79 Rapid and Intense Induction of Apoptosis in Human Eosinophils by Anti-CD30 Antibody Treatment In Vitro Maki Terakawa*, Kenji Matsumoto§, Kenju Miura*, Hirohisa Saito¥ *Suntory Biomed Research Limited, Osaka, Japan §National Children's Medical Research Center, Tokyo, Japan ~National Children's Hospital, Tokyo, Japan Apoptosis is an important cellular mechanism for controlling cell viability and proliferation. With respect to eosinophils, cytokines prolong their survival while corticosteroids reduce their survival in vitro. CD30, a member of the TNF/NGF receptor family, is reported to be expressed on the surface of many cell types including Hodgkin's lymphoma cells. CD30 is capable of inducing apoptosis after antibody treatment on some cell lines but not all. To determine whether this surface structure is involved in apoptosis of eosinophils, we examined the effect of anti-CD30 antibody treatment on the viability of human eosinophil. Eosinophils were purified from human peripheral blood with percoll density gradient centrifugation and negative selection with immunomagnetic beads. Purified eosinophils (> 99%) were suspended in RPMI-1640 medium supplemented with 10% FCS and 1 ng/ml IL-5 at a cell density of 5x105/ml. Expression of CD30 on eosinophils was examined by flow cytometry. Eosinophils were cultured in 24-well culture plate previously coated with various concentrations of anti-CD30 antibodies (1 to 1,000 ng/ml) or control antibody (irrelevant IgG 1 control mAb, 100 ng/ml), and cell viability was examined by FITC-conjugated Annexin V and propium iodide after 4 hrs of culture. Resting eosinophils expressed low but consistent amounts of CD30 (Mean Fluorescent Intensity: 8.9_+ 1.4 vs 2.6 _+ 0.2 for anti-CD30 (AC10) and irrelevant IgG1 mAb, respectively, n = 5, p 1 ng/ml anti-CD30 antibody in a concentrationand time-dependent manner. %Annexin V ÷ cells at 4 hrs was: 5.3 +0.9, 58.7 -+ 6.7, 64.2 _+ 4.6, or 3.8 _+ 0.7, for IgG1 control mAb, anti-CD30 (HRS-4), anti-CD30 (Bet-H8), or anti-CD30 (Ber-H2), respectively (Mean -+ SEM, n=6-8, * p < 0.001). By DNA fragmentation assay, there was evidence of ladder formation in anti-CD30 (HRS-4 or Ber-H8)-treated eosinophils, consistent with apoptosis. We conclude that eosinophil express low level of CD30, and that treatment with some anti-CD30 antibody shortened eosinophil survival. This suggests that CD30 on eosinophil regulates their survival, perhaps by inducing apoptosis.