Simvastatin abrogates inflamed neutrophil adhesive properties, in association with the inhibition of Mac-1 integrin expression and modulation of Rho kinase activity

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Leukocytes play a primary role in vascular inflammation, and thus an understanding of the pathways involved in the activation of these cells and means to inhibit their consequent adhesion to the vessel wall is of significant interest. This study aimed to determine whether statins have a direct effect upon neutrophil adhesive properties under inflammatory conditions. Neutrophils from healthy individuals were subjected to adhesion assays (with fibronectin as ligand) and flow cytometry. In the presence of a TNF-alpha inflammatory stimulus, neutrophils displayed a rapid and substantial enhancement in their adhesive properties that was abrogated by preincubation of cells with simvastatin. Neutrophil surface expression of the Mac-1 integrin subunit, CD11b, was augmented by TNF-alpha, and this increased expression was also inhibited by simvastatin. TNF-alpha also induced neutrophil LFA-1 and Mac-1 activation, but this activation was not blocked by simvastatin. Interestingly, while addition of the isoprenoids, geranygerayl pyrophosphate and farnesyl pyrophosphate, to cells did not alter the effect of simvastatin on TNF-alpha-stimulated adhesion, concurrent incubation of cells with the Rho kinase (ROCK) inhibitor reversed the effects of simvastatin on neutrophil adhesion and CD11b expression. Simvastatin appears to have direct anti-inflammatory effects in neutrophils that may be mediated by modulation of ROCK activity.622127132Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq

Neutrophils of Rheumatoid Arthritis Patients on Anti-TNF-alpha Therapy and in Disease Remission Present Reduced Adhesive Functions in Association with Decreased Circulating Neutrophil-Attractant Chemokine Levels

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)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-alpha 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-alpha 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.734309318Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq

Inhibition of phosphodiesterase 9A reduces cytokine-stimulated in vitro adhesion of neutrophils from sickle cell anemia individuals

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)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. Effects of Interleukin 8 (IL-8), tumor necrosis factor-alpha (TNF-alpha), 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. SCA neutrophils demonstrated increased adhesive properties, compared to CON neutrophils; IL-8, TNF-alpha 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. 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.607633642Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq

Endothelial Activation by Platelets from Sickle Cell Anemia Patients

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Sickle cell anemia (SCA) is associated with a hypercoagulable state. Increased platelet activation is reported in SCA and SCA platelets may present augmented adhesion to the vascular endothelium, potentially contributing to the vaso-occlusive process. We sought to observe the effects of platelets (PLTs) from healthy control (CON) individuals and SCA individuals on endothelial activation, in vitro. Human umbilical vein endothelial cells (HUVEC) were cultured, in the presence, or not, of washed PLTs from CON or steady-state SCA individuals. Supernatants were reserved for cytokine quantification, and endothelial adhesion molecules (EAM) were analyzed by flow cytometry; gene expressions of ICAM1 and genes of the NF-kappa B pathway were analyzed by qPCR. SCA PLTs were found to be more inflammatory, displaying increased adhesive properties, an increased production of IL-1 beta and a tendency towards elevated expressions of P-selectin and activated alpha(IIbb)beta(3). Following culture in the presence of SCA PLTs, HUVEC presented significant augmentations in the expressions of the EAM, ICAM-1 and E-selectin, as well as increased IL-8 production and increased ICAM1 and NFKB1 (encodes p50 subunit of NF-kappa B) gene expressions. Interestingly, transwell inserts abolished the effects of SCA PLTs on EAM expression. Furthermore, an inhibitor of the NF-kappa B pathway, BAY 11-7082, also prevented the induction of EAM expression on the HUVEC surface by SCA PLTs. In conclusion, we find further evidence to indicate that platelets circulate in an activated state in sickle cell disease and are capable of stimulating endothelial cell activation. This effect appears to be mediated by direct contact, or even adhesion, between the platelets and endothelial cells and via NF kappa B-dependent signaling. As such, activated platelets in SCD may contribute to endothelial activation and, therefore, to the vaso-occlusive process. Results provide further evidence to support the use of anti-platelet approaches in association with other therapies for SCD.92Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)INCT do SangueFundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)FAPESP [2008/57441-0, 2009/54279-0