Epidermal langerhans cells are resistant to the permeabilizing effects of extracellular ATP: In vitro evidence supporting a protective role of membrane ATPase

Extracellular adenosine 5′-triphosphate (ATP_o) can induce pore formation in cell membranes, leading to cell permeabilization and eventual cell death. In this study, we examined the sensitivity of human epidermal Langerhans cells to ATP-induced permeabilization and tested the possibility that the Mg⁺⁺ or Ca⁺⁺-dependent plasma membrane ectonucleotidase (mATPase) on Langerhans cells provides protection against the cytotoxic effects of ATP_o. Membrane permeability was assessed by using the fluorescent tracer propidium iodide, which confers red nuclear fluorescence to permeabilized cells. Langerhans cells were identified within human epidermal cell suspensions with fluorescein isothiocyanateconugated MoAb against CD1a or human leukocyte antigen-DR (HLA-DR) antigens. Cultured human keratinocytes and J774 macrophages were both highly sensitive to permeabilization induced by incubation with ATP (0.5 to 20 mM at 37°C), whereas Langerhans cells were relatively resistant. The non-hydrolyzable ATP analog, adenosine 5′-β, γ imido) triphosphate, but not other nucleotides such as ADP, AMP, GTP, or UTP, was also able to induce permeabilization comparable to that of ATP, thereby suggesting that ATP hydrolysis is not required for this effect. ATP^4- is the moiety most likely responsible for permeabilization, because propidium iodide uptake occurred only when the pH of the medium was ≥ 7.4. Permeabilization induced by ATP was augmented by chelation of divalent cations with ethylenediaminetetraacetic acid and by the addition of lanthanum or cerium (0.01 to 1 mM). Finally, incubation with the adenosine analog, 5′-p-fluorosulfonylbenzoyl-adenosine (1 mM), inhibited mATPase staining of Langerhans cells in human epidermal sheets, but markedly augmented ATP-induced permeabilization of Langerhans cells. The results indicate that epidermal LC are resistant to the lytic effects of ATP_o and that mATPase is involved in such resistance.