frequent finding in platelets from ET patients.