RESUMO
OBJECTIVES: Intravascular hemolysis may have important pathophysiological consequences, such as the induction of cellular adhesion and vasculopathy. We compared the adhesive properties of red cells (RBC) and platelets in hereditary spherocytosis (HS), paroxysmal nocturnal hemoglobinuria (PNH) and sickle cell disease (SCD) patients. DESIGN AND METHODS: The adhesion of RBC and platelets, from patients and healthy subjects, was determined using static adhesion assays. RBC surface markers were characterized by flow cytometry and lactate dehydrogenase (LDH), plasma hemoglobin (pHb) and TNF-α were assayed in serum/plasma samples. RESULTS: pHb levels were elevated in all three hemolytic diseases, indicating the incidence of intravascular hemolysis. RBC adhesion and TNF-α were augmented in HS and SCD, but not in PNH. Reticulocyte counts were raised in the three diseases, but were higher in HS and SCD than in PNH; high expressions of CD71, CD36 and CD49d were observed on SCD RBC, while CD71 alone was increased on HS and PNH RBC. Splenectomy was associated with reversals of increased pHb, RBC adhesion, reticulocytes, RBC marker expression and inflammation in HS. In contrast, platelet adhesion was elevated in SCD and PNH, but not HS. Platelet adhesion correlated significantly with serum LDH, but not pHb, in the hemolytic disease cohort; interestingly, LDH did not correlate with reticulocytes or pHb levels. CONCLUSIONS: Results indicate that extravascular, rather than intravascular, hemolysis (and ensuing RBC production) may contribute to elevations in RBC adhesive properties in HS and SCD, while mechanisms peculiar to each disease may augment platelet adhesion in SCD and PNH.
Assuntos
Anemia Falciforme/sangue , Eritrócitos/patologia , Hemoglobinúria Paroxística/sangue , Adesividade Plaquetária , Esferocitose Hereditária/sangue , Adolescente , Adulto , Idoso , Antígenos CD/metabolismo , Plaquetas/patologia , Antígenos CD36/metabolismo , Estudos de Casos e Controles , Criança , Eritrócitos/fisiologia , Feminino , Humanos , Integrina alfa4/metabolismo , L-Lactato Desidrogenase/sangue , Masculino , Pessoa de Meia-Idade , Proibitinas , Receptores da Transferrina/metabolismo , Valores de Referência , Contagem de Reticulócitos , Fator de Necrose Tumoral alfa/sangue , Adulto JovemRESUMO
OBJECTIVE: Leukocyte adhesion to vessel walls may initiate vaso-occlusion in sickle cell anemia (SCA); however, the extent to which inflammation participates in this mechanism is not understood. This in vitro study investigated whether inflammatory molecules, commonly augmented in SCA, can affect neutrophil adhesive properties and whether cyclic guanosine monophosphate (cGMP)-elevating agents can inhibit such adhesion. SUBJECTS AND METHODS: Effects of Interleukin 8 (IL-8), tumor necrosis factor-α (TNF-α), granulocyte macrophage-colony stimulating factor (GM-CSF) cytokines, BAY 73-6691 [phosphodiesterase (PDE)-9A-inhibitor], and BAY 41-2271 (guanylate-cylase stimulator) on the adhesive properties of neutrophils from healthy control (CON) and steady-state SCA individuals were determined using static-adhesion assays. RESULTS: SCA neutrophils demonstrated increased adhesive properties, compared to CON neutrophils; IL-8, TNF-α and GM-CSF increased CON neutrophil adhesion and further increased SCA neutrophil adhesion to fibronectin (FN). The PDE9A inhibitor, BAY-73-6691, significantly reduced basal CON neutrophil and SCA neutrophil adhesion; this was accompanied by decreased SCA neutrophil surface expressions of the L-selectin and CD11b adhesion molecules. BAY-73-6691 also significantly reduced cytokine-stimulated CON neutrophil and SCA neutrophil adhesion to FN; however, this was not accompanied by alterations in adhesion-molecule presentation. CONCLUSIONS: The chronic inflammatory nature of SCA may contribute to leukocyte adhesive functions in SCA. Furthermore, elevation of leukocyte cGMP may be an interesting approach for inhibition of leukocyte adhesion to the vessel wall, even in the presence of inflammatory stimuli.
Assuntos
3',5'-AMP Cíclico Fosfodiesterases/antagonistas & inibidores , Anemia Falciforme/fisiopatologia , Adesão Celular/efeitos dos fármacos , Citocinas/farmacologia , Neutrófilos/efeitos dos fármacos , Neutrófilos/fisiologia , Adolescente , Adulto , GMP Cíclico/metabolismo , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Neutrófilos/citologia , Adulto JovemRESUMO
BACKGROUND: Pharmacological approaches to inhibit increased leukocyte adhesive interactions in sickle cell disease may represent important strategies for the prevention of vaso-occlusion in patients with this disorder. We investigated, in vitro, the adhesion molecules involved in endothelial-sickle cell disease neutrophil interactions and the effect of simvastatin on sickle cell disease neutrophil adhesion to tumor necrosis factor-α-activated endothelial monolayers (human umbilical vein endothelial cells), and neutrophil chemotaxis. DESIGN AND METHODS: Sickle cell disease patients in steady state and not on hydroxyurea were included in the study. Endothelial cells treated, or not, with tumor necrosis factor-α and simvastatin were used for neutrophil adhesion assays. Neutrophils treated with simvastatin were submitted to interleukin 8-stimulated chemotaxis assays. RESULTS: Sickle cell disease neutrophils showed greater adhesion to endothelial cells than control neutrophils. Adhesion of control neutrophils to endothelial cells was mediated by Mac-1 under basal conditions and by the Mac-1 and LFA-1 integrins under inflammatory conditions. In contrast, adhesion of sickle cell disease neutrophils to endothelium, under both basal and tumor necrosis factor-α-stimulated conditions, was mediated by Mac-1 and LFA-1 integrins and also by VLA-4. Under stimulated inflammatory conditions, simvastatin significantly reduced sickle cell disease neutrophil adhesion, and this effect was reversed by inhibition of nitric oxide synthase. Furthermore, intercellular adhesion molecule-1 expression was significantly abrogated on tumor necrosis factor-α-stimulated endothelium incubated with simvastatin, and statin treatment inhibited the interleukin-8-stimulated migration of both control and sickle cell disease neutrophils. CONCLUSIONS: The integrins Mac-1, LFA-1 and, interestingly, VLA-4 mediate the adhesion of sickle cell disease leukocytes to activated endothelial cell layers, in vitro. Our data indicate that simvastatin may be able to reduce endothelial activation and consequent leukocyte adhesion in this in vitro model; future experiments and clinical trials may determine whether simvastatin therapy could be employed in patients with sickle cell disease, with beneficial effects on vaso-occlusion.
Assuntos
Anemia Falciforme/metabolismo , Endotélio Vascular/efeitos dos fármacos , Integrinas/metabolismo , Neutrófilos/efeitos dos fármacos , Neutrófilos/metabolismo , Sinvastatina/farmacologia , Adulto , Anticorpos Monoclonais/metabolismo , Adesão Celular/efeitos dos fármacos , Adesão Celular/genética , Moléculas de Adesão Celular/antagonistas & inibidores , Células Cultivadas , Quimiotaxia/efeitos dos fármacos , Células Endoteliais/efeitos dos fármacos , Células Endoteliais/metabolismo , Feminino , Humanos , Hipolipemiantes/farmacologia , Masculino , Pessoa de Meia-Idade , Fator de Necrose Tumoral alfa/farmacologia , Adulto JovemRESUMO
Modulation of intracellular cyclic guanosine monophosphate (cGMP) may characterize a therapeutic target for sickle cell disease (SCD); cGMP-dependent signalling may be important for erythroid foetal haemoglobin induction and exert anti-inflammatory functions in leucocytes. As the inhibition of phosphodiesterases (PDEs), which regulate intracellular cGMP, can result in tissue-specific elevation of cGMP, we studied the gene expressions of cGMP-specific PDEs (-1A, -5A and -9A) in the reticulocytes and neutrophils of healthy controls, steady-state SCD patients and SCD patients on hydroxycarbamide therapy (SCDHC). PDE9A gene expression was found in numerous cell types; however, high expression was found in neutrophils, reticulocytes, CD34(+)-derived erythroid cells and K562 erythroleukaemic cells, indicating a high haematopoietic cell expression. PDE9A gene expression was, however, significantly higher in the reticulocytes and neutrophils of SCD individuals, compared to control cells; Western blotting confirmed the production of PDE9A protein in SCD neutrophils and K562 cells. Inhibition of PDE9A enzyme with the specific inhibitor, BAY73-6691, significantly increased production of the gamma-globin gene (HBG) in K562 cells and reversed the increased adhesive properties of SCD neutrophils. Since elevation of haematopoietic intracellular cGMP may be beneficial in SCD, the relatively limited tissue distribution of PDE9A suggests that it could represent a novel drug target worthy of further study.
Assuntos
3',5'-AMP Cíclico Fosfodiesterases/metabolismo , 3',5'-GMP Cíclico Fosfodiesterases/metabolismo , Anemia Falciforme/enzimologia , 3',5'-AMP Cíclico Fosfodiesterases/antagonistas & inibidores , 3',5'-GMP Cíclico Fosfodiesterases/antagonistas & inibidores , Adolescente , Adulto , Anemia Falciforme/tratamento farmacológico , Antidrepanocíticos/farmacologia , Antidrepanocíticos/uso terapêutico , Brasil , Estudos de Casos e Controles , Células Eritroides/enzimologia , Feminino , Humanos , Hidroxiureia/farmacologia , Hidroxiureia/uso terapêutico , Masculino , Pessoa de Meia-Idade , Neutrófilos/enzimologia , Pirazóis/farmacologia , Pirimidinas/farmacologia , Reticulócitos/enzimologia , gama-Globinas/metabolismoRESUMO
Increased leukocyte adhesion to vascular endothelium contributes to vaso-occlusion in sickle cell disease. Since nitric oxide bioavailability is decreased in sickle cell disease and nitric oxide may inhibit leukocyte adhesion, we investigated whether stimulation of NO-signaling pathways can reduce the adhesive properties of neutrophils from sickle cell disease individuals (sickle cell diseaseneu). sickle cell diseaseneu presented greater adhesion in vitro to both fibronectin and ICAM-1 than control neutrophils. Co-incubation of sickle cell diseaseneu with the nitric oxide-donor agents, sodium nitroprusside and dietheylamine NONOate (DEANO), and the guanylate cyclase stimulator, BAY41-2272, all significantly reduced the increased adhesion to fibronectin/ICAM-1. Oxadiazolo[4,3-a]quinoxalin-1-one, a guanylate cyclase inhibitor, reversed sodium nitroprusside/DEANO-diminished adhesion to fibronectin, implicating cGMP-dependent signaling in this mechanism. Interestingly, intracellular cGMP was significantly higher in neutrophils from sickle cell disease individuals on hydroxyurea (sickle cell diseaseHUneu). Accordingly, sickle cell diseaseHUneu adhesion to fibronectin/ICAM-1 was significantly lower than that of sickle cell diseaseneu. Agents that stimulate the nitric oxide/cGMP-dependent pathway may have beneficial effects on leukocyte function if used in these subjects.
Assuntos
Anemia Falciforme/sangue , Adesão Celular/efeitos dos fármacos , Hidrazinas/farmacologia , Neutrófilos/efeitos dos fármacos , Doadores de Óxido Nítrico/farmacologia , Óxido Nítrico/fisiologia , Nitroprussiato/farmacologia , Pirazóis/farmacologia , Piridinas/farmacologia , Adulto , Anemia Falciforme/tratamento farmacológico , Anemia Falciforme/patologia , Antígeno CD11a/análise , Antígeno CD11b/análise , GMP Cíclico/fisiologia , Células Endoteliais/patologia , Feminino , Fibronectinas/metabolismo , Humanos , Hidroxiureia/farmacologia , Hidroxiureia/uso terapêutico , Integrina alfa4/análise , Molécula 1 de Adesão Intercelular/metabolismo , Masculino , Pessoa de Meia-Idade , Neutrófilos/patologia , Oxidiazóis/farmacologia , Quinoxalinas/farmacologia , Traço Falciforme/sangue , Traço Falciforme/tratamento farmacológico , Traço Falciforme/genética , Traço Falciforme/patologia , Talassemia alfa/sangue , Talassemia alfa/genética , Talassemia alfa/patologiaRESUMO
The significance of the leukocyte in sickle cell disease (SCD) pathophysiology is becoming increasingly recognised; we sought to examine whether the chemotactic properties of neutrophils of SCD individuals may be altered and, further, to better understand the signalling events that mediate altered SCD neutrophil function. Adhesion to immobilised fibronectin (FN) and chemotaxis of control and SCD neutrophils were assessed using in vitro static adhesion assays and 96-well chemotaxis chamber assays. Adhesion assays confirmed a significantly higher basal adhesion of SCD neutrophils to FN, compared with control neutrophils. Chemotaxis assays established, for the first time, that SCD neutrophils demonstrate greater spontaneous migration and, also, augmented migration in response to IL-8, when compared with control neutrophils. Co-incubation of SCD neutrophils with KT5720 (an inhibitor of PKA) abrogated increased basal SCD neutrophil adhesion, spontaneous chemotaxis and IL-8-stimulated chemotaxis. Stimulation of SCD neutrophils with IL-8 also significantly augmented SCD neutrophil adhesion to FN with a concomitant increase in cAMP levels and this increase in adhesion was abolished by KT5720. Interestingly, the adhesive properties of neutrophils from SCD individuals on hydroxyurea therapy were not significantly altered and results indicate that a reduction in intracellular cAMP may contribute to lower the adhesive properties of these cells. Data indicate that up-regulated cAMP signalling plays a significant role in the altered adhesive and migratory properties in SCD neutrophils. Such alterations may have important implications for the pathophysiology of the disease and the cAMP-PKA pathway may represent a therapeutic target for the abrogation of altered leukocyte function.
Assuntos
Anemia Falciforme/enzimologia , Quimiotaxia , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Neutrófilos/enzimologia , Transdução de Sinais , Adolescente , Adulto , Anemia Falciforme/tratamento farmacológico , Anemia Falciforme/patologia , Anemia Falciforme/fisiopatologia , Antidrepanocíticos/farmacologia , Antidrepanocíticos/uso terapêutico , Carbazóis/farmacologia , Adesão Celular/efeitos dos fármacos , Quimiotaxia/efeitos dos fármacos , AMP Cíclico/metabolismo , Proteínas Quinases Dependentes de AMP Cíclico/antagonistas & inibidores , Inibidores Enzimáticos/farmacologia , Feminino , Fibronectinas , Humanos , Hidroxiureia/farmacologia , Hidroxiureia/uso terapêutico , Indóis/farmacologia , Interleucina-8/farmacologia , Masculino , Pessoa de Meia-Idade , Neutrófilos/patologia , Pirróis/farmacologia , Transdução de Sinais/efeitos dos fármacosRESUMO
Propagation of the vaso-occlusive process in sickle cell anaemia (SCA) is a complex process involving the adhesion of steady-state SCA patients red cells and reticulocytes to the vascular endothelium. The effect of hydroxyurea therapy (HUT) on the adhesive properties of sickle cells and the expression of adhesion molecule genes by erythroid cells of SCA individuals is not yet fully understood. The expressions of the CD36 gene and the VLA-4-integrin subunit genes, CD49d (alpha-subunit) and CD29 (beta-subunit), were compared in the reticulocytes of steady-state SCA patients and patients on HUT using real-time PCR. Basal adhesion of red cells from these subjects was also compared using static adhesion assays, as was surface protein expression, using flow cytometry. Basal sickle red cell adhesion to fibronectin was significantly greater than that of normal cells (P < 0.01); in contrast, HUT was associated with significantly lower levels (P < 0.01) of red cell adhesion that were similar to those of control cells; this decrease could not be justified solely by altered reticulocyte numbers in this population. Accordingly, flow cytometry demonstrated that reticulocytes from patients on HUT had significantly lower CD36 and CD49d surface expressions (P < 0.01) and, importantly, significantly lower expressions of the CD36, CD49d and CD29 genes (P < 0.05) than reticulocytes of SCA patients not on HUT. Taken together, data support the hypothesis that HUT reduces the adhesive properties of sickle cells and that this decrease appears to be mediated, at least in part, by a decrease in the gene and, consequently, surface protein expression of adhesion molecules such as VLA-4 and CD36.
Assuntos
Anemia Falciforme/tratamento farmacológico , Moléculas de Adesão Celular/biossíntese , Adesão Celular/efeitos dos fármacos , Regulação da Expressão Gênica/efeitos dos fármacos , Hidroxiureia/uso terapêutico , Adulto , Anemia Falciforme/patologia , Antígenos CD36/biossíntese , Antígenos CD36/genética , Moléculas de Adesão Celular/genética , Avaliação de Medicamentos , Feminino , Fibronectinas/metabolismo , Perfilação da Expressão Gênica , Humanos , Hidroxiureia/farmacologia , Integrina alfa4/biossíntese , Integrina alfa4/genética , Integrina alfa4beta1/biossíntese , Integrina alfa4beta1/genética , Integrina beta1/biossíntese , Integrina beta1/genética , Sistema do Grupo Sanguíneo Lutheran , Masculino , Pessoa de Meia-Idade , Proteínas de Neoplasias/biossíntese , Proteínas de Neoplasias/genética , RNA Mensageiro/biossíntese , Reticulócitos/metabolismo , Reticulócitos/patologiaRESUMO
Migration of eosinophil (Eo) into tissues is a hallmark of chronic eosinophilic leukaemia (CEL), but the exact mechanism involved in cell migration is unknown. We report on a patient with CEL who presented high expressions of VLA-4, LFA-1 and Mac-1 integrins on the Eo surface, increased chemotaxis of Eo to eotaxin, decreased chemotaxis of Eo after inhibition of the cyclic guanosine monophosphate (cGMP), and a high level of intracellular cGMP. These findings suggest that an upregulation of expression of these integrins on Eo surface and a high intracellular level of cGMP may be involved in the increased Eo migration observed in our CEL patient. However, the definitive roles of these molecules in the proliferation and migration of Eo in CEL disease require wider confirmation by analysis of additional patients with the disease.
Assuntos
Movimento Celular , Eosinófilos/metabolismo , Síndrome Hipereosinofílica/metabolismo , Integrina alfa4beta1/metabolismo , Antígeno-1 Associado à Função Linfocitária/metabolismo , Antígeno de Macrófago 1/metabolismo , Fatores Quimiotáticos de Eosinófilos , Quimiotaxia de Leucócito , Doença Crônica , Eosinófilos/patologia , Feminino , Humanos , Síndrome Hipereosinofílica/diagnóstico , Síndrome Hipereosinofílica/terapia , Pessoa de Meia-IdadeRESUMO
Aberrant DNA methylation of multiple promoter associated CpG islands is a frequent phenomenon in acute lymphocytic leukemia (ALL). Recently, methylation of a cell cycle control pathway composed of P73, P15 and P57KIP2 has been shown to confer poor prognosis to adult patients with ALL. Using bisulfite PCR methods, we have explored the prevalence of methylation of this pathway in a cohort of children with ALL (N=20), and compared these results with those observed in a group of adult patients (N=53). P73 was methylated in 4 (20%) pediatric patients, P15 in 3 (15%), and P57KIP2 in 2 (10%). These compared to 14 (26%), p=0.5, 16 (30%), p=0.04 and 20 (37%), p=0.04, respectively in adult patients. Methylation of two or more genes was not observed in any pediatric patient, but in 15 (28%) adult patients (p=0.003). Poor survival of adult patients was associated with methylation of > or =2 genes (p=0.003). These results indicate that differences in DNA methylation of specific molecular pathways may contribute to the prognostic differences known to occur between pediatric and adult patients with ALL.
Assuntos
Proteínas de Ciclo Celular/genética , Metilação de DNA , Proteínas de Ligação a DNA/genética , Proteínas Nucleares/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , Proteínas Supressoras de Tumor/genética , Adolescente , Adulto , Idoso , Ciclo Celular/genética , Ciclo Celular/fisiologia , Proteínas de Ciclo Celular/metabolismo , Criança , Pré-Escolar , Estudos de Coortes , Ilhas de CpG , Inibidor de Quinase Dependente de Ciclina p15 , Inibidor de Quinase Dependente de Ciclina p57 , DNA/genética , DNA/metabolismo , Proteínas de Ligação a DNA/metabolismo , Feminino , Genes Supressores de Tumor , Humanos , Masculino , Pessoa de Meia-Idade , Proteínas Nucleares/metabolismo , Leucemia-Linfoma Linfoblástico de Células Precursoras/metabolismo , Prognóstico , Regiões Promotoras Genéticas/genética , Proteína Tumoral p73 , Proteínas Supressoras de Tumor/metabolismoRESUMO
A role for leukocytes in sickle cell vaso-occlusive crisis is becoming increasingly recognized. Neutrophil counts are higher in sickle cell patients and neutrophils from these patients demonstrate increased adhesion to endothelial monolayers under certain circumstances. The effects of selected cytokines on the adhesion mechanisms of normal neutrophils and neutrophils from sickle cell anaemia patients (SCA neutrophils) were investigated. Neutrophils were separated from the blood of homozygous (HbSS) SCA patients and healthy controls. Following pre-incubation (25 min, 37 degrees C) of the cells with cytokines, the adhesion of the cells to fibronectin (FN)-coated plates (20 micro) was determined (60 min, 37 degrees C, 5% CO2). Basal adhesion of normal and SCA neutrophils to FN was not statistically different. Pretreatment of normal neutrophils with either IL-6 (10-100 pg/ml), GCSF (1- 10 ng/ml) or IL-8 (1-100 ng/ml) had no significant effect upon their adhesion to FN. In contrast, SCA neutrophil adhesion to FN was increased significantly following pre-incubation with IL-6, G-CSF and IL-8 (p < 0.01). RANTES (1-100 ng/ml) had no significant effect on either normal or SCA neutrophil adhesion to FN. Flow-cytometric analyses demonstrated that IL-8 (10 ng/ml) significantly augments CD11b (Mac-1 integrin subunit) expression on SCA neutrophils, but not normal neutrophils. IL-6 and G-CSF (10 pg/ml and 10 ng/ml, respectively), however, had no effect on SCA neutrophil adhesion molecule expression. In conclusion, SCA neutrophil adhesion mechanisms may increase in the presence of certain cytokines, in vivo, and this activation may contribute to the physiopathology of sickle cell disease.
Assuntos
Anemia Falciforme/patologia , Antígenos CD11/biossíntese , Quimiocinas/farmacologia , Ativação de Neutrófilo/efeitos dos fármacos , Neutrófilos/patologia , Adolescente , Adulto , Anemia Falciforme/metabolismo , Adesão Celular/efeitos dos fármacos , Separação Celular , Células Cultivadas , Quimiocina CCL5/farmacologia , Criança , Relação Dose-Resposta a Droga , Células Endoteliais/metabolismo , Feminino , Fibronectinas/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Regulação da Expressão Gênica/fisiologia , Fator Estimulador de Colônias de Granulócitos/farmacologia , Hemoglobina Falciforme/metabolismo , Homozigoto , Humanos , Interleucina-6/farmacologia , Interleucina-8/farmacologia , Masculino , Pessoa de Meia-Idade , Proteínas Recombinantes/farmacologiaRESUMO
A hemoglobina S (HbS) está presente na populaçäo brasileira com prevalência variável, dependente dos grupos raciais formadores de cada regiäo. A migraçäo eletroforética em pH alcalino apresenta similaridade com outras hemoglobinas, e estudos complementares para sua correta caracterizaçäo säo necessários. No presente estudo objetivamos traçar um fluxograma com as metodologias disponíveis para a caracterizaçäo da hemoglobina S e das hemoglobinas que apresentam migraçäo semelhante em pH alcalino. No período de janeiro a junho de 2000, analisamos amostras de sangue com suspeita de hemoglobina S encaminhadas ao Laboratório de Hemoglobinas da Unesp. Caracterizamos diferentes mutantes e formas interativas com hemoglobina S, por procedimentos eletroforéticos, em variados pH, análises citológicas e testes bioquímicos específicos. Os procedimentos de análise aplicados resultaram em orientaçäo fornecida aos laboratórios de rotina sobre como proceder no diagnóstico laboratorial destas alteraçöes de hemoglobina. Desta forma contribuímos para um melhor conhecimento sobre a variabilidade genética das hemoglobinas em nossa populaçäo, auxiliando no acompanhamento clínico e no aconselhamento genético das hemoglobinopatias com fisiopatologia relacionada à alteraçäo