COMPARISON OF 2 MODIFIED TECHNIQUES FOR PURIFYING BLOOD EOSINOPHILS

As there is much heterogeneity in the morphology and function of blood eosinophils, comparison of their properties between groups of subject s requires recovering the majority of these cells. In two currently us ed techniques to isolate eosinophils, blood granulocytes are processed either on Percoll gradients after an incubation of granulocytes with 10(-8) M N-formyl-methionyl-leucyl-phenylalanine (fMLP) or on a magnet ic cell sorter (MACS). In this study, these techniques were modified t o increase the efficiency of eosinophil recovery. With the Percoll gra dients, using 1.078 g/ml as the top gradient instead of 1.082 g/ml dou bled the eosinophil recovery from 43 +/- 5.3% (mean +/- SEM) to 86.9 /- 2.9%, without decreasing the purity (96.1 +/- 1.4% versus 96.2 +/- 0.9%). With a MACS, the neutrophils in granulocytes obtained on Ficoll -Paque (1.077 g/ml) instead of on Percoll gradient 1.082-1.094 g/ml, w ere tagged with anti-CD16 antibodies and eliminated by passing them th rough a magnetic field. When blood eosinophils of the same subjects we re isolated using the two techniques, similar recovery and purity leve ls were obtained: Percoll gradients, 72.7 +/- 6.8% and 92.5 +/- 2.2%; MACS, 80.2 +/- 5.1% and 90.4 +/- 3.8%. Eosinophils isolated through th e two techniques were also compared for their production of superoxide anion and leukotriene (LT) C4, with and without pre-incubation with c ytokines interleukine-3, interleukine-5 and granulocyte-macrophage col ony stimulating factor. The release of these products was similar betw een the two eosinophil preparations under all conditions tested except for interleukine-3 where eosinophils isolated with a MACS produced mo re LTC4. These results show that both techniques efficiently recover p ure eosinophils. Furthermore, cell incubation with 10(-8) M fMLP did n ot enhance superoxide anion and LTC4 production nor modify the respons e to cytokines. The two modified techniques are therefore suitable for comparative studies of eosinophils from different groups of subjects.