Bim deficiency and

bcl-2 transgene expression delay spontaneous, chemotherapeutic drug-induced, and stress-induced apoptosis of granulocytes. Granulocytes from bone marrow (A-D) of wt,vav-bcl-2 transgenic, bim^−/−,bax^−/−,bcl-w^−/−, lpr mice, andbcl-2^−/− reconstituted mice (see “Materials and methods”) were sorted by flow cytometry and cultured in simple medium.(A-B) Survival under conditions of cytokine withdrawal was determined after 24, 48, and 72 hours by staining with annexin V plus propidium iodide and flow cytometric analysis. (C-D) Granulocytes of the indicated genotypes were either cultured in simple medium, the presence of FasL (100 ng/mL) multimerized with M2 anti-Flag mAb (1 μg/mL), the cytotoxic drugs VP-16 (10 μg/mL), taxol (1 μM), or the calcium ionophore ionomycin (200 ng/mL). Survival was assessed after 48 hours of incubation as mentioned in panel A. Data shown represent arithmetic means ± SDs of 4 independent experiments. Analysis was performed in duplicate on 4 to 8 animals of each genotype. Analysis of mobilized granulocytes from the peritoneal cavity (E-F). Mice of the genotypes indicated in panel A were injected intraperitoneally with 0.5% casein in PBS (2 mL). Peritoneal exudate cells were harvested by lavage 3 hours later, and granulocytes were sorted by flow cytometry as indicated in panel A. (E-F) Survival under conditions of cytokine withdrawal was determined after 16, 24, and 48 hours as described in panel A. (G-J) Granulocytes of the indicated genotypes were cultured in simple medium or treated with FasL (100 ng/mL) multimerized with M2 anti-Flag mAb (1 μg/mL) or the cytotoxic drug VP-16 (20 μg/mL). Survival was assessed after 8 (G-H) or 16 (I-J) hours of incubation as described in panel A. Data shown represent arithmetic means ± SDs of 2 to 3 independent experiments performed in duplicate on 2 to 5 animals of each genotype.