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Neutrophils participate in the initiation and progression of rheumatoid arthritis (RA) although the exact mechanisms responsible for neutrophil accumulation in rheumatoid joints are not understood. This study compared the adhesive and chemotactic functions of neutrophils from RA patients in activity (DAS28 > 3.2) and not in activity (DAS28 < 2.6) and observed the effects of different treatment approaches on these functions. Neutrophils were isolated from healthy controls (CON), and patients with active or inactive RA in use of therapy not specific for RA (NSAIDs), in use of DMARDs and in use of anti-TNF-α therapy. Adhesive and chemotactic properties were evaluated using in vitro assays; adhesion molecule expression was assessed by flow cytometry and real-time PCR and circulating chemokines were determined by ELISA. No significant alterations in the adhesive and chemotactic properties of neutrophils from active RA were observed when compared to CON neutrophils, independently of treatment regimen. In contrast, neutrophils from RA patients in disease remission presented reduced adhesive properties and a lower spontaneous chemotactic capacity, in association with decreased adhesion molecule expression, although profiles of alterations differed for those patients on DMARDs and those on anti-TNF-α therapy. Circulating levels of the major neutrophilic chemokines, IL-8 and epithelial neutrophil activating peptide-78, were also significantly decreased in those patients demonstrating a clinical response. Remission of RA appears to be associated with ameliorations in aspects important for neutrophil adhesion and chemotaxis; whether these alterations contribute to decrease neutrophil migration to the synovial fluid, with consequent improvements in the clinical manifestations of RA, remains to be determined. [ABSTRACT FROM AUTHOR]

Haemoglobin S polymerization in the red blood cells (RBCs) of individuals with sickle cell anaemia (SCA) can cause RBC sickling and cellular alterations. Piezo1 is a mechanosensitive protein that modulates intracellular calcium (Ca 2+ ) influx, and its activation has been associated with increased RBC surface membrane phosphatidylserine (PS) exposure. Hypothesizing that Piezo1 activation, and ensuing Gárdos channel activity, alter sickle RBC properties, RBCs from patients with SCA were incubated with the Piezo1 agonist, Yoda1 (0.1-10 μM). Oxygen-gradient ektacytometry and membrane potential measurement showed that Piezo1 activation significantly decreased sickle RBC deformability, augmented sickling propensity, and triggered pronounced membrane hyperpolarization, in association with Gárdos channel activation and Ca 2+ influx. Yoda1 induced Ca 2+ -dependent adhesion of sickle RBCs to laminin, in microfluidic assays, mediated by increased BCAM binding affinity. Furthermore, RBCs from SCA patients that were homo-/heterozygous for the rs59446030 gain-of-function Piezo1 variant demonstrated enhanced sickling under deoxygenation and increased PS exposure. Thus, Piezo1 stimulation decreases sickle RBC deformability, and increases the propensities of these cells to sickle upon deoxygenation and adhere to laminin. Results support a role of Piezo1 in some of the RBC properties that contribute to SCA vaso-occlusion, indicating that Piezo1 may represent a potential therapeutic target molecule for this disease., (© 2023 British Society for Haematology and John Wiley & Sons Ltd.)

The presence of high levels of fetal haemoglobin (HbF) provides well-validated clinical benefits to patients with sickle cell anaemia (SCA). Nevertheless it has been difficult to show clear direct effects of the known genetic HbF modifiers, such as the enhancer polymorphisms for haematopoietic transcription factors BCL11A and MYB, on SCA severity. Investigating SCA patients from Brazil, with a high degree of European genetic admixture, we have detected strong effects of these variants on HbF levels. Critically, we have shown, for the first time, that the presence of such HbF-promoting variants leads to a reduced rate of SCA complications, especially stroke., (© 2016 John Wiley & Sons Ltd.)

Inflammation, leucocyte and red cell adhesion to the endothelium contribute to the pathogenesis of sickle cell anaemia. Neutrophils appear to be important for vaso-occlusion, however, eosinophils may also participate in this phenomenon. The role of eosinophils in the pathophysiology of sickle cell anaemia (SCA) and the effect of hydroxycarbamide (HC) therapy on the functional properties of these cells are not understood. Patients with SCA and those on HC therapy (SCAHC) were included in the study. SCAHC individuals presented significantly lower absolute numbers of eosinophils than SCA. Furthermore, SCAHC eosinophils demonstrated significantly lower adhesive properties, compared to SCA eosinophils. SCA and SCAHC eosinophils presented greater spontaneous migration when compared with control eosinophils. Baseline eosinophil peroxidase and reactive oxygen species release was higher for SCA individuals than for control individuals, as were plasma levels of eosinophil derived neurotoxin. SCAHC eosinophil degranulation was lower than that of SCA eosinophil degranulation. Eotaxin-1 and RANTES levels were higher in the plasma of SCA and SCAHC individuals, when compared with controls. These data suggest that eosinophils exist in an activated state in SCA and indicate that these cells play a role in the vaso-occlusive process. The exact mechanism by which HC may alter SCA eosinophil properties is not clear., (© 2013 John Wiley & Sons Ltd.)

Chronic vascular inflammation and endothelial activation may initiate vaso-occlusion in sickle cell disease (SCD). TNFSF14 (CD258; LIGHT), a recently-identified pro-thrombotic and pro-inflammatory tumour necrosis factor (TNF)-superfamily cytokine, has a potent activating effect on endothelial cells. We evaluated whether TNFSF14 production is altered in SCD and whether platelets contribute to this production. TNFSF14 was measured in platelet-free plasma from healthy-control individuals (CON), steady-state sickle cell anaemia (SCA), SCA on hydroxycarbamide therapy (SCAHC) and haemoglobin SC (HbSC) patients. Mean plasma TNFSF14 was significantly increased in SCA, SCAHC and HbSC, compared to CON individuals. In SCA/SCAHC patients, plasma TNFSF14, showed no correlation with haematological variables, but was significantly correlated with serum lactate dehydrogenase and inflammatory markers (CD40LG , IL8 and ICAM1). Platelet-membrane TNFSF14 expression was significantly augmented on SCA platelets, and correlated with platelet activation; furthermore, measurement of platelet TNFSF14 release indicated that platelets may be a major source of circulating TNFSF14 in SCA. Interestingly, high plasma TNFSF14 was significantly associated with elevated tricuspid regurgitant velocity (≥2·5 m/s) in a population of SCA/SCAHC patients. The pro-inflammatory and atherogenic cytokine, TNFSF14, could contribute to endothelial activation and inflammation in SCA; future investigations may confirm whether this protein contributes to major clinical complications of the disease, such as pulmonary hypertension, and represents a potential therapeutic target., (© 2012 Blackwell Publishing Ltd.)

Whilst high pro-coagulant activity is reported in sickle cell disease (SCD), the precise role of platelets (PLTs) in SCD inflammatory and vaso-occlusive processes is unclear. Adhesion of PLTs from healthy controls (CON), SCD individuals (SCD) and SCD patients on hydroxycarbamide (SCDHC) to fibrinogen (FB) was compared using static adhesion assays. PLT adhesion molecules and intraplatelet cyclic adenosine monophosphate (icAMP) were observed by flow cytometry and enzyme-linked immunosorbent assay. SCD-PLTs demonstrated significantly greater adhesion than CON-PLTs to FB. Participation of the alpha(IIb)beta(3)-integrin in SCD-PLT adhesion was implicated by increased alpha(IIb)beta(3) activation and data showing that an alpha(IIb)beta(3)-function-inhibiting antibody significantly diminished SCD-PLT adhesion to FB. Platelet activation was potentiated by reductions in icAMP; cAMP levels were decreased in SCD-PLTs, being comparable to those of thrombin-stimulated CON-PLTs. Furthermore, SCD-PLT adhesion to FB was significantly reduced by cilostazol, an inhibitor of cAMP-hydrolyzing phosphodiesterase 3A (PDE3A). Both alpha(IIb)beta(3)-integrin activation and icAMP correlated significantly with fetal haemoglobin in SCD. Accordingly, hydroxycarbamide therapy was associated with lower PLT adhesion and higher icAMP. SCD-PLTs may be capable of adhering to proteins encountered on the inflamed vascular wall and, potentially, participate in vaso-occlusive processes. Hydroxycarbamide and, speculatively, nitric oxide donor or cyclic-nucleotide-targeted therapies may aid in the reversal of PLT adhesive properties in SCD.

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.

Sickle cell disease (SCD) is a chronic inflammatory condition characterized by high leucocyte counts, altered cytokine levels and endothelial cell injury. As the removal of inflammatory cells by apoptosis is fundamental for the resolution of inflammation, we aimed to determine whether the leucocyte apoptotic process is altered in SCD. Neutrophils from SCD individuals showed an inhibition of spontaneous apoptosis when cultured in vitro, in the presence of autologous serum for 20 h. Intracellular cyclic adenosine monophosphate (cAMP) levels were approximately twofold increased in SCD neutrophils; possible cAMP-upregulating factors present in SCD serum include interleukin-8, granulocyte-macrophage colony-stimulating factor and prostaglandin. Accordingly, co-incubation of SCD neutrophils with KT5720, a cAMP-dependent protein kinase (PKA) inhibitor, abrogated increased SCD neutrophil survival. Caspase-3 activity was also significantly diminished in SCD neutrophils cultured for 16 h and this activity was restored when cells were co-incubated with KT5720. BIRC2 (encoding cellular inhibitor of apoptosis protein 1, cIAP(1)), MCL1 and BAX expression were unaltered in SCD neutrophils; however, BIRC3 (encoding the caspase inhibitor, cIAP(2)), was expressed at significantly higher levels. Thus, we report an inhibition of spontaneous SCD neutrophil apoptosis that appears to be mediated by upregulated cAMP-PKA signalling and decreased caspase activity. Increased neutrophil survival may have significant consequences in SCD; contributing to leucocytosis, tissue damage and exacerbation of the chronic inflammatory state.

The present study found that the cyclic adenosine monophosphate (cAMP)-dependent pathway efficiently induced gamma-globin expression in adult erythroblasts, and this pathway plays a role in gamma-globin gene (HBG) expression in beta-thalassaemia. Expression of HBG mRNA increased to about 46% of non-HBA mRNA in adult erythroblasts treated with forskolin, while a cyclic guanosine monophosphate (cGMP) analogue induced HBG mRNA to levels <20% of non-HBA mRNA. In patients with beta-thalassaemia intermedia, cAMP levels were elevated in both red blood cells and nucleated erythroblasts but no consistent elevation was found with cGMP levels. The transcription factor cAMP response element binding protein (CREB) was phosphorylated in nucleated erythroblasts and its phosphorylation levels correlated with HBG mRNA levels of the patients. Other signalling molecules, such as mitogen-activated protein kinases and signal transducers and activators of transcription proteins, were phosphorylated at variable levels and showed no correlations with the HBG mRNA levels. Plasma levels of cytokines, such as erythropoietin, stem cell factor and transforming growth factor-beta were increased in patients, and these cytokines induced both HBG mRNA expression and CREB phosphorylation. These results demonstrate that the cAMP-dependent pathway, the activity of which is augmented by multiple cytokines, plays a role in regulating HBG expression in beta-thalassaemia.

Increased adhesive events between the blood vessel endothelium and red and white cells play a central role in the initiation of vasoocclusive crisis in sickle cell disease (SCD). Soluble VCAM-1 levels are increased in the plasma of sickle cell patients and may be reduced during hydroxyurea (HU) therapy. Reports regarding any changes in soluble ICAM-1 (sICAM-1) levels in sickle cell patients, however, are conflicting, and as yet no beneficial effect of HU upon levels has been observed. Thus, we sought to thoroughly investigate changes in sICAM-1 levels in SCD patients and the effect of HU therapy (20-30 mg/kg/day). Plasma sVCAM-1 levels were significantly higher in steady-state SCD patients than in normal controls (766 +/- 86 ng/mL vs. 325 +/- 38 ng/mL, respectively, P < 0.0001). sVCAM-1 levels were decreased in patients on HU therapy (543 +/- 69 ng/mL) compared to those not taking HU; however, this difference was not significant. Plasma sICAM-1 levels were significantly increased in steady-state SCD patients compared to normal individuals (285 +/- 20 ng/mL vs. 202 +/- 16 ng/mL, respectively, P = 0.002), and HU therapy significantly reduced sICAM-1 levels in patients (217 +/- 12, P = 0.027) to levels approaching those of healthy individuals. sVCAM-1 levels inversely correlated with fetal hemoglobin levels in SCD patients, while a nonsignificant inverse trend was observed between sICAM-1 levels and fetal hemoglobin. In conclusion, plasma sICAM-1 levels were significantly increased in SCD patients, and this increase was reversed by hydroxyurea therapy, possibly reflecting reduced endothelial activation in patients taking HU. Such an event may benefit patients by reducing adhesive interactions between white cells and the endothelium., (Copyright 2004 Wiley-Liss, Inc.)

Activation of soluble guanylate cyclase (sGC) has been reported to up-regulate gamma-globin gene transcription in erythroid cell lines and primary erythroblasts. sGC is activated by nitric oxide (NO), subsequently catalysing the conversion of guanosine triphosphate to cyclic guanosine monophosphate (cGMP), which mediates various physiological responses. To study the importance of this mechanism in the erythroid cells of sickle cell patients, cGMP levels were measured in the red blood cells (RBC) of normal individuals, steady-state sickle cell patients (SS) and SS patients on hydroxyurea (HU) therapy (SS + HU). cGMP levels were found to be significantly higher in RBC of SS patients (SS RBC) than in RBC of normal individuals, and were further increased in RBC of SS + HU patients. cGMP levels correlated with fetal haemoglobin (HbF) levels in SS/SS + HU patients, but not with reticulocyte count. Furthermore, NO-stimulated sGC activity, following incubation of cells with a NO donor, was significantly greater in SS RBC than in normal RBC. These results demonstrate, for the first time, an increased metabolism of NO mediated by sGC in the SS RBC, which is further increased by hydroxyurea. Augmentation of cGMP levels by NO in erythroid cells may constitute a mechanism for induction of HbF and other erythrocyte functions and represent a possible therapeutic target for treatment of sickle cell disease.