Isolation of transcriptionally active umbilical cord blood-derived basophils expressing Fc epsilon RI, HLA-DR and CD203c.
BACKGROUND
Basophils are inflammatory cells associated with allergy and parasite infections. Investigation of their true biological function has long been hampered by the difficulty in obtaining sufficient amounts of pure basophils and by the lack of phenotypic markers. Moreover, it has been very difficult to clone and identify basophil-specific granule proteins, partially because of an almost complete lack of mRNA in mature circulating basophils.


METHODS
To obtain transcriptionally active immature basophils, umbilical cord blood cells were cultured in the presence of interleukin (IL)-3. The cells were analysed by flow cytometry and by histological staining.


RESULTS
The continuous presence of IL-3 in cord blood cultures resulted in the expansion of basophil precursors co-expressing FcepsilonRI and the recently described mast cell/basophil marker, 97A6 (CD203c). Several nonbasophil markers (i.e. CD3, CD14, CD15, CD16, CD19 and CD21) were absent on the cultured basophils. However, we show that in early cultures, almost 60% of the CD203c+ cells co-express human leukocyte antigen (HLA)-DR, a marker that is absent on mature circulating basophils. The presence of HLA-DR on basophil precursors may explain the low recovery (24+/-5.2%) obtained after isolation of cultured basophils, when using a conventional basophil isolation kit that removes HLA-DR+ cells. A novel purification method was developed, including a two-step cocktail of antibodies against selected markers, which resulted in both high purity (95+/-0.5%) and recovery (59+/-1.5%) of cultured basophils.


CONCLUSIONS
We here establish cord blood cultures as a source from which transcriptionally active basophil precursors can be isolated in reasonable quantities for thorough biochemical characterization.
Reimer
J M
JM
Department of Cell and Molecular Biology, Uppsala University, Uppsala, Sweden.
Magnusson
S
S
Juremalm
M
M
Nilsson
G
G
Hellman
L
L
Wernersson
S
S
D001491:Basophils / Q000276:immunology* / Q000378:metabolism*; D002469:Cell Separation; D002478:Cells, Cultured; D005312:Fetal Blood / Q000166:cytology* / Q000276:immunology* / Q000378:metabolism; D006684:HLA-DR Antigens / Q000096:biosynthesis / Q000235:genetics*; D006801:Humans; D010727:Phosphoric Diester Hydrolases / Q000096:biosynthesis / Q000235:genetics*; D011755:Pyrophosphatases / Q000096:biosynthesis / Q000235:genetics*; D017455:Receptors, IgE / Q000096:biosynthesis* / Q000235:genetics*; D013234:Stem Cells / Q000276:immunology / Q000378:metabolism; D015533:Transcriptional Activation / Q000276:immunology; D014471:Umbilical Veins
C427944:ENPP3 protein, human; D006684:HLA-DR Antigens; D017455:Receptors, IgE; D010727:Phosphoric Diester Hydrolases; D011755:Pyrophosphatases