Basogranulin as a novel marker of basophil activation and mediator of allergic disease

Abstract

Allergic diseases are among the most common chronic diseases with significant financial burden and impairment of quality of life. Basophils have long been implicated in these conditions through their activation and subsequent release of mediators, and there has been speculation of contributions to diverse other conditions. The unique granule constituent basogranulin is secreted upon cell activation and may be detected on the cell membrane. Our aim has been to investigate the physiochemical properties of basogranulin and to examine differential gene expression from activated basophils. To develop a new basophil activation test, we have investigated alterations in the cellular expression of basogranulin following basophil activation in cases of drug allergy. As basophil numbers are often ignored in full blood counts (FBC), we have sought to evaluate their associations with various diseases and other indices.
Immunofluorescence staining of peripheral blood indicated the binding of basogranulin to lymphocytes following basophil activation. Measurement of increased membrane expression of basogranulin was associated with other basophil activation markers. It was employed in a basophil action test that compared favourably with an established test based on the detection of CD63. Transcriptomic analysis of basophils stimulated in vitro using anti-FcRI was associated with the upregulation of genes for peptide receptor binding, with CYSLTR1 being the most upregulated gene. At the same time, stimulation with the bacterial peptide fmlp was associated with the upregulation of genes related to chemotaxis, FPR3 being the most upregulated. In addition, the proteomic analysis of an immunoprecipitated of basophil lysate using BB1 revealed a molecule of 220 kDa. Liquid chromatography-mass spectrometry (LCMS) sequencing of this molecule indicated the presence of proteins of the desmosome family together with annexin and keratin.
There was some success in developing indirect ELISA and dot blot procedures for measuring basogranulin in biological fluids. However, attempts to validate new monoclonal antibodies specific for basogranulin that could be used in a more sensitive sandwich ELISA proved problematic. A flow cytometric assay for basogranulin was optimised and validated to detect basophil activation in drug allergies. Increased expression of basogranulin, as well as CD63, was observed following basophil activation by drugs and other stimuli.
When full blood count data was collected from hospitals in the UK (n=51,442), Nigeria (n =3,250), and Egypt (n= 1,612) analysed, some associations were found between basophil numbers and clinical conditions, other cell types and features of the disease. Significantly raised basophil numbers were observed in atopic diseases, inflammatory conditions, infection and some malignant diseases. Numbers of basophils correlated with those of eosinophils in some of the cases.
Basophils deserve consideration for their roles in allergy and various other conditions. Methods based on the detection of basogranulin should provide new research tools and open the way for new diagnostic procedures. Elucidating the role of basogranulin should indicate its unique contributions to health and disease.

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Identifiers

ORCID for Bashir Kurawa Ado: orcid.org/0000-0002-3998-249X

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

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