Lidia Michalec

Comparison of platelet aggregability and P-selectin surface expression on platelets isolated by different methods.

Three methods commonly used for isolation of blood platelets from plasma were compared. Platelets were isolated by: 1) a washing method; 2) a method of metrizamide-gradient centrifugation; 3) a modified method of gel-filtration. The last method employed BSA-Sepharose gel instead of routinely used Sepharose gel saturated with BSA. BSA-Sepharose gel was prepared by covalent binding of thermally deactivated BSA to CNBr-activated Sepharose 2B. In contrast to platelets isolated by the other methods, an aggregability of the gel-filtered platelets and control platelets in plasma, both activated with ADP, were comparable. When expression of P-selectin on the surface of freshly isolated platelets was examined, the gel-filtered platelets exhibited the same extent of fluorescence signal as platelets in the citrated blood, whereas platelets isolated by the other methods exhibited twice the extent of the signal. The methods involving the centrifugation process cause a low but a significant platelet activation.

MICROPLATE READER - A CONVENIENT TOOL IN STUDIES OF BLOOD COAGULATION

Results of platelet aggregation measured with a dual channel aggregometer and with a microplate reader are compared. Platelets in plasma were activated by ADP and by collagen, and thrombin was used for the aggregation study of gel filtered platelets. The results obtained with both instruments were quantitatively and qualitatively similar. A microplate reader allowed a simultaneous measurement of a high number of samples with a high degree of reproducibility. The same instrument can be useful in other coagulology studies. Results of citrated plasma clotting by thrombin or by recalcination together with results of platelet counting, both obtained with a microplate reader, are presented in this report as well.

The role of low-level lactate production in airway inflammation in asthma

Warburg and coworkers (Warburg O, Posener K, Negelein E. Z Biochem 152: 319, 1924) first reported that cancerous cells switch glucose metabolism from oxidative phosphorylation to aerobic glycolysis, and that this switch is important for their proliferation. Nothing is known about aerobic glycolysis in T cells from asthma. The objective was to study aerobic glycolysis in human asthma and the role of this metabolic pathway in airway hyperreactivity and inflammation in a mouse model of asthma. Human peripheral blood and mouse spleen CD4 T cells were isolated by negative selection. T cell proliferation was measured by thymidine incorporation. Cytokines and serum lactate were measured by ELISA. Mouse airway hyperreactivity to inhaled methacholine was measured by a FlexiVent apparatus. The serum lactate concentration was significantly elevated in clinically stable asthmatic subjects compared with healthy and chronic obstructive pulmonary disease controls, and negatively correlated with forced expiratory volume in 1 s. Proliferating CD4 T cells from human asthma and a mouse model of asthma produced higher amounts of lactate upon stimulation, suggesting a heightened glycolytic activity. Lactate stimulated and inhibited T cell proliferation at low and high concentrations, respectively. Dichloroacetate (DCA), an inhibitor of aerobic glycolysis, inhibited lactate production, proliferation of T cells, and production of IL-5, IL-17, and IFN-γ, but it stimulated production of IL-10 and induction of Foxp3. DCA also inhibited airway inflammation and hyperreactivity in a mouse model of asthma. We conclude that aerobic glycolysis is increased in asthma, which promotes T cell activation. Inhibition of aerobic glycolysis blocks T cell activation and development of asthma.

Ero1α Is Expressed on Blood Platelets in Association with Protein-disulfide Isomerase and Contributes to Redox-controlled Remodeling of αIIbβ3

Recent evidence supports a role of protein-disulfide isomerase (PDI) in redox-controlled remodeling of the exofacial domains of αIIbβ3 in blood platelets. The aim of this study was to explain whether Ero1α can be responsible for extracellular reoxidation of the PDI active site. We showed that Ero1α can be found on platelets and is rapidly recruited to the cell surface in response to platelet agonists. It is physically associated with PDI and αIIbβ3, as suggested by colocalization analysis in confocal microscopy and confirmed by immunoprecipitation experiments. Apart from monomeric oxidized Ero1α, anti-αIIbβ3 immunoprecipitates showed the presence of several Ero1α-positive bands that corresponded to the complexes αIIbβ3-PDI-Ero1α, PDI-Ero1α, and Ero1α-Ero1α dimers. It binds more efficiently to the activated αIIbβ3 conformer, and its interaction is inhibited by RGD peptides. Ero1α appears to be involved in the regulation of αIIbβ3 receptor activity because of the following: (a) blocking the cell surface Ero1α by antibodies leads to a decrease in platelet aggregation in response to agonists and a decrease in fibrinogen and PAC-1 binding, and (b) transfection of MEG01 with Ero1α increases αIIbβ3 receptor activity, as indicated by increased binding of fibrinogen.

The Specificity and Function of the Metal-binding Sites in the Integrin β3 A-domain

The A-domains within integrin β subunits contain three metal sites termed the metal ion-dependent adhesion site (MIDAS), site adjacent to the metal ion-dependent adhesion site (ADMIDAS), and ligand-induced metal-binding site (LIMBS), and these sites are involved in ligand engagement. The selectivity of these metal sites and their role in ligand binding have been investigated by expressing a fragment corresponding to the β3 A-domain, β3-(109-352), and single point mutants in which each of the cation-binding sites has been disabled. Equilibrium dialysis experiments identified three Mn2+- and two Ca2+-binding sites with the LIMBS being the site that did not bind Ca2+. Although the ADMIDAS could bind Ca2+, it did not bind Mg2+. These results indicate that the Ca2+-specific site that inhibits ligand binding is the ADMIDAS. Two different assay systems, surface plasmon resonance and a microtiter plate assay, demonstrated that the β3 A-domain fragment bound fibrinogen in the presence of 0.1 mm Ca2+ but not in 3 mm Ca2+. This behavior recapitulated the effects of Ca2+ on fibrinogen binding to αvβ3 but not αIIbβ3. Disabling any of the three cation-binding sites abrogated fibrinogen binding. These results indicate that the specificities of the three metal-binding sites for divalent cations are distinct and that each site can regulate the ligand binding potential of the β3 A-domain.

Steroid resistance of airway type 2 innate lymphoid cells from patients with severe asthma: The role of thymic stromal lymphopoietin

Background: Type 2 innate lymphoid cells (ILC2s) represent an important type 2 immune cell. Glucocorticoid regulation of human ILC2s is largely unknown. Objective: We sought to assess steroid resistance of human blood and airway ILC2s from asthmatic patients and to examine its mechanism of induction. Methods: We studied human blood and lung ILC2s from asthmatic patients and control subjects using flow cytometry and ELISA. Results: Dexamethasone inhibited (P = .04) chemoattractant receptor–homologous molecule expressed on TH2 lymphocytes and type 2 cytokine expression by blood ILC2s stimulated with IL‐25 and IL‐33. However, it did not do so when ILC2s were stimulated with IL‐7 and thymic stromal lymphopoietin (TSLP), 2 ligands of IL‐7 receptor &agr;. Unlike blood ILC2s, bronchoalveolar lavage (BAL) fluid ILC2s from asthmatic patients were resistant to dexamethasone. BAL fluid from asthmatic patients had increased TSLP but not IL‐7 levels. BAL fluid TSLP levels correlated (r = 0.74) with steroid resistance of ILC2s. TSLP was synergistically induced in epithelial cells by IL‐13 and human rhinovirus. Mechanistically, dexamethasone upregulated ILC2 expression of IL‐7 receptor &agr;, which augmented and sustained signal transducer and activator of transcription (STAT) 5 signaling by TSLP. TSLP induced mitogen‐activated protein kinase kinase (MEK), c‐Fos, inhibitor of DNA binding 3, phosphorylated signal transducer and activator of transcription (pSTAT) 3, and pSTAT5, molecules linked to steroid resistance. Dexamethasone inhibited c‐Fos, inhibitor of DNA binding 3, and pSTAT3 but not pSTAT5 and MEK. The MEK inhibitor trametinib, the Janus kinase–STAT inhibitor tofacitinib, and the STAT5 inhibitor pimozide reversed steroid resistance of BAL ILC2s. Conclusions: Dexamethasone inhibited type 2 cytokine production by blood ILC2s. IL‐7 and TSLP abrogated this inhibition and induced steroid resistance of ILC2s in a MEK‐ and STAT5‐dependent manner. BAL fluid ILC2s from asthmatic patients with increased TSLP levels were steroid resistant, which was reversed by clinically available inhibitors of MEK and STAT5.

Platelet membrane fluidity and intraplatelet Ca2+ mobilization are affected in uraemia

Abstract:  In present investigations, platelet membrane fluidity and intraplatelet Ca2+ mobilization were analysed in uraemic platelets by fluorescence techniques. Thirteen non‐dialyzed uraemic patients and 16 control subjects were examined. Anisotropy of DPH‐probe, measured at 37°C, was significantly higher in control (0.2236 ± 0.0050) than in uraemic platelets (0.1969 ± 0.0082; p < 0.01). There was no difference between control (109.8 ± 6.0 nm) and uraemic platelets (100.0 ± 7.3 nm) when the basal [Ca2+]i in resting platelets was determined. Activation of platelets by ADP (12.5 μm) or by thrombin (0.1 U/ml) resulted in an increase in [Ca2+]i. It was significantly higher (p* < 0.003 for ADP and p* < 0.009 for thrombin, respectively) in control platelets (383.6 ± 56.3 nm and 2031.0 ± 298.8 nm, respectively) than in uraemic ones (191.0 ± 21.3 nm and 838.7 ± 144.1 nm, respectively). The amount of released Ca2+ was higher in control platelets activated by both ADP and thrombin (157.6 ± 21.4 nm and 409.3 ± 71.0 nm, respectively) than in uraemic platelets (76.7 ± 15.7 nm and 203.0 ± 29.3 nm, respectively) and the differences were significant (p < 0.01 and p* < 0.01, respectively). These results indicate an abnormal intracellular Ca2+ mobilization in uraemic platelets. Both increased membrane fluidity and decreased Ca2+ mobilization should be considered as a possible reason of reduced fibrinogen receptor exposure on uraemic platelets.

Experimental asthma persists in IL-33 receptor knockout mice because of the emergence of thymic stromal lymphopoietin–driven IL-9+ and IL-13+ type 2 innate lymphoid cell subpopulations

Background IL‐33 plays an important role in the development of experimental asthma. Objective We sought to study the role of the IL‐33 receptor suppressor of tumorigenicity 2 (ST2) in the persistence of asthma in a mouse model. Methods We studied allergen‐induced experimental asthma in ST2 knockout (KO) and wild‐type control mice. We measured airway hyperresponsiveness by using flexiVent; inflammatory indices by using ELISA, histology, and real‐time PCR; and type 2 innate lymphoid cells (ILC2s) in lung single‐cell preparations by using flow cytometry. Results Airway hyperresponsiveness was increased in allergen‐treated ST2 KO mice and comparable with that in allergen‐treated wild‐type control mice. Peribronchial and perivascular inflammation and mucus production were largely similar in both groups. Persistence of experimental asthma in ST2 KO mice was associated with an increase in levels of thymic stromal lymphopoietin (TSLP), IL‐9, and IL‐13, but not IL‐5, in bronchoalveolar lavage fluid. Expectedly, ST2 deletion caused a reduction in IL‐13+ CD4 T cells, forkhead box P3–positive regulatory T cells, and IL‐5+ ILC2s. Unexpectedly, ST2 deletion led to an overall increase in innate lymphoid cells (CD45+lin−CD25+ cells) and IL‐13+ ILC2s, emergence of a TSLP receptor–positive IL‐9+ ILC2 population, and an increase in intraepithelial mast cell numbers in the lung. An anti‐TSLP antibody abrogated airway hyperresponsiveness, inflammation, and mucus production in allergen‐treated ST2 KO mice. It also caused a reduction in innate lymphoid cell, ILC2, and IL‐9+ and IL‐13+ ILC2 numbers in the lung. Conclusions Genetic deletion of the IL‐33 receptor paradoxically increases TSLP production, which stimulates the emergence of IL‐9+ and IL‐13+ ILC2s and mast cells and leads to development of chronic experimental asthma. An anti‐TSLP antibody abrogates all pathologic features of asthma in this model.

The role of Protein Disulfide Isomerase and thiol bonds modifications in activation of integrin subunit alpha11

Integrins belong to a family of transmembrane receptors that mediate cell migration and adhesion to ECM. Extracellular domains of integrin heterodimers contain cysteine-rich regions, which are potential sites of thiol-disulfide exchanges. Rearrangements of extracellular disulfide bonds regulate activation of integrin receptors by promoting transition from an inactive state into a ligand-binding competent state. Modifications of integrin disulfide bonds dependent on oxidation-reduction can be mediated by Protein Disulfide Isomerse (PDI). This paper provides evidences that binding to integrin ligands initiate changes in free thiol pattern on cell surface and that thiol-disulfide exchange mediated by PDI leads to activation of integrin subunit α11. By employing co-immunoprecipitation and confocal microscopy analysis we showed that α11β1 and PDI create complexes bounded by disulfide bonds. Using surface plasmon resonance we provide biochemical evidence that PDI can interact directly with integrin subunit α11.