The secretory route of the leaderless protein interleukin 1beta involves exocytosis of endolysosome-related vesicles - PubMed (original) (raw)

C Andrei et al. Mol Biol Cell. 1999 May.

Free PMC article

Abstract

Interleukin 1beta (IL-1beta), a secretory protein lacking a signal peptide, does not follow the classical endoplasmic reticulum-to-Golgi pathway of secretion. Here we provide the evidence for a "leaderless" secretory route that uses regulated exocytosis of preterminal endocytic vesicles to transport cytosolic IL-1beta out of the cell. Indeed, although most of the IL-1beta precursor (proIL-1beta) localizes in the cytosol of activated human monocytes, a fraction is contained within vesicles that cofractionate with late endosomes and early lysosomes on Percoll density gradients and display ultrastructural features and markers typical of these organelles. The observation of organelles positive for both IL-1beta and the endolysosomal hydrolase cathepsin D or for both IL-1beta and the lysosomal marker Lamp-1 further suggests that they belong to the preterminal endocytic compartment. In addition, similarly to lysosomal hydrolases, secretion of IL-1beta is induced by acidotropic drugs. Treatment of monocytes with the sulfonylurea glibenclamide inhibits both IL-1beta secretion and vesicular accumulation, suggesting that this drug prevents the translocation of proIL-1beta from the cytosol into the vesicles. A high concentration of extracellular ATP and hypotonic medium increase secretion of IL-1beta but deplete the vesicular proIL-1beta content, indicating that exocytosis of proIL-1beta-containing vesicles is regulated by ATP and osmotic conditions.

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Figures

Figure 1

Figure 1

ProIL-1β is contained in part in vesicles cofractionating with lysosomes. Western blot analysis of PNS (5% of total, lanes 1 and 4), P1 (lanes 2 and 5), and P2 (lanes 3 and 6) or supernatants from 106 cells (lanes 7 and 8) obtained from activated monocytes cultured in the absence (−) or presence (+) of 100 μM glibenclamide (glib). After removal of 5% PNS, PNS were treated with proteinase K (PK) before the ultracentrifugation. Lanes 9 and 10, P1 from undigested PNS was untreated (lane 9) or solubilized (+TX100, lane 10) with Triton X-100 before proteinase K (PK) digestion. Filters were hybridized with rabbit anti-CD (A), melted, and rehybridized with mouse anti-IL-1β (B) antibodies. The migration of the three molecular forms of CD and proIL-1β and IL-1β is indicated.

Figure 2

Figure 2

Kinetic analyses of cytosolic and particulated IL-1β. LPS-activated monocytes were pulsed with 1 mCi/ml methionine-cysteine Promix [35S] for 15 min (time of chase, 0) and chased in complete medium for 1, 3, 6, or 15 h in the absence (open symbols) or presence (closed symbols) of 100 μg of pepstatin and leupeptin. At the end of each period of chase subcellular fractionation was carried out as above, P1 and P2 were treated with proteinase K, followed by protease inhibitors and solubilization, and the IL-1β present in the pooled P1 and P2 fractions (A), in the cytosol (B), and in the supernatants (C) was analyzed by immunoprecipitation with anti-IL-1β antiserum followed by SDS-PAGE and autoradiography as described (Rubartelli et al., 1990). Data are expressed as densitometric areas (AU, arbitrary units). One representative experiment of three performed is shown.

Figure 3

Figure 3

Immunoelectron microscopy analysis of the colocalization of IL-1β and CD (A–C, E, and F) and of IL1β and Lamp-1 (D and G) in P1 fraction. Double immunolabeling with anti-IL-1β (10-nm gold particles) and anti-CD (18-nm gold particles) antibodies reveals the presence of both molecules in organelles (>200 nm in diameter), which display the typical morphology of late endosomes and early lysosomes (A, arrow) or more dense, mature lysosomes (B, arrow). These organelles appear double immunolabeled also for IL-1β (10-nm gold particles) and Lamp-1 (18-nm gold particles) (D, arrow). Mature lysosomes showing positive staining for IL-1β alone are also found (C, arrow). Dense vesicles, <200 nm in diameter, appear either positively immunolabeled for IL-1β only (E, arrowheads), for both IL-1β and CD (F, arrowhead), or for both IL-1β and Lamp-1 (G, arrowhead). Bars, 200 nm.

Figure 4

Figure 4

Migration of proIL-1β on Percoll density gradient. P1 and P2 fractions were pooled and further fractionated by centrifugation on a 25% Percoll density gradient under conditions that separate heavy-density lysosomes from lower-density endosomes. Membranes were collected from the individual fractions and tested for β-hexosoaminidase activity or analyzed by Western blot using anti-CD, anti-IL-1β, or anti-Rab7 antibodies. Fraction 1 represents the lowest density; fraction 18 is the highest density. The reactivity of proCD and CD (A), IL-1β (B), and Rab7 (C) was quantified by image digitalization and plotted as arbitrary units (a.u.); β-hexosoaminidase activity (C) is expressed as percent of total. The Western blots hybridized with anti-CD and anti-IL-1β are shown as insets in A and B, respectively. One representative experiment four is shown.

Figure 5

Figure 5

Effects of lysosomotropic drug treatment on secretion and vesicular accumulation of IL-1β. A, Monocytes were stimulated 1 h with LPS without (nil; lanes 1, 4, and 7) or with 50 mM NH4Cl (NH4Cl pre; lanes 2, 5, and 8) and cultured 2.5 h in the absence or presence of the same drug as indicated. Alternatively, monocytes were stimulated 1 h with LPS without NH4Cl, followed by 2.5 h of culture with the drug (NH4Cl chase; lanes 3, 6, and 9). Filters were hybridized with anti-IL-1β (upper panels) or anti-CD (lower panels) antibodies. Cyt, cytosolic fractions (lanes 1–3); P1, P1 pellet (lanes 4–6); sec, supernatants (lanes 7–9). (B) Supernatants from monocytes untreated (nil, lane 1), chased (lanes 2–4), or pretreated (lanes 5–7) as in A with 50 mM NH4Cl (lanes 2 and 5), 50 μM chloroquine (chlor; lanes 3 and 6), or 1 μM BafA1 (lanes 4 and 7). Filters were hybridized with anti-IL-1β antibody.

Figure 6

Figure 6

Exocytosis of IL-1β–containing vesicles is regulated by extracellular [Mg2+]. (A) Supernatants from monocytes incubated 2.5 h with LPS in medium alone (lanes 1 and 5) or plus 5 mM EDTA (lanes 3 and 7), 5 mM MgCl2 (lanes 2 and 6), or 5 mM EDTA plus 5 mM MgCl2 (lanes 4 and 8). When indicated (+ATP, lanes 5–8), 1 mM ATP was added during the last 30 min. (B) PNS (5%) and P1 from activated monocytes cultured 1 h in the absence (−, lanes 1 and 2) or presence of 5 mM MgCl2 (Mg2+, lanes 3 and 4) or 5 mM EDTA (lanes 5 and 6). Filters were hybridized with anti-IL-1β (upper panels) or anti-CD (lower panels).

Figure 7

Figure 7

Exocytosis of IL-1β–containing vesicles is regulated by extracellular osmotic conditions. (A) Supernatants from monocytes incubated 2.5 h with LPS in isotonic conditions (medium alone, iso, lanes 1 and 4) or plus 0.1 M sucrose (hyper, lanes 2 and 5) or diluted 1:2 with water (hypo, lanes 3 and 6) in the absence (−, lanes 1–3) or presence of 1 mM ATP (+, lanes 4–6) for the last 30 min. Filters were hybridized with anti-IL-1β (upper panels) or anti-CD (lower panels). (B) PNS and P1 from monocytes incubated 2.5 h with LPS in isotonic conditions (medium alone, iso, lanes 1 and 2) or plus 0.1 M sucrose (hyper, lanes 3 and 4) or diluted 1:2 with water (hypo, lanes 5 and 6).

Figure 8

Figure 8

Two step model for IL-1β secretion, showing vesicle-mediated transport from the cytosol to the extracellular space. The translocation step is blocked by glibenclamide and when the ΔpH between cytosol and vesicles is abolished; the exocytosis is induced by high extracellular ATP and by hypotonic medium, whereas it is inhibited by low extracellular ATP and by hypertonic conditions.

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