Cytoprotective response of A1, a Bcl-2 homologue expressed in mature human neutrophils and promyelocytic HL-60 cells, to oxidant stress-induced cell death.

The ability to generate reactive oxidative intermediates is one of the quintessential properties of mature human neutrophils. Endogenously generated oxidants have been shown to be an important mechanism underlying neutrophil cell death. In acute lung inflammation, newly recruited neutrophils further encounter external oxidants, including reactive oxygen and nitrogen intermediates. In our present study, we showed that A1, a constitutive and inducible Bcl-2 homologue expressed in mature circulating human neutrophils, might confer the protection from hydrogen peroxide (H(2)O(2))- and peroxynitrite (ONOO)-induced cell death. Utilizing the myeloid precursor cell line, HL-60, we further examined the hypothesis that A1 was capable of conferring cytoprotective activity against these oxidative stresses. Whereas the control-transfected HL-60 cells expressed small amounts of A1 and were sensitive to the biologically relevant, cell death-inducing oxidants, H(2)O(2) and ONOO, the stable transfectants that overexpressed A1 were significantly more tolerant. Furthermore, there was a correlation between the level of A1 expression and the antiapoptotic activity. Thus, our results suggest a cytoprotective role of A1 in mature human neutrophils under oxidant stresses in host defense and inflammation.

Broad-spectrum caspase inhibition paradoxically augments cell death in TNF-alpha -stimulated neutrophils.

It is increasingly clear that there are caspase-dependent and -independent mechanisms for the execution of cell death and that the utilization of these mechanisms is stimulus- and cell type-dependent. Intriguingly, broad-spectrum caspase inhibition enhances death receptor agonist-induced cell death in a few transformed cell lines. Endogenously produced oxidants are causally linked to necroticlike cell death in these instances. We report here that broad-spectrum caspase inhibitors effectively attenuated apoptosis induced in human neutrophils by incubation with agonistic anti-Fas antibody or by coincubation with tumor necrosis factor-alpha (TNF-alpha) and cycloheximide ex vivo. In contrast, the same caspase inhibitors could augment cell death upon stimulation by TNF-alpha alone during the 6-hour time course examined. Caspase inhibitor-sensitized, TNF-alpha-stimulated, dying neutrophils exhibit apoptoticlike and necroticlike features. This occurred without apparent alteration in nuclear factor-kappaB (NF-kappaB) activation. Nevertheless, intracellular oxidant production was enhanced and sustained in caspase inhibitor-sensitized, TNF-alpha-stimulated neutrophils obtained from healthy subjects. However, despite reduced or absent intracellular oxidant production following TNF-alpha stimulation, cell death was also augmented in neutrophils isolated from patients with chronic granulomatous disease incubated with a caspase inhibitor and TNF-alpha. These results demonstrate that, in human neutrophils, TNF-alpha induces a caspase-independent but protein synthesis-dependent cell death signal. Furthermore, they suggest that TNF-alpha activates a caspase-dependent pathway that negatively regulates reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidase activity.

Perturbation of B-cell development in mice overexpressing the Bcl-2 homolog A1.

Decisions about cell survival or death are central components of adaptive immunity and occur at several levels in immune system development and function. The Bcl-2 family of homologous proteins plays an important role in these decisions in lymphoid cells. Bcl-2, Bcl-xL, and A1 are differentially expressed during B- and T-cell development, and they have shared and distinct roles in regulating cell death. We sought to gain insight into the role of A1 in immune system development and function. A murine A1-a transgene was expressed under the control of the Emu enhancer, and mice with A1 overexpression in B- and T-cell lineages were derived. Thymocytes and early B cells in Emu-A1 mice showed extended survival. B-lineage development was altered, with expansion of the pro-B cell subset at the expense of pre-B cells, suggesting an impairment of the pro- to pre-B-cell transition. This early B-cell phenotype resembled Emu-Bcl-xL mice but did not preferentially rescue cells with completed V(D)J rearrangements of the immunoglobulin heavy chain. In contrast to Emu-Bcl-2 transgenes, A1 expression in pro-B cells did not rescue pre-B-cell development in SCID mice. These studies indicate that A1 protects lymphocytes from apoptosis in vitro but that it has lineage- and stage-specific effects on lymphoid development. Comparison with the effects of Bcl-2 and Bcl-xL expressed under similar control elements supports the model that antiapoptotic Bcl-2 homologs interact differentially with intracellular pathways affecting development and apoptosis in lymphoid cells.