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

OBJECTIVES: 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. METHODS: Neutrophils from healthy individuals were subjected to adhesion assays (with fibronectin as ligand) and flow cytometry. RESULTS: In the presence of a TNF-α 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-α, and this increased expression was also inhibited by simvastatin. TNF-α 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-α-stimulated adhesion, concurrent incubation of cells with the Rho kinase (ROCK) inhibitor reversed the effects of simvastatin on neutrophil adhesion and CD11b expression. CONCLUSION: Simvastatin appears to have direct anti-inflammatory effects in neutrophils that may be mediated by modulation of ROCK activity.

Chagas disease and gynecologic neoplasias.

The inflammation caused by Trypanosoma cruzi produces irritation and cell proliferation and may contribute to the development of cancer. The objective was to determine the occurrence of gynecologic neoplasia (GN) and demographic characteristics in patients with Chagas disease (CD). We used protocols of 671 autopsies between 1976 and 2008. The patients were divided into 3 groups: with GN and CD, only with CD, and only with GN. The 2 diseases were observed in 4.5% of patients with a mean age of 47.6 years and who were predominantly white. The megaesophagus and megacolon were more frequent in the group with only CD. The most common benign neoplasm was uterine leiomyoma, and malignant, carcinoma of the cervix. We conclude that the epidemiological profile of patients with CD and GN was similar to the other groups, and the CD was found not to be a risk factor or protective against the development of GN.

Evaluation of Canonical Inflammasome Activation in Human Monocytes by Imaging Flow Cytometry.

Canonical inflammasome activation is a tightly regulated process that has been implicated in a broad spectrum of inflammatory disorders. Inflammasome formation requires assembly of a cytosolic sensor protein with the adapter, ASC (apoptosis-associated speck-like protein containing a caspase activating and recruitment domain). Once formed, this multimeric protein structure allows for the activation of caspase-1, responsible for IL-1ß/IL-18 release. During this process, cytoplasmic dispersed ASC molecules cluster in one condensed micrometric-sized complex named ASC "speck," which is traditionally assessed by fluorescence microscopy and widely accepted as a readout for canonical inflammasome activation. However, equally reliable but less time-consuming quantitative methods have emerged as a significant need in order to improve clinical assessment of inflammasome-related conditions. Multispectral imaging flow cytometry (MIFC) combines the qualitative power of fluorescence microscopy with high throughput capabilities and multiplexing potential of flow cytometry into one single system. Here we explored the optimal imaging-based tools to measure ASC speck formation via imaging flow cytometry by using peripheral blood mononuclear cells (PBMCs) stimulated with the NLRP3 agonist Nigericin, as a positive control. We demonstrate that this technique is also able to detect the distribution of active caspase-1 within the ASC aggregates by incubating cells with FAM-FLICATM, a fluorochrome inhibitor of caspase-1. By applying these tools in PBMCs from patients with distinct inflammatory disorders we demonstrate that MIFC is able to assess canonical inflammasome activation in a quantitative and statistically robust manner in clinically relevant samples. Therefore, we propose that accurate assessment of specks by MIFC could help guide preventive or therapeutic strategies in an array of human inflammatory diseases in which inflammasomes play an important role.

TNF induces neutrophil adhesion via formin-dependent cytoskeletal reorganization and activation of ß-integrin function.

Although essential for inflammatory responses, leukocyte recruitment to blood vessel walls in response to inflammatory stimuli, such as TNF-α, can contribute to vascular occlusion in inflammatory diseases, including atherosclerosis. We aimed to further characterize the mechanisms by which TNF stimulates adhesive and morphologic alterations in neutrophils. Microfluidic and intravital assays confirmed the potent effect that TNF has on human and murine neutrophil adhesion and recruitment in vitro and in vivo, respectively. Inhibition of actin polymerization by cytochalasin D significantly diminished TNF-induced human neutrophil adhesion in vitro and abolished TNF-induced membrane alterations and cell spreading. In contrast, TNF-induced increases in ß2-integrin (Mac-1 and LFA-1) expression was not significantly altered by actin polymerization inhibition. Consistent with a role for cytoskeletal rearrangements in TNF-induced adhesion, TNF augmented the activity of the Rho GTPase, RhoA, in human neutrophils. However, inhibition of the major RhoA effector protein, Rho kinase (ROCK), by Y-27632 failed to inhibit TNF-induced neutrophil adhesion. In contrast, the formin FH2 domain inhibitor, SMIFH2, abolished TNF-induced human neutrophil adhesion and diminished leukocyte recruitment in vivo. SMIFH2 also inhibited TNF-induced cytoskeletal reorganization in human neutrophils and abolished the alterations in ß2-integrin expression elicited by TNF stimulation. As such, Rho GTPase/mDia formin-mediated cytoskeletal reorganization appears to participate in the orchestration of TNF-induced neutrophil-adhesive interactions, possibly mediated by formin-mediated actin nucleation and subsequent modulation of ß2-integrin activity on the neutrophil surface. This pathway may represent a pharmacologic target for reducing leukocyte recruitment in inflammatory diseases.

Attenuation of TNF-induced neutrophil adhesion by simvastatin is associated with the inhibition of Rho-GTPase activity, p50 activity and morphological changes.

Neutrophil adhesion to the vasculature in response to potent inflammatory stimuli, such as TNF-α (TNF), can contribute to atheroprogression amongst other pathophysiological mechanisms. Previous studies have shown that simvastatin, a statin with known pleiotropic anti-inflammatory properties, can partially abrogate the effects of TNF-induced neutrophil adhesion, in association with the modulation of ß2-integrin expression. We aimed to further characterize the effects of this statin on neutrophil and leukocyte adhesive mechanisms in vitro and in vivo. A microfluidic assay confirmed the ability of simvastatin to inhibit TNF-induced human neutrophil adhesion to fibronectin ligand under conditions of shear stress, while intravital imaging microscopy demonstrated an abrogation of leukocyte recruitment by simvastatin in the microvasculature of mice that had received a TNF stimulus. This inhibition of neutrophil adhesion was accompanied by the inhibition of TNF-induced RhoA activity in human neutrophils, and alterations in cell morphology and ß2-integrin activity. Additionally, TNF augmented the activity of the p50 NFκB subunit in human neutrophils and TNF-induced neutrophil adhesion and ß2-integrin activity could be abolished using pharmacological inhibitors of NFκB translocation, BAY11-7082 and SC514. Accordingly, the TNF-induced elevation of neutrophil p50 activity was abolished by simvastatin. In conclusion, our data provide further evidence of the ability of simvastatin to inhibit neutrophil adhesive interactions in response to inflammatory stimuli, both in vivo and in vitro. Simvastatin appears to inhibit neutrophil adhesion by interfering in TNF-induced cytoskeletal rearrangements, in association with the inhibition of Rho A activity, NFκB translocation and, consequently, ß2-integrin activity.

Endothelial activation by platelets from sickle cell anemia patients.

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-κB pathway were analyzed by qPCR. SCA PLTs were found to be more inflammatory, displaying increased adhesive properties, an increased production of IL-1ß and a tendency towards elevated expressions of P-selectin and activated αIIbß3. 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-κB) gene expressions. Interestingly, transwell inserts abolished the effects of SCA PLTs on EAM expression. Furthermore, an inhibitor of the NF-κB 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 NFκB-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.