Assessing basophil activation in allergic disease by the measurement of the unique marker basogranulin: Release into cell supernatants and biological fluids, and alterations in intracellular and membrane expression

Abstract

Allergic conditions affect increasing numbers of the population, are associated with considerable morbidity and in their most severe forms can be life-threatening. A prominent feature in allergic conditions is the activation of mast cells and basophils by allergen resulting in the release of inflammatory mediators. A unique product of basophils is basogranulin, a protein stored and released from the secretory granules on cell activation. Our aim has been to develop new assays for assessing basophil activation based on basogranulin measurements, and applying these assays for measuring basophil sensitivity in samples from allergic patients. In addition, we have investigated basogranulin in saliva and BAL fluid as a marker for basophil activation in vivo. Basophils stimulated with fmlp, anti-IgE and grass pollen in vitro and the basogranulin released into supernatants was quantified by dot blotting. In addition, flow cytometric assays were developed for measuring alterations of intracellular and surface basogranulin expression following basophil activation in vitro with fmlp, anti-IgE anti-FcɛRI or specific allergens. There was an apparent depletion in intracellular basogranulin stores in activated basophils when compared with that in non-stimulated basophils. The depletion of intracellular basogranulin was inversely associated with increased expression of CD63. Surface basogranulin expression was barely detected in nonstimulated basophils but was increased following stimulation. Membrane expression of basogranulin was correlated with that of CD63 in nonstimulated basophils (0.829, P< 0.005, n=10), and in basophils stimulated with anti-IgE (r=0.877, P< 0.001, n=51), anti-FcɛRI (r=0.680, P< 0.0001, n=20), or fmlp (r=0.914, P< ii 0.0001, n=9). When basophils were stimulated with extracts of various allergens including D. pteronyssinus, D. farinae, crab, shrimp and oyster increased basogranulin expression wasobserved in cases for where this was not detected for CD63. Alterations in intracellular and surface basogranulin expression were also visualised by confocal microscopy, and by this technique considerable heterogeneity in marker expression was observed between individual cells. Basogranulin could be detected in saliva samples of peanut allergic children, suggesting a potential role in assessing the contribution of basophils in clinical disease. In BAL fluid from patients with atopic asthma basogranulin levels were higher than that from healthy subjects, but not from those with severe asthma. Basogranulin measurement within basophils, on the cell membrane and in biological fluids represent novel approaches for assessing basophil activation and for establishing the contribution of basophils in clinical disease. They show promise as new means for the diagnosis of allergic sensitivity and confirmation of allergic reactions mediated by basophils.

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ORCID for Andrew Walls: orcid.org/0000-0003-4803-4595

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