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Seminars in Immunology 25 (2013) 425– 429

Contents lists available at ScienceDirect

Seminars in Immunology

jo u r nal homepage: www.elsev ier .com/ locate /ysmim

eview

he secretion of IL-1� and options for release

atrizia Piccioli, Anna Rubartelli ∗

ell Biology Unit, IRCCS AOU San Martino IST, 16132 Genova, Italy

r t i c l e i n f o

eywords:L-1�nflammasome

a b s t r a c t

Unlike most cytokines, IL-1� lacks a secretory signal sequence raising the question of how is this cytokineprocessed and delivered outside the producing cells. After the seminal observation that IL-1� is actively

eaderless secretionecretory lysosomesicrovesicles

utophagy

secreted by human monocytes through a route alternative to the classic endoplas mic reticulum–Golgi,several different pathways have been proposed for IL-1� secretion in different cell types and cultureconditions, some of which are unique to macrophage cell lines. Here we describe the most credited ofthese pathways. In particular, we will focus on IL-1� secretion from primary human blood monocytes.In fact, although data from macrophages or macrophage cell lines are predominant, secretion of IL-1� bymonocytes is the most clinically relevant.

© 2013 Elsevier Ltd. All rights reserved.

. Introduction

IL-1� is one of the most potent proinflammatory cytokines,nducing inflammatory responses in virtually all tissues and organs.nitially discovered as the major fever-producing endogenous pyro-en, IL-1� was demonstrated to possess multiple and diverseroperties in the response to infection, injury and immune chal-

enge [1]. The broad role of IL-1� in health and disease wasonfirmed when the recombinant form of the cytokine becamevailable. Indeed, IL-1� is essential for a proper acute inflam-atory response to an infectious challenge, and therefore to

nsure the recovery from infection or trauma. However, theytokine also plays a pathogenic role in many chronic diseases,ncluding diabetes, atherosclerosis and cancer [2]. In spite of itsxquisite role as a cytokine, of the presence of specific receptorsn target cells, and of the existence of a complex extracellularechanism of control, including soluble decoy receptors [3] and

eceptor antagonist such as IL-1Ra [4], IL-1� lacks any knownignal sequence and thus is not a classical secretory protein.lthough it is clear that an active mechanism of secretion for

L-1� exists, and is different from the classical ER–Golgi one,he definition of this (or these) mechanism(s) remains incom-lete.

∗ Corresponding author at: Cell Biology Unit, IRCCS AOU San Martino IST,argo Rosanna Benzi 10, 16132 Genova, Italy. Tel.: +39 0105558379;ax: +39 0105558560.

E-mail addresses: patrizia.piccioli@hsanmartino.itP. Piccioli), anna.rubartelli@hsanmartino.it (A. Rubartelli).

044-5323/$ – see front matter © 2013 Elsevier Ltd. All rights reserved.ttp://dx.doi.org/10.1016/j.smim.2013.10.007

2. IL-1� is activated by proteolytic processing

Although several years of research on IL-1 documented anactive cytokine with a molecular weight of 15–17,000 Da, when theIL-1� gene was cloned in 1984 [5] the cDNA coded for a precursorprotein (pro-IL-1�) with a molecular weight of 31,000. In thefollowing years, caspase-1 was identified as the IL-1� convertingenzyme (ICE) able to proteolytically process pro-IL-1� into theactive cytokine IL-1� [6]. Also caspase-1 is present in cells as aprecursor, and the mechanism of its activation remained obscurefor many years, until Jurg Tschopp and his group in 2002 dis-covered the inflammasome. The inflammasome is a multiproteinintracellular complex that, upon assembly, activates caspase-1 andprocessing of IL-1� [7]. Today, it is widely accepted that pro-IL-1�is expressed by inflammatory cells after activation of patternrecognition receptors (PRR), including Toll-like receptors (TLR),by pathogen products or factors released by damaged tissues, andthat the precursor accumulates intracellularly. Then, other signals,including exogenous ATP, pathogenic fibers or crystals trigger theassembly of the inflammasome leading to activation of caspase-1that in turn processes pro-IL-1� to its mature form [8]. However,although in murine macrophages second signals are strictlyrequired to induce processing and secretion, in primary humanmonocytes second signals such as ATP enhances secretion of IL-1�but are dispensable as PRR stimulation alone induces secretion ofIL-1�, to a lower extent and with slower kinetics but sustained intime: in vitro, LPS-stimulated monocytes secrete IL-1� for morethan 24 h [9]. The possibility of human monocytes to do without a

second signal is due to the fact that PRR-triggering induces releaseof low but effective amounts of ATP by these cells. Externalized ATPstimulates the monocyte purinergic P2X7 receptor, triggering thecascade of events that lead to inflammasome activation [9]. This

4 rs in Immunology 25 (2013) 425– 429

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Fig. 1. Non-classical pathways of IL-1� secretion. The schematic representationof the different mechanisms proposed to mediate the unconventional secretionof IL-1� is shown. (1) Secretory lysosomes mediate IL-1� secretion. A fraction ofcaspase-1 and pro-IL-1� is incorporated into secretory lysosomes that undergoCa2+-dependent exocytosis with release of mature IL-1�. Intracellular Ca2+ rise isrequired for exocytosis but not for processing; at variance, a low intracellular K+

concentration, due to the K+ efflux induced by P2X7R triggering by exogenousATP, mediates inflammasome activation and pro-IL-1� processing. (2) Secretoryautophagy mediates IL-1� secretion. Amphisomes derived from the fusion betweenautophagosomes containing pro-IL-1� (and possibly inflammasome components)with endosomes (E) or multivescicular bodies (MVB, which also may store pro-IL-1�, see point (3) below) merge with the plasma membrane, leading to exocytosisof IL-1� soluble or within exosomes. Pro-IL-1� containing autophagosomes caninstead fuse with lysosomes (L) with consequent proteolytic degradation of thecytokine. Degradative autophagy would explain the inhibitory effects of autophagyon IL-1� secretion described in several studies (see text). (3) Exosomes mediateIL-1� secretion. MVB containing pro-IL-1� positive vesicles (exosomes) gener-ated by invagination of MVB membrane may undergo exocytosis with release ofIL-1�-containing exosomes. IL-1� is likely solubilized following lysis of exosomemembranes. (4) Microvescicle shedding mediates IL-1� secretion. Activation ofP2X7R by ATP induces an immediately shedding of membrane-derived microvesi-cles containing IL-1� and possibly inflammasome components; the cytokine isthen released in the extracellular compartment after microvescicle lysis. (5) Directmembrane translocation mediates IL-1� secretion. Cells undergoing pyroptosis dueto sustained activation of the inflammasomes externalize cytosolic IL-1� directly

26 P. Piccioli, A. Rubartelli / Semina

echanism allows monocytes to process and secrete IL-1� verylowly, a behaviour most likely relevant in the pathophysiology ofhronic IL-1�-mediated diseases.

. IL-1� is a leaderless secretory protein

A second unexpected finding revealed by the cDNA of pro-IL-� gene sequence was the lack the N-terminal secretory signalequence (also called leader sequence) that allows the targetingf secretory proteins to the endoplasmic reticulum (ER) and theirranslocation to the ER lumen [10]. Secretory proteins completehe biosynthesis into the ER lumen, are transported along the exo-ytotic pathway to the Golgi apparatus and the secretory vesiclesnd reach the extracellular space when secretory vesicles fuse withhe plasma membrane releasing their content outside the cells.he finding that a cytokine, which localizes and works extracel-ularly, lacks the secretory signal sequence raised the question ofow such a protein can reach the extracellular space. In principle,

“leaderless” protein should be synthesized on free ribosomes andtay in the soluble cytosol. At first, the simplest hypothesis pro-osed to explain the externalization of IL-1� was that the cytokineas just released by dying cells at the site of inflammation [5].owever, a few years later, IL-1� was demonstrated to be actively

ecreted by human monocytes activated by LPS through a novelecretory pathway that avoids the ER–Golgi one [11,12]. The exist-nce of a leaderless pathway of secretion for IL-1� was based onhe following evidence: (i) IL-1� is selectively released by activated

onocytes: the mature cytokine, but not pro-IL-1� or other cyto-olic proteins is found in culture supernatants; (ii) viable cells areequired for secretion of IL-1�: when activated monocytes wereilled by freezing and thawing before incubating at 37 ◦C, pro-IL-� and other cytosolic protein were released, but not mature IL-1�;iii) drugs blocking the transit along the exocytotic pathway inhibitecretion of classical secretory protein but not of IL-1� [11].

It must be stressed, however, that some extracellular proteases,ncluding neutrophil-derived protease-3, are able to process pro-L-1� in the extracellular compartment to low molecular weightorms endowed with biologic activity [13,14]. Thus, in addition tohe active secretion of mature IL-1�, deriving from inflammasomerocessing, also the passive externalization of pro-IL-1� in certain

nflammatory microenvironments where neutrophil proteases areresent and active may result in generation of IL-1 activity.

. Mechanisms of IL-1� secretion

In spite of advancements into the mechanism of IL-1�rocessing, and the discovery that other leaderless proteins play

role in innate immunity, how IL-1� is actually secreted from cellsemains elusive. This is largely due to technical reasons, such as theifficulties in reconstituting this secretory pathway. In addition, IL-� and the other leaderless secretory proteins do not share anybvious specific sequence or other features that could help iden-ify their binding partners and understand the mechanism of theirntracellular route from the cytosol to the extracellular space. Sev-ral mechanisms have been proposed in the last 23 years, some ofhich are restricted to IL-1�, but others possibly shared by other

eaderless secretory proteins (Fig. 1).

.1. IL-1 is actively secreted through a mechanism that avoidshe classical ER–Golgi pathway

As anticipated, the first evidence of an active secretory mecha-ism for IL-1� dates back at 1990 when our group demonstratedhat IL-1� is actively secreted by activated human monocytes via

pathway different from the classical ER–Golgi route [11]. Drugs

through the permeabilized plasma membrane. The mechanisms illustrated are notmutually exclusive.

such as Brefeldin A or Monensin, which block the transport alongthe exocytotic pathway of IL-6, of tumor necrosis factor and ofother secretory proteins produced by monocytes, did not inhibitsecretion of IL-1�, rather which was increased by these agents. Wealso observed that IL-1� secretion is induced by stress. Moreover,we observed that IL-1� is contained, in part, within intracellularvesicles that protect the cytokine from protease digestion and sug-gested that these intracellular vesicles may be part of the IL-1�secretory pathway [11]. In the following years, before the discov-ery of inflammasome, several studies reported that secretion ofmature IL-1� requires, or benefits from, a second stimulus in addi-tion to the first one provided by PRR triggering. Extracellular ATPwas identified as a powerful stimulus to induce IL-1� maturationand secretion by LPS-stimulated macrophages [15]. In 1997, Ferrariet al. [16] showed that ATP-driven IL-1� processing and secretion ismediated by activation of the purinergic P2X7 receptor, a findingsconfirmed by several studies in the following decades [17]. P2X7receptor stimulation promotes inflammasome activation by induc-ing K+ exit with resultant drop of the intracellular concentration ofK+. In addition, P2X7 receptor activation mediate IL-1� secretion

by inducing a sustained rise in cytosolic Ca2+ concentration [18].The requirement of calcium rise in IL-1� secretion was also shownin other cell systems, such as microglia [19] and astrocytes [20].

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.2. IL-1 secretion via exocytosis of secretory lysosomes

Some years later a secretory lysosome-dependent pathwayor IL-1� secretion was characterized in our laboratory [21,22]Fig. 1). We observed that a portion of the pro-IL-1� and caspase-

of LPS-activated primary human monocytes is found in vesicleselonging to the endolysosomal compartment that, unlike classicalndosomes and lysosomes, undergo exocytosis, i.e., the secretoryysosomes. According to our model, the mature form of IL-1� isroduced within the vesicles by caspase-1 cleavage, after whichhe endolysosomes fuse with the plasma membrane and matureL-1� is released into the extracellular space. Since only pro-IL-�, but not mature IL-1�, is found intracellularly, processing is

ikely to occur immediately before lysosome exocytosis and IL-1�xternalization.

Secretory lysosomes display features of both classical endolyso-omes and of secretory granules responsible for regulated secretionn specialized cells [23]. They are abundant in hemopoietic cells

here they participate to inflammatory and immune responsey delivering their cargoes extracellularly in a Ca2+-dependentay. Lysosome-mediated secretion is a regulated process in that

triggering signal is required to induce exocytosis [23]. In thease of IL-1�, the triggering signal is exogenous ATP that inducesysosome exocytosis with IL-1� release [22]. Interestingly, likeecretory granules in specialized epithelial or neuronal cells, secre-ory lysosomes may undergo polarized secretion. For instance, inytotoxic T lymphocytes, cytoskeleton rearrangement mediates theecruitment of the lytic granules (secretory lysosomes) toward thenteracting target cell, followed by the delivery of the cytotoxicnzymes restricted to the intercellular space, thereby avoidingilling of “innocent bystander” cells. In human dendritic cells, IL-1�and IL-18) secretion is induced by antigen specific T cells [24–27]r NK cells [28]. Interaction between DCs and CD8+ T cells [27]r NK cells [28] is associated with recruitment of IL-1� or IL-8-containing secretory lysosomes in the area of contact amonghe cells followed by lysosome exocytosis at the immunologicalynapse, the small intercellular space between interacting immuneells. This restricts the area of cytokine release to the immunologi-al synapse and allows activation of target cells without spreadingf the cytokine.

Like in cytotoxic T lymphocytes, cytoskeleton rearrangement isnvolved: ATP-induced IL-1� release from monocytes was impairedpon disruption or stabilization of microtubules with nocodazoler taxol, respectively [29]. Moreover, like in regulated secretion,a2+ signaling is strictly required [22,30]. However, cytosolic Ca2+

ise induces exocytosis of lysosomes but not pro-IL-1� processing:xposure of LPS-primed monocytes to the calcium ionophore iono-ycin results in rapid and abundant externalization of pro-IL-1� asell as of other molecular forms of intermediate molecular weight,

ut not of mature, 17 Kd IL-1� [22].

.3. IL-1 secretion via microvesicle shedding from the plasmaembrane

In 2001, a different mechanism of IL-1� secretion was pro-osed by Mckenzie and co-workers [31] (Fig. 1). They showed thatells from the THP-1 myelomonocytic cell line shed microvesiclesrom their plasma membrane within a few seconds of activationf P2X7 receptors by exogenous ATP. Two minutes after stimula-ion, extracellular bioactive IL-1� was only found into the released

icrovesicles, but no soluble IL-1� was found in supernatants. Onlyater IL-1� appeared in the vesicle-free supernatant, possibly due to

ysis of shed vesicles. ATP-induced microvesicle shedding was alsohown to mediate IL-1� externalization by microglia [32]. In thisystem, ATP released by astrocytes played an important role notnly in triggering microvesicle shedding by microglia but also in

munology 25 (2013) 425– 429 427

inducing the P2X7 receptor-dependent pores on the vesicle mem-brane that allow the exit of the cytokine.

4.4. IL-1 secretion via exosomes

A model of IL-1� release alternative to those described abovewas identified by Qu and colleagues in 2007 [33] (Fig. 1). Thismodel proposes that the P2X7R stimulation induces formation ofmultivesicular bodies that contain exosomes with entrapped IL-1�, caspase-1, and other inflammasome components. Interestingly,inflammasome assembly and active caspase-1 were simulta-neously required to trigger the export of IL-1�, supporting theprevious observation that IL-1� processing and secretion are atleast temporarily associated [21,22].

4.5. IL-1 secretion across the plasma membrane

An additional, non-vesicular pathway of IL-1� secretion hasbeen described to occur in monocyte/macrophages under par-ticular conditions such as sustained activation of the NLRP3 orNLRC4 inflammasome cascade. These conditions induce caspase-1-mediated pyroptotic death [34–36]. As a consequence, a directefflux of cytosolic mature IL-1� occurs across hyper-permeableplasma membranes (Fig. 1).

5. Secretory autophagy and IL-1� secretion

Autophagy is generally defined as a degradative process thatdelivers intracellular material to lysosomes. The function ofautophagy is to maintain or restore the cell homeostasis throughcytosolic autodigestion during starvation, thus ensuring cell-autonomous provision of energy and nutrients, and removal of oldorganelles and aggregates [37]. Recently, however, the involvementof autophagy in the process of secretion of leaderless secretoryproteins has been demonstrated in yeast [38,39], leading to theproposal that a “secretory autophagy” also exists [40,41]. Whilein the case of degradative autophagy, autophagosomes fuse withthe vacuole, resulting in degradation of their contents, in the caseof secretory autophagy, autophagosomes fuse with endosomes toform amphisomes that undergo exocytosis with extracellular deliv-ery of the secretory protein [41,42].

Interestingly, a role for autophagy has been proposed also inmammals for IL-1� secretion. However, although in yeast all thestudies agree that autophagy induces secretion of leaderless secre-tory proteins, the literature reports discrepant results on whetherautophagy promotes [43] or inhibits [44–48] IL-1� secretion. Inprinciple, the former hypothesis is attractive since it is knownthat TLR triggering, which in human monocytes drives both IL-1�synthesis and secretion [9], induces autophagy in myelomono-cytic cells [49,50]. Moreover, the hypothesis is consistent with thevesicular models of IL-1� secretion [21,33]. Lysosomes and mul-tivesicular bodies like autophagosomes may remain intracellularas degradative compartments or fuse with the plasma membraneand release their cargoes outside (Fig. 1). However, autophagy asa blocking mechanism for IL-1� secretion is supported by clearin vitro data, such as the strong inhibition of IL-1� secretioninduced by substances that promote autophagy and, conversely,the enhancement of IL-1� secretion with compounds that blockautophagy [45,46,48] and by in vivo evidence of impaired IL-1�production in mice deficient of autophagy genes [44,51].

A possible explanation is that the delivery of pro-IL-1� into

autophagosomes is indeed required for IL-1� secretion, and isinduced by TLR activation that, as reported [49,50], inducesautophagy (Fig. 2). Depending on the type and potency of thestimuli that trigger IL-1� producing cells, autophagosomes may

428 P. Piccioli, A. Rubartelli / Seminars in Immunology 25 (2013) 425– 429

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ig. 2. IL-1� and LC3 colocalize in autophagosomes after LPS stimulation of monocyfter 3 h of exposure to 1 �g of LPS, cells were fixed, permeabilized and double stainrovided by NCI BRB Preclinical repository, Rockville, MD, USA, panel B) and analyz

use with endosomes and undergo exocytosis, with delivery of IL-� out of the cell, or with lysosomes, with degradation of pro-IL-1�52]. Remarkably, in LPS-stimulated monocytes, we observed colo-alization of IL-1� in intracellular vesicles with LC3, an autophagyarker important for transport and maturation of the autophago-

ome (Fig. 2), followed by secretion of high levels of IL-1� (data nothown), thereby supporting a secretory autophagy. However, thisxperiment does not exclude that both secretory and degradativeutophagy take place in TLR-stimulated monocytes.

. Conclusions

Many different pathways have been proposed for IL-1� secre-ion: IL-1� secretion may involve the externalization of theytokine directly at the plasma membrane [34–36] or via vesi-les [21,31,33]. Secretory lysosomes, microvesicles shed from thelasma membrane, and exosomes have been identified as vesiclesble to carry IL-1� out of the cell in different cell types (primaryonocytes, monocyte continuous cell lines, mouse macrophages).hy so many secretory pathway for IL-1�? Although not explored

o date, it is likely that different mechanisms of secretion are usedor the control of inflammation in different inflammatory condi-ions. For instance, pyropotosis may indeed mediate IL-1� secretionut only under conditions of excessive cell stimulation [36], inhich case the clinical relevance would be limited. Differences

n the cell type, in the functional state of the cells or in the cul-ure conditions, as well as strength and duration of the stimulus53,54] may account for the various vesicular or non-vesicular IL-� secretory pathways used. Some of the proposed mechanisms ofecretion may also depend on artificial experimental conditions. Ineneral, adaptation of primary cells to continuous growth in vitroesults in many non-physiologic changes in such cells. First, cellsre chronically exposed to an O2 tension much higher than thehysiologic one. High O2 is pro-oxidant and induces a strong andersistent overexpression of antioxidant genes to detoxify the freeadicals produced. As the redox response is a main player in IL-1�rocessing and secretion [17], it is conceivable that the change inhe redox state and response in cultured cells affects the process of

aturation and secretion of the cytokine. Thus, the results obtainedn continuous cell lines, which dominate the field of IL-1� secre-ion and inflammasome activation, could be due at least in parto culture-related artifacts [53]. Actually, blood monocytes are the

ajor producers of IL-1�: they secrete high levels of the cytokinen response to concentrations of LPS of 1 or 10 ng/mL. This is true

lso in vivo: humans are highly sensitive to LPS, as 3 ng/kg injectedntravenously induces fever, TNF�, IL-1� and IL-6 in the circula-ion and blood monocytes are responsible of these effects. Likewise,lood monocytes are the cell population most likely involved in the

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uman monocytes from healthy donors were purified and cultured as described [9].th LC3 antibody (Novus Biologicals, Littleton, CO, USA, panel A) and IL-1� mAb (3ZD

confocal microscopy. Panel C: merge.

pathogenesis of many inflammatory conditions, including autoin-flammatory diseases [55].

In any case, all the pathways described are incompletely char-acterized and many questions remain unanswered. For instance, aspecific transporter for IL-1� on the cell surface or on the mem-brane of intracellular vesicles has not been identified. How IL-1�is recognized among the other cytosolic proteins and targeted tothe vesicles or to the plasma membrane is also still unknown. It ispossible, but not demonstrated, that intracellular proteins such asHMGB1 bind IL-1� and “chaperone” it out of the cell [43]. Hopefullyin the near future novel techniques will be developed allowing theunquestionable identification of the essential components of theIL-1� secretory pathway(s).

Acknowledgments

We apologize to authors whose work could not be cited in thisassay due to space limitations. We like to thank Dr. Patrizia Castel-lani for the confocal microscopy analysis and all members of ourlaboratory for discussion. This work was supported in part by grantsfrom Compagnia di San Paolo and Ricerca Corrente by Italian HealthMinistry to A.R.

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