Summary 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, cIAP1), MCL1 and BAX expression were unaltered in SCD neutrophils; however, BIRC3 (encoding the caspase inhibitor, cIAP2), 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.

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A chronic inflammatory state is now thought to underlie much of the pathophysiology of sickle cell disease (SCD). Increased leukocyte numbers are associated with increased morbidity and mortality in SCD and the adhesion of leukocytes to the vascular endothelium and sickle red cells may play an initiating role in the vaso-occlusive process. We sought to better understand the signaling events that lead to alterations in SCD neutrophil adhesive and chemotactic abilities. Neutrophils were isolated from the peripheral blood of healthy controls and SCD individuals in steady state by separation over a ficoll-paque gradient. Following washing and lysis of contaminating red cells, adhesion to fibronectin (FN) and chemotaxis of control and SCD neutrophils (4×106 cells/ml in RMPI medium) were assessed using static adhesion assays and a 96-well chemotaxis chamber assay (ChemoTX, Neuroprobe). Cell adhesion to FN (20mg/ml; 30 min, 37°C, 5% CO2) or IL-8-induced migration (2h, 37°C, 5% CO2) was calculated using a standard curve of the original cell suspension and expressed as the percentage of cells adhered or the number of cells migrated. As previously shown, SCD neutrophils demonstrate a greater ability to adhere to FN-coated plates than control individual leukocytes (13.1±1.48 % neutrophils adhered compared to 5.0±0.6 %; n=11; P<0.001 Mann-Whitney test). Interestingly, SCD neutrophils demonstrate significantly higher levels of the secondary messenger, cAMP, than control neutrophils (4.76±0.38 pMol/106 cells compared to 1.91±0.34 pMol/106 cells; n≥12; P<0.001). When neutrophils were incubated on FN-coated plates in the presence of the cAMP-dependent kinase (PKA) inhibitor, KT5720 (3μM), increased SCD neutrophil adhesion was significantly reversed to levels approaching those of control neutrophils (7.73±1.3 %; n=11; P=0.001). In contrast, control neutrophil adhesion to FN was not significantly affected by the PKA inhibitor (P>0.05). Furthermore, activation of primed SCD neutrophils by the chemokine, IL-8 (500ng/ml), resulted in a significant increase in SCD neutrophil adhesion to FN (19.3±2.2 %; P=0.02; n=7) with a concomitant increase in intracellular cAMP levels (335.8±17.1 % increase, P<0.05, n=3) that could also be reverted by co-incubation with the PKA inhibitor, KT5720 (11.8 ± 0.8 %; P=0.02; n=7). Interestingly, in vitro chemotaxis of SCD neutrophils in response to a stimulus of IL-8 (100 ng/ml) was found to be significantly increased when compared to control neutrophil IL-8-stimulated chemotaxis (14.3±1.1 x105 cells/ml migrated compared to 8.8±0.8x105 cells/ml; P=0.005; n=8). Once again, KT5720 (3 μM, pre-incubation for 15 min, 37°C), was able to significantly reverse (8.5±1.1 x105/ml; P=0.03; n=6) this enhanced IL-8-dependent SCD neutrophil chemotaxis. Results suggest that an up-regulated PKA-dependent pathway may play an important role in the altered adhesive and chemotactic properties of SCD neutrophils and their further activation by cytokines/chemokines such as IL-8, found in increased levels in the serum of SCD patients. Thus, upregulation of the cAMP-PKA pathway in SCD neutrophils may have important consequences for the chronic inflammatory state in SCD patients and may characterize a novel target for the control of altered neutrophil function in SCD.

Sickle cell disease (SCD) is a chronic inflammatory disease that results in hemolytic anemia and vaso-occlusive events, the principal cause of morbidity in SCD patients. Whilst roles for red cells and leukocytes in the vaso-occlusive process in SCD are well established, the contribution of platelets to this process remains unclear. Increased platelet activation has been described in SCD patients, contributing to the hemostatic activation observed in the disease. Since the adhesion of platelets to the vascular endothelium and extracellular matrix (ECM) could potentially contribute to vaso-occulusion, this study compared the adhesive properties of platelets (PLTs) from healthy control subjects (CON), steady-state SCD patients (SCD) and SCD patients on hydroxyurea therapy (SCDHU; 20–30 mg/kg/day). Washed PLTs were ressuspended in Krebs solution (1.2×108 PLT/ml) and their adhesion to fibrinogen (FB, 50 μg/ml) coated 96-well plates evaluated utilizing static adhesion assays (15 min, 37°C). SCD PLTs demonstrated a significantly greater adhesion to FB than CON PLTs (26.0±4.0%; 15.8±2.0%, respectively, n≥13; p<0.05, Mann-Whitney test). In contrast, SCDHU PLT adhesion was significantly lower than that of SCD PLTs, being similar to that of CON PLTs (11.7±1.5%, n=13; p<0.01 comp. SCD). A thrombin stimulus (50 mU/ml) significantly increased CON, SCD and SCDHU adhesion to FB (26.2±2.1%; 33.8±3.7%; 23.5±3.7%, respectively, n≥13; p<0.001 comp. to basal, paired t test). Levels of the second messenger, cyclic adenosine monophosphate (cAMP), the reduction of which potentiates platelet activation, were found to be significantly decreased in the PLTs of SCD individuals compared to those of CON and SCDHU (5.4±0.6; 8.0±0.7; 7.1±0.4 pmol/108 PLT, respectively, n=7; P<0.05 for SCD comp. to CON and SCDHU). Interestingly, thrombin significantly reduced cAMP levels in CON and SCDHU (6.0±0.9; 5.4±0.5 pmol/108 PLTs, respectively, n=7, P<0.05 comp. to basal), but not SCD PLTs (4.7±0.6 pmol/108 PLT, n=7; P>0.05), providing further evidence that PLTs circulate in an activated state in SCD individuals. Flow cytometry studies demonstrated no alterations in the expressions of CD42b (GPIb molecule), CD62P (P-selectin), CD49b (α2 integrin subunit) and CD41a (αIIb integrin subunit) on the surface of SCD PLTs compared to CON and SCDHU (P<0.05, data not shown). However, binding of the PAC-1 antibody, which specifically recognizes activated αIIbβ3 integrin, was significantly higher on SCD PLTs than on CON and SCDHU PLTs (40.7±8.9%; 17.1±6.5%; 20.3±5.4%, respect., n≥8, p<0.05). The αIIbβ3 integrin, in its activated affinity, can bind to numerous soluble ligands that in turn may bridge the interaction between PLTs, endothelial cells and other cell types. As such, PLTs from SCD individuals in steady state circulate in an activated state and present an increased ability to adhere to the blood component, FB, compared to control PLTs. Increased adhesive properties were found associated with decreased intraplatelet cAMP and high affinity αIIbβ3 integrin expression. Interestingly, HU therapy was associated with a reversal in increased PLT adhesion coupled with augmented intraplatelet cAMP and reduced αIIbβ3 activation. Thus, SCD platelets may have an increased capacity for adhesion to the vascular wall and ECM components, where the presence of adhered and activated platelets may contribute to the vaso-occlusive process by facilitating leukocyte accumulation at the endothelium and the release of platelet factors that may stimulate further inflammation at the site.

Gostaria de agradecer à Dra. Nicola por me aceitar como sua aluna, sua experiência e profissionalismo contribuíram para a minha formação, tanto profissional como pessoal.Ela é uma pessoa muito humana e generosa, que nunca mediu esforços para conseguir recursos e apoio nas horas que mais precisei. Tenho muito que agradecê-la. Meus agradecimentos ao prof. Dr. Fernando que me recebeu em seu laboratório como estagiária, e a toda equipe de alunos que apostaram e acreditaram no meu trabalho. À Flávia Pinho, pois ela foi a primeira pessoa que tive contato no laboratório e que se tornou uma grande amiga. Obrigada por tudo! À Carol e à Dri que me ensinaram como trabalhar numa sala de cultura, pois a cultura de células foi o meu primeiro aprendizado no laboratório, muito obrigada! À Dul que sempre me ajudou com os documentos para que eu conseguisse transporte e bolsa de estudos, sempre muito prestativa para minha permanência no laboratório. Muito obrigada! À minha grande amiga Ana Flávia, pois há muitos anos nossos caminhos estão se cruzando. É com grande prazer que declaro o meu amor por essa amiga que sempre esteve ao meu lado nas horas mais felizes e mais tristes da minha vida. Muito obrigada, Ana! Também gostaria de agradecer às funcionárias Lena e Simone que sempre trouxeram alegria e quitutes maravilhosos ao laboratório! Obrigada! Muito obrigada à Camila,