Impaired P2X1 Receptor–Mediated Adhesion in Eosinophils from Asthmatic Patients

Abstract

Eosinophils play an important role in the pathogenesis of asthma and can be activated by extracellular nucleotides released following cell damage or inflammation. For example, increased ATP concentrations were reported in bronchoalveolar lavage fluids of asthmatic patients. Although eosinophils are known to express several subtypes of P2 receptors for extracellular nucleotides, their function and contribution to asthma remain unclear. In this article, we show that transcripts for P2X1, P2X4, and P2X5 receptors were expressed in healthy and asthmatic eosinophils. The P2X receptor agonist α,β-methylene ATP (α,β-meATP; 10 μM) evoked rapidly activating and desensitizing inward currents (peak 18 ± 3 pA/pF at −60 mV) in healthy eosinophils, typical of P2X1 homomeric receptors, which were abolished by the selective P2X1 antagonist NF449 (1 μM) (3 ± 2 pA/pF). α,β-meATP–evoked currents were smaller in eosinophils from asthmatic patients (8 ± 2 versus 27 ± 5 pA/pF for healthy) but were enhanced following treatment with a high concentration of the nucleotidase apyrase (17 ± 5 pA/pF for 10 IU/ml and 11 ± 3 pA/pF for 0.32 IU/ml), indicating that the channels are partially desensitized by extracellular nucleotides. α,β-meATP (10 μM) increased the expression of CD11b activated form in eosinophils from healthy, but not asthmatic, donors (143 ± 21% and 108 ± 11% of control response, respectively). Furthermore, α,β-meATP increased healthy (18 ± 2% compared with control 10 ± 1%) but not asthmatic (13 ± 1% versus 10 ± 0% for control) eosinophil adhesion. Healthy human eosinophils express functional P2X1 receptors whose activation leads to eosinophil αMβ2 integrin–dependent adhesion. P2X1 responses are constitutively reduced in asthmatic compared with healthy eosinophils, probably as the result of an increase in extracellular nucleotide concentration.

Evidence for shear-mediated Ca2+ entry through mechanosensitive cation channels in human platelets and a megakaryocytic cell line

“…The specificity of the antibodies for their intended target in Western blots (Fig. 6) has been previously validated using knockdown of endogenous expression, heterologous expression, or tissue from receptor-deficient mice (63–68). The widely used α-tubulin mouse anti-human monoclonal antibody (CP06; Calbiochem), 1:1000 was used as a control.…”

Purinergic Signalling: Therapeutic Developments

“…ATP in sputum was significantly elevated in patients with asthma, which was correlated with the percentage of neutrophils in sputum ( Soma et al, 2016 ). Impaired P2X1R-mediated adhesion in eosinophils from asthmatic patients has been reported ( Wright A. et al, 2016 ). P2Y 6 R activation was effective for treatment of a mouse model of asthma ( Chetty et al, 2016 ).…”

Human group 2 innate lymphoid cells do not express the IL-5 receptor

“…Human eosinophils were purified from whole blood using CD16 microbead depletion of neutrophils over CS columns (both Miltenyi Biotec, Surrey, UK) to a purity of 97% as described previously. E2 Purified eosinophils were immediately placed into Qiazol (Qiagen, Manchester, UK) for downstream RNA isolation and used as a positive control for the detection of transmembrane and soluble mRNA variants of the IL-5Rα gene. ILC2s were isolated from peripheral blood by flow cytometric cell sorting and cultured in vitro as described in detail previously.…”