Abstract
Background: ATP triggers cells to release microvesicles (MVs), which play crucial roles in cellular communication by ferrying molecular cargo between cells. MVs may protect its contents from dilution, degradation or consumption, yet there is a paucity of literature examining TNF release within intra-alveolar MVs.
Aims/Objectives: To investigate if ATP causes alveolar macrophages (AM) to preferentially release TNF within MVs.
Methods: Primary mouse AMs were activated with 1µg/ml LPS, and then exposed to a 'second hit' of ATP 6mM/ecto-ATPase inhibitor 1mM for 1 hour to produce MVs (identified on flow cytometry as CD11c+, <1µm, detergent-sensitive events). TNF/IL-6 content of MV pellets and MV-free supernatants (separated by high-speed centrifugation) were analyzed by ELISA/western blotting and compared to control (LPS stimulation alone).
Results: ATP treatment eradicated soluble TNF release from AMs (Control 107±13 pg vs ATP 4.8±6.1 pg, p<0.01 N=5-7) but substantively upregulated TNF content within MVs (0.5±3.6vs 26.8±8.5, p<0.01 N=5-7) as measured by ELISA. Western blotting confirmed these findings including 17kDa TNF within MVs. ATP did not significantly alter the profile of IL-6 release in the soluble (Control 14.53±6.0 pg vs ATP 6.0±0.5 pg, N=3-4) or MV fraction (3.1±0.7 vs 2.4±0.3 pg, N=3-4), suggesting this 'switch' may be specific to TNF.
Conclusion: These data show that a danger signal such as ATP, superimposed onto a pro-inflammatory stimulus, suppresses TNF release from AMs in the ordinary soluble form but preferentially packages TNF within MVs. This switch phenomenon indicates a novel mechanism of long-range TNF signalling in inflammatory lung diseases such as acute lung injury.
- Copyright ©the authors 2016