HMGB1, a non-histone nuclear factor, acts extracellularly as a mediator of delayed endotoxin lethality, which raises the question of how a nuclear protein can reach the extracellular space. We show that activation of monocytes results in the redistribution of HMGB1 from the nucleus to cytoplasmic organelles, which display ultrastructural features of endolysosomes. HMGB1 secretion is induced by stimuli triggering lysosome exocytosis. The early mediator of inflammation interleukin (IL)-1β is also secreted by monocytes through a non-classical pathway involving exocytosis of secretory lysosomes. However, in keeping with their respective role of early and late inflammatory factors, IL-1β and HMGB1 respond at different times to different stimuli: IL-1β secretion is induced earlier by ATP, autocrinally released by monocytes soon after activation; HMGB1 secretion is triggered by lysophosphatidylcholine, generated later in the inflammation site. Thus, in monocytes, non-classical secretion can occur through vescicle compartments that are at least partially distinct.

Interleukin 1␤ (IL-1␤), 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-1␤ out of the cell. Indeed, although most of the IL-1␤ precursor (proIL-1␤) 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-1␤ and the endolysosomal hydrolase cathepsin D or for both IL-1␤ 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-1␤ is induced by acidotropic drugs. Treatment of monocytes with the sulfonylurea glibenclamide inhibits both IL-1␤ secretion and vesicular accumulation, suggesting that this drug prevents the translocation of proIL-1␤ from the cytosol into the vesicles. A high concentration of extracellular ATP and hypotonic medium increase secretion of IL-1␤ but deplete the vesicular proIL-1␤ content, indicating that exocytosis of proIL-1␤-containing vesicles is regulated by ATP and osmotic conditions. INTRODUCTIONInterleukin 1 (IL-1) 1 is a multifunctional cytokine and a major soluble mediator of inflammation (Dinarello, 1991). Although its biological activity is extracellular, this protein lacks a secretory signal sequence (Rubartelli and Sitia, 1997), raising the question of how it can be transported out of the cell. Two forms of IL-1 exist, ␣ and ␤; however, studies on IL-1 secretion mostly focused on IL-1␤, which is the major extracellular form in humans (Dinarello, 1991). IL-1␤ is synthesized by monocytes upon activation as a 35-kDa precursor, which accumulates in the cytosol (Singer et al., 1988) and is proteolytically processed to the mature form of 17 kDa by the caspase IL-1␤-converting enzyme (ICE) (Cerretti et al., 1992;Thornberry et al., 1992). ICE is present in the cell cytosol (Ayala et al., 1994;Singer et al., 1995) as a p45 inactive polypeptide. Where and how maturation of ICE to the active (p10/p20) form and the processing of IL-1␤ take place are unclear; however, the mature form of IL-1␤ seems to be either ¶ Corresponding author. E-mail address: annarub@hp380.ist. et al., 1990). Although the levels of basal release of IL-1␤ are quite low (Rubartelli et al., 1990), secretion is dramatically induced by extracellular ATP (Hogquist et al., 1991;Rubartelli et al., 1993;Perregaux and Gabel, 1994), which can be autocrinally produced by activated monocytes (Ferrari et al., 1997) and interact with P2Z purinoreceptors expressed on their membrane (Hickman et al., 1994;Di Virgilio, 1995). The cellular pathway underlying this ATP-driven secretion is, however, unknown. We have previously shown that IL-1␤ is ...

Blocking the activity of IL-1␤ has entered the clinical arena of treating autoimmune diseases. However, a successful outcome of this approach requires a clear definition of the mechanisms controlling IL-1␤ release. These are still unclear as IL-1␤, lacking a secretory signal peptide, follows a nonclassical pathway of secretion. Here, we analyze the molecular mechanism(s) undergoing IL-1␤ processing and release in human monocytes and provide a unifying model for the regulated secretion of the cytokine. Our data show that in a first step, pro-caspase-1 and endotoxininduced pro-IL-1␤ are targeted in part to specialized secretory lysosomes, where they colocalize with other lysosomal proteins.

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Here we show that dendritic cells accumulate the precursor form of the leaderless secretory protein interleukin-18 (pro-interleukin-18) in the cell cytosol and in organelles cofractionating with endolysosomes. Upon antigen specific contact with T lymphocytes, particulated pro-interleukin-18 decreases rapidly, and the cytokine appears extracellularly, suggesting that exocytosis of pro-interleukin-18-containing organelles is induced. Exocytosis of secretory lysosomes is modulated by calcium: in agreement with this, calcium influx results in secretion of prointerleukin-18. In turn, pro-interleukin-18 secretion induced by T cells is prevented by the calcium channel blocker nifedipine. Our results demonstrate a novel, calcium-mediated mechanism of post-translational regulation of secretion for interleukin-18, that allows a fast release of the cytokine. ß

We recently reported that human dendritic cells release the leaderless secretory protein interleukin-1␤ (IL-1␤) following specific interaction with alloreactive T lymphocytes. To clarify the molecular mechanism underlying this secretion, this study investigated the intracellular trafficking of IL-1␤ in dendritic cells and the signal ( IntroductionRegulated secretion is traditionally considered as a specialized process occurring in only a few polarized cell types, namely, exocrine, endocrine, and neuronal cells. 1 However, former observations that, on binding to antigen-presenting B cells, T-helper cells release lymphokines preferentially over the membrane area where T-cell receptor cross-linking is occurring, 2,3 led to the hypothesis that a regulated polarized secretion may exist also in nonpolarized cells.It is now clear that many hemopoietic cells use regulated secretion; examples are mast cells and granulocytes, which degranulate in response to Fc receptor cross-linking, 4 or platelets, which respond to vascular lesions by releasing small molecules and proteins from intracellular granules. 5 Unlike conventional secretory cells, endowed with specific structures for storage and release, hemopoietic cells use secretory lysosomes, a mixture organelle between lysosomes and secretory granules. 6 Interestingly, other cell types, including fibroblasts, are able to transform conventional lysosomes into a secretory organelle underlying inducible exocytosis 7 ; thus, regulated secretion seems to be a more widespread phenomenon than previously thought. However, even if secretory lysosomes are quite ubiquitous, the ability of directing their content toward a given target, resulting in polarized secretion, has been described so far only for a few immune cells, namely, T lymphocytes or natural killer cells. 8,9 This may depend on how exocytosis is induced. Indeed, in most cell types exocytosis is driven by stimuli triggering structures distributed uniformly on the plasma membrane (such as IgE receptors on mast cells); in contrast, in the case of T and natural killer cells, binding to target cells with engagement of a single or few receptor complexes results in local activation leading to polarized degranulation.Although it is known that increases in intracellular free calcium concentration ([Ca ϩϩ ] i ) and cytoskeleton rearrangement occur during the process, 7 the molecular mechanisms underlying regulated lysosome exocytosis are still unclear.We have recently demonstrated in monocytes an adenosine triphosphate (ATP)-dependent exocytosis of secretory lysosomes, resulting in release of lysosomal enzymes and of the mature form of interleukin-1␤ (IL-1␤). 10 The latter belongs to the family of leaderless secretory proteins, which despite their extracellular localization and function, lack a secretory signal peptide and are externalized through nonclassical pathways, avoiding the endoplasmic reticulum-Golgi exocytotic route. 11,12 IL-1␤ is synthesized on free cytosolic ribosomes as a 35-kd precursor (pro-IL-1␤), which undergoe...

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To analyze the intracellular trafficking of two IL-15 isoforms bearing 48- or 21-amino acid leader peptides (L), we have generated cDNA encoding the two proteins fused at the C terminus with green fluorescent protein (GFP). Confocal microscopy analyses showed that, when transfected in CHO cells, 48L IL-15/GFP was localized in the Golgi apparatus and in early endosomes, while 21L IL-15/GFP was detectable only in the cytosol. The presence of 48L IL-15/GFP in endosomes was confirmed by enzyme-linked immunosorbent assay on endosome-enriched subcellular fractions. Exogenous IL-15 was bound and taken up in endosomes by untransfected CHO cells, indicating that endosomal localization was, at least in part, related to a receptor-mediated uptake. The 48L IL-15/GFP fusion protein was efficiently secreted by COS-7 or CHO cell transfectants, while IL-15 secretion was less efficient in transfectants expressing 21L IL-15/GFP or untagged 48L or 21L IL-15. Treatment with brefeldin A or with inhibitors of N-linked glycosylation further indicated that the 48L IL-15/GFP is secreted through the endoplasmic reticulum/Golgi pathway. Our data suggest a different trafficking of the two IL-15 isoforms and multiple mechanisms controlling IL-15 secretion.