Abstract
Basophils are blood cells of low abundance associated with allergy, inflammation and parasite infections. To study the transcriptome of mature circulating basophils cells were purified from buffy coats by density gradient centrifugations and two-step magnetic cell sorting. However, after extensive analysis the cells were found to be transcriptionally inactive and almost completely lack functional mRNA. In order to obtain transcriptionally active immature basophils for analysis of their transcriptome, umbilical cord blood cells were therefore cultured in the presence of interleukin (IL)-3 for 9 days and basophils were enriched by removing non-basophils using magnetic cell sorting. The majority of purified cells demonstrated typical metachromatic staining with Alcian blue dye (95%) and expression of surface markers FcεRI and CD203c, indicating a pure population of cells with basophil-like phenotype. mRNA was extracted from these cells and used to construct a cDNA library with approximately 600 000 independent clones. This library served as tool to determine the mRNA frequencies for a number of hematopoietic marker proteins. It was shown that these cells express basophil/mast cell-specific transcripts, i.e. β-tryptase, serglycin and FcεRI α-chain, to a relatively low degree. In contrast, the library contained a high number of several eosinophil-associated transcripts such as: major basic protein (MBP), charcot leyden crystal (CLC), eosinophil cationic protein (ECP), eosinophil derived neurotoxin (EDN) and eosinophil peroxidase (EPO). Out of these transcripts, MBP and EPO were the most frequently observed, representing 8% and 3.2% of the total mRNA pool, respectively. Moreover, in a proteome analysis of cultured basophils we identified MBP and EPO as the two most prominent protein bands, suggesting a good correlation between protein and mRNA analyses of these cells. The mixed phenotype observed for these cells strengthens the conclusion that eosinophils and basophils are closely linked during human hematopoietic development. The dual phenotype also indicates that other cytokines than IL-3 or cell surface interactions are needed to obtain the full basophil specific phenotype in vivo.
Highlights
Basophils have historically often been described as blood mast cells (MCs) since they both stain positive for basic dyes and contain histamine
Buffy coats were used as starting material to obtain sufficient numbers of human blood basophils for mRNA analysis and cDNA library construction
A cDNA library with a total of 14 000 clones was obtained. These clones were plated on two plates and plaque lifts to two hybond N+ filters were performed which were hybridized with 32P-labelled probes against bactin, the mast cell tryptase, the IgE high affinity receptor a-chain, and a mononuclear cell cDNA probe
Summary
Basophils have historically often been described as blood mast cells (MCs) since they both stain positive for basic dyes and contain histamine. Basophils normally constitute less than 1% of the circulating leukocytes. MCs are much more common, are rarely found in peripheral blood and are essentially resident tissue cells [1]. The basophilic granulocyte was first described by Paul Ehrlich in his workBeitrage zur Kenntnis der granulierten Bindegewellzellen und der eosinophilien Leukocytenin the late 19th century [2]. They were given their name due to the fact that they stain with basic dyes like Toluidine Blue and Alcian blue. Recently basophils have attracted major interest as a potential key initiator for the induction of T-helper type 2 (Th2)
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