Purification of human blood basophils and leukotriene C4 generation following calcium ionophore stimulation.
A simple method for purification of basophils from a relatively small volume of blood has been developed, which enables us to collect basophils with a purity of over 80%. Basophils were partially purified from 10-20 ml of citrated whole blood using the discontinuous Percoll density gradient centrifugation technique and then contaminant cells were removed using monoclonal antibodies against CD2, CD19, CD14 and CD16. At the end of the procedure, basophils were > 80% pure with lymphocytes accounting for most of the contaminating cells. When stimulated with anti-IgE or fMLP, histamine release from purified basophils was similar to that from mixed leukocytes. When highly purified basophils were challenged with calcium ionophore A23187, generation of leukotriene C4 (LTC4) was not significantly different between asthmatic patients and normal subjects (45.6 +/- 22.6 vs 52.7 +/- 25.6 ng/10(6) cells). Basophils were capable of generating LTC4 in approximately the same quantities as eosinophils (46.5 +/- 11.7 ng/10(6) cells, n = 3). Furthermore, it has been shown that incubation of basophils and eosinophils with calcium ionophore generates only small quantities of thromboxane B2 (TXB2).
Mita
H
H
Clinical Research Center, National Sagamihara Hospital, Kanagawa, Japan.
Akiyama
K
K
Hayakawa
T
T
Yamada
T
T
Ikeda
Y
Y
Shida
T
T
D000888:Antibodies, Anti-Idiotypic / Q000494:pharmacology; D001249:Asthma / Q000097:blood; D001491:Basophils / Q000187:drug effects* / Q000378:metabolism; D001773:Blood Cells; D000001:Calcimycin / Q000494:pharmacology*; D002469:Cell Separation / Q000379:methods*; D002499:Centrifugation, Density Gradient; D006636:Histamine Release / Q000187:drug effects; D006801:Humans; D017997:Leukotriene C4 / Q000096:biosynthesis*; D009240:N-Formylmethionine Leucyl-Phenylalanine / Q000494:pharmacology; D013929:Thromboxane B2 / Q000096:biosynthesis
D000888:Antibodies, Anti-Idiotypic; C026408:anti-IgE antibodies; D017997:Leukotriene C4; D000001:Calcimycin; D013929:Thromboxane B2; D009240:N-Formylmethionine Leucyl-Phenylalanine