CONICET | Buscador de Institutos y Recursos Humanos

Interleukin-1beta (IL-1beta) is a pro-inflammatory cytokine synthesized in the cytoplasm as a precursor, pro-IL-1beta, which has to be proteolytically processed to acquire biological activity. In human neutrophils (PMN) IL-1beta processing is dependent of caspase-1 and elastase and/or proteinase-3. The release of IL-1beta does not follow the conventional ER-Golgi route of secretion, being secreted by poorly-defined non-conventional secretory pathways. The current study was undertaken to test the hypothesis that a non conventional autophagy (Au) mechanism is required for neutrophil IL-1beta secretion. We have previously determined that the Au inhibitors 3-MA, wortmannin and Bafilomycin A1 inhibit IL-1beta secretion induced by LPS and LPS+ATP. In this study we determined that in response to these agonists, the secretion of IL-8, a cytokine whose release follows the canonical ER-Golgi pathway, was not regulated by the above mention inhibitors. We also observed that Au stimulation by starvation promoted IL-1beta secretion induced by both agonists (n=4; p<0.01) but did not affect IL-8 secretion (n=4). We also found that neither the Au inhibitors nor starvation significantly affected PMN viability evaluated by annexin V-FITC/IP staining. Quantification of confocal microscopy (CM) images indicated that 74±5% of LPS+ATP-stimulated PMN exhibited autophagy vesicles (LC3B+) vs 3±1% of unstimulated cells and in 39±4% of the stimulated cells, IL-1â colocalized with LC3B vs 4±1% in unstimulated cells (n=3; p<0.001). Kinetic studies performed in PMN stimulated for 3, 4 and 5 h that in parallel evaluated Au and IL-1beta secretion showed that lipidated-LC3B increased upon LPS+ATP stimulation and then decreased in currently to the secretion of IL-1beta to supernatants. Altogether, our findings confirm that Au is part of the pathway involved in IL-1beta exportation from PMN.