Modulation of cell death of both phagocytes and alveolar epithelium may play a critical role in the progression and resolution of acute lung injury. The Fas (CD95)/Fas ligand system plays a putative role in induction of apoptotic cell death in neutrophils (PMNs), but has not been well described in human alveolar epithelium. Interaction of PMNs with Fas-bearing cell types may also induce apoptosis (Liles et al., J Exp Med, 184:429, 1996) via release of soluble Fas ligand. We hypothesized that Fas antigen is present on alveolar epithelial cell lines as well as on PMNs, and that interaction with PMNs may induce epithelial apoptosis. Methods: Isolated human neutrophils and human lung adenocarcinoma cell line (A549, ATCC) were stained with FITC-labeled antibodies to Fas antigen and evaluated by flow cytometry. Cells were incubated with a crosslinking functional anti-CD95 antibody (10-20 mcg/ml, Pharmingen) for 24-72 hours and assessed for viability by vital dyes. DNA fragmentation typical of apoptosis was measured by TdT-mediated fluorescence-labeled dUTP nick end labeling assay (TUNEL). For coculture experiments, A549 cells were grown to confluence in Transwell chambers. Cells were then exposed for 72 hours to isolated PMNs placed on a microporous membrane insert which prevents PMN contact. After exposure, A549 cells and PMNs were evaluated for viability and DNA fragmentation. Results: Both human PMNs and A549 showed constitutive surface Fas expression. Functional CD95 Ab (20 mcg/ml) induced cell death (10±7% viability vs. 98±1% control by vital dye assay, n=7, p<.05 by ANOVA) and DNA fragmentation (TUNEL MFI 354±98% of control, n=6, p<.05) in A549 cultures. CD95 also enhanced PMN apoptosis (TUNEL MFI 464±75 vs. 253±24 in control; p=.05). Coincubation of A549 with PMNs undergoing apoptosis over 72 hours decreased A549 viability (68±12% survival vs. 99±1% in control, n=7, p<.05) and increased DNA fragmentation (TUNEL MFI 118±13% of control, n=7, p<.05). Conclusions: Fas antigen crosslinking induces DNA fragmentation and cell death in both PMNs and A549 cell lines. Neutrophils undergoing apoptosis induce A549 cell death and DNA fragmentation, possibly by PMN release of soluble Fas ligand.