Figure 5.

The features of gp120/anti-gp120-induced apoptosis in human PBL are identical to anti-CD4- or anti-CXCR4-induced apoptosis. (A) gp120/anti-gp120-induced cell death in human PBL induced changes in FSC/SSC typical of apoptosis (□). Furthermore, loss of asymmetry in plasma membrane phospholipids as determined by MC540 staining (▨) preceded loss of cell viability as determined by propidium iodide staining (■). After preincubation with 100 ng/ml gp120 (HIV-1 IIIB, Neosystem, Strasbourg, France) for 60 min at 4°C, PBL (5 × 10⁵ cells per sample) were incubated with diluted rabbit anti-gp120 serum (1/500). Cell death was determined after 30 min. The experiment was done in triplicate and is representative of three. (B) gp120/anti-gp120-induced cell death in human PBL does not result in formation of subdiploid nuclei. Cell death was induced in activated CD95-sensitive PBL as described in A. Anti-APO-1 (10 μg/ml) was used as a positive control. Cell death of an aliquot of cells was measured by FSC/SSC analysis (10⁴ cells counted) after 2 h (gp120/anti-gp120) or 24 h (anti-APO-1) (□). The remaining cells were incubated overnight in propidium iodide-lysis buffer and formation of subdiploid nuclei (■) was determined in a FACScan cytometer (10⁴ cells counted) (20). The experiment shown is a representative of two experiments done. (C) gp120/anti-gp120-induced apoptosis did not involve the known caspases. Before induction of cell death as described in B human PBL were pretreated in the absence (□) or presence (■) of the caspase inhibitor zVAD-fmk (20 μM) for 30 min at 37°C. Cell death was determined by FSC/SSC analysis 2 h (gp120/αgp120) or 24 h (αAPO-1) after induction. One experiment representative of three, done in triplicates, is shown.