CHARACTERIZATION OF THE SUBCELLULAR COMPARTMENT INVOLVED IN HUMAN NEUTROPHIL INTERLEUKIN-1Β (IL-1 Β) SECRETION

KEITELMAN, IRENE A.; SABBIONE, FLORENCIA; FEDERICO FUENTES; SHIROMIZU, MAIUMI; GUZMAN, MAURICIO; MIGLIO, M.; GALLETTI, JEREMÍAS GASTÓN; JANCIC, CAROLINA C.; TREVANI, ANALÍA S.

Buenos Aires

Congreso; REUNIÓN CONJUNTA DE SOCIEDADES DE BIOCIENCIAS; 2017

IL-1b is a major proinflammatory cytokine synthesized in the cytoplasmas an inactive precursor that is activated by proteolytic cleavage.It is a leaderless cytosolic protein that is secreted by unconventionalmechanisms different from the classical RE-Golgi pathway.We previously determined that human neutrophil IL-1β processingis dependent on caspase-1 and elastase and/or proteinase-3. Ourstudies also indicated that neutrophil IL-1b is secreted by an unconventionalsecretory autophagic mechanism. Here we aim to getinsights of the subcellular mechanisms that control IL-1b secretionfrom human neutrophils by employing immunofluorescence stainingand confocal microscopy. We determined that after 3.5 h post-LPS+ATP stimulation part of the neutrophil IL-1b colocalized withelastase and myeloperoxidase in a vesicular compartment (n=3).Neutrophil stimulation with LPS+ATP also induced colocalizationof caspase-1 with IL-1b (Mander´s coefficient -MC-: 0.52; n=2; atleast 80 cells analyzed/experiment), and this effect was potentiatedwhen neutrophils were also subjected to starvation (MC: 0.59).We also found colocalization of IL-1b with the chaperone proteinHSP90 (MC: 0.55 and 0.47; full nutrient and starvation, respectively;n=2). Noteworthy, wide vesicles with IL-1b overlapping with HSP90signals were found protruding from the cell membrane. By employingthe fluorescent probe FLICA to track the presence of activatedcaspase-1 in live cell imaging assays, we detected FLICA signal asdefined spots inside cells with a pattern similar to that observed withcaspase-1. Additionally, IL-1b did not colocalize with LAMP-2 at either3.5 or 5 h post-LPS+ATP stimulation (MC: 0.21 and 0.22; n=4,at least 50 cells analyzed/experiment). Our results suggest that IL-1b is packaged in vesicular compartments which can also includecaspase-1 and/or elastase where it might be processed or degraded.HSP90 might contribute to IL-1b entry to vesicles different tolysosomes.