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Review article
Cytokine release from innate immune cells: association with diverse membrane
trafficking pathways
Paige Lacy1 and Jennifer L. Stow2
1Pulmonary
Research Group, Department of Medicine, University of Alberta, Edmonton, AB; and 2Institute for Molecular Bioscience, University of Queensland,
St Lucia, Australia
Cytokines released from innate immune
cells play key roles in the regulation of
the immune response. These intercellular
messengers are the source of soluble
regulatory signals that initiate and constrain inflammatory responses to pathogens and injury. Although numerous studies describe detailed signaling pathways
induced by cytokines and their specific
receptors, there is little information on
the mechanisms that control the release
of cytokines from different cell types.
Indeed, the pathways, molecules, and
mechanisms of cytokine release remain a
“black box” in immunology. Here, we
review research findings and new approaches that have begun to generate
information on cytokine trafficking and
release by innate immune cells in response to inflammatory or infectious
stimuli. Surprisingly complex machinery,
multiple organelles, and specialized membrane domains exist in these cells to
ensure the selective, temporal, and often
polarized release of cytokines in innate
immunity. (Blood. 2011;118(1):9-18)
Introduction
The production and release of cytokines from innate immune cells are
critical responses to inflammation and infection in the body. Innate
immune cells comprise populations of white blood cells such as
circulating dendritic cells (DCs), neutrophils, natural killer (NK) cells,
monocytes, eosinophils, and basophils, along with tissue-resident mast
cells and macrophages.1 Residing at the frontline of defense in immunity, these cells control opportunistic invasion by a wide range of viral,
fungal, bacterial, and parasitic pathogens, in part by releasing a plethora
of cytokines and chemokines to communicate with other cells and
thereby to orchestrate immune responses. This array of soluble mediators secreted by different innate immune cells includes TNF, IFN␥,
interleukins IL-1␤, IL-4, IL-6, IL-10, IL-12, IL-18, CCL4/RANTES,
and TGF␤. Cytokine release can be directly evoked by immunoglobulin- or complement receptor-mediated signaling or by pathogens through a diverse array of cellular receptors, including pattern
recognition receptors such as TLRs.1,2 The Gram-negative bacterial
coat component lipopolysaccharide (LPS), the main culprit behind
toxic shock syndrome and sepsis, is a highly potent trigger of
cytokine secretion through TLR4.
For the immune system to function appropriately, the synthesis and
release of cytokines must be highly regulated and sequentially and
temporally orchestrated. Thus, cascades of cytokines released by innate
immune cells initially mount inflammatory or allergic responses and
then later ensure that the responses subside in a timely fashion.3
Proinflammatory cytokines also serve to recruit and active T lymphocytes and other cells to mount adaptive immune responses.1
However, even with the overwhelming and detailed literature on
cytokine actions, just how cytokines are released or secreted by
innate immune cells remains a significant “black box” in immunology. Most diagrams in textbooks and reviews show a simple arrow
indicating cytokine release from a given cell type after activation of
a signaling cascade in response to receptor stimulation. Perhaps
surprisingly, the precise mechanisms of cytokine trafficking and
release remain obscure in most cell types.
Our knowledge of cytokine secretion is superimposed on the field of
intracellular trafficking, and both have advanced, thanks to new
technologies that allow newly synthesized proteins to be visualized and
tracked through cells as fluorescently tagged proteins (eg, with green
fluorescent protein [GFP] and its many derivatives) with the use of
imaging in live and fixed cells.4,5 The functional components of the
trafficking machinery can now be studied in vitro with RNAinterference
and expression of mutated proteins, as well as through the use of
knockout and transgenic mice.
We discuss here new findings showing that cytokine secretion is
a complex and tightly controlled process, and we show that the
intracellular pathways available for release are often uniquely tailored to
each cytokine and cell type. In classical secretory pathways (Figure 1),
cytokines with signal peptides are cotranslationally inserted into the
endoplasmic reticulum (ER) for synthesis as either soluble or transmembrane precursors. They are then trafficked in vesicles to the Golgi
complex for further processing, and at the trans-Golgi network (TGN)
they are loaded into vesicles or carriers for constitutive delivery to the
cell surface or other organelles. In specialized cell types, additional
modes of secretion are proffered by loading cytokines and other cargo
into granules for storage and later release. Herein, studies on the release
of CCL5/RANTES from eosinophils and the secretion of TNF by
macrophages are described as examples of classical secretory pathways
via granule-mediated and constitutive routes, respectively. Finally, we
consider the possible mechanisms for nonclassical release (Figure 1) of
cytoplasm-derived cytokines such as IL-1␤ and IL-18.
Submitted August 24, 2010; accepted April 13, 2011. Prepublished online as
Blood First Edition paper, May 11, 2011; DOI 10.1182/blood-2010-08-265892.
© 2011 by The American Society of Hematology
BLOOD, 7 JULY 2011 䡠 VOLUME 118, NUMBER 1
Trafficking machinery
Like all newly synthesized proteins, most cytokines rely on a host
of membrane-bound and cytoplasmic cellular proteins, the socalled trafficking machinery, to mediate their transport through the
cell. Trafficking machinery molecules are needed to shepherd
9
10
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BLOOD, 7 JULY 2011 VOLUME 118, NUMBER 1
LACY and STOW
䡠
Figure 1. Different secretory pathways offer variable modes of release for cytokines. All cells have ⱖ 1 variations on the classic secretory pathways depicted in the top
panel, whereby proteins (eg, cytokines) synthesized in the ER and Golgi complex are transported in membrane-bound vesicles, granules, or both to the cell surface for release.
Although all cells have constitutive pathways, specialized cell types additionally have regulated (granule-mediated) pathways, and some cells have a variation of this process
(piecemeal degranulation) in which membrane-bound vesicles are used to selectively transport cytokines from secretory granules to the cell surface. All of these pathways
have multiple transport steps, each requiring sets of trafficking machinery molecules to execute carrier budding, movement, and membrane fusion. The bottom panel shows
nonclassical secretory pathways, which involve movement of proteins (eg, cytokines) directly from their point of synthesis in the cytoplasm to the external milieu. Various
mechanisms proposed for crossing the plasma membrane, as the single transport step required, include the use of membrane transporters, exosome release, microvesicle
shedding, or cell lysis for cytokine release.
newly synthesized proteins into secretory carriers, then for the
budding, movement, and fusion of the membrane-bound carriers at
each transport step (Figure 1).6-8 This complex machinery includes
large families of proteins from which individual family members
operate in distinct combinations to provide specificity for each
transport step (Table 1). Much of this machinery resides in the
membrane and is assembled at specific sites on organelle or carrier
membranes to facilitate the transport of newly synthesized proteins.
For bidirectional transport between the ER and Golgi, members
of the ADP ribosylation factor (ARF)/Sar family of small guanosine triphosphatase (GTPases) and their accessory proteins help to
sort and load secretory cargo into vesicles coated with coating
protein I or II.23 At the TGN, ADP ribosylation factor proteins and
adaptor complexes sort some proteins into clathrin-coated vesicles
for transport to late endosomes, whereas secretory proteins are
commonly loaded into uncoated tubulovesicular carriers for constitutive transport to endosomes or directly to the cell surface.7
Vesicle budding at each step requires membrane curvature to form
“buds” or tubules and fission to release them as carriers. These
actions involve series of proteins, lipid kinases, and phosphatases,
along with the GTPase dynamin, as well as other fission proteins.7
Both actin- and microtubule-based motors assist in the movement
of carriers through the cell.24,25 The docking and fusion of carriers
at target membranes then involve specific combinations of tethering
complexes, Rab GTPases8 and SNARE (soluble N-ethylmaleimide–
sensitive factor attachment protein [SNAP] receptors) fusion proteins.26
Small GTPases of the 60-member Rab family associate with and
demarcate the membranes of different organelles8; for instance, Rabs 5,
7, and 11 associate with early, late, and recycling endosomes, respectively. Rabs operate through multiple effectors to bring about vesicle
formation, movement, and prefusion docking.8
Among the best studied of the trafficking machinery proteins in
cytokine release are the membrane fusion proteins known as
SNAREs.26 The SNAREs include subfamilies of vesicle-associated
membrane proteins (VAMPs) and syntaxins which are classified by
the amino acid composition of their core SNARE domains as
R-SNAREs and Q-SNAREs, respectively. Typically, Q- and RSNAREs on opposing target and vesicle membranes unite as a
4-helix coiled-coil bundle that winches tethered membranes together for fusion (Figure 2).27 SNARE-mediated fusion has to
occur for each intracellular transport step, but the fusion of
granules, vesicles, or endosomes with the plasma membrane is the
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Table 1. Trafficking machinery involved in cytokine secretion from innate immune cells
Molecule type
Transport step mediated
Cytokine
Cell type
Reference
R-SNAREs
VAMP-2
Secretory vesicles, plasma membrane
CCL5
Eosinophils
VAMP-3
Recycling endosome, plasma membrane
IL-6, TNF
Macrophages
9
Recycling endosomes, plasma membrane
IFN␥, TNF
NK cells
12
VAMP-8
Secretory vesicle, plasma membrane
TNF
Macrophages
13
Vti1b
Golgi-recycling endosomes
TNF
Macrophages
11,14
Secretory vesicles, plasma membrane
CCL5
Eosinophils
Secretory granules, plasma membrane
TNF
Mast cells
Syntaxin-4
Recycling endosomes, plasma membrane
TNF
Macrophages
Secretory vesicles, plasma membrane
CCL5
Eosinophils
Syntaxin-6
Golgi-recycling endosomes
TNF
Macrophages
11,14
Syntaxin-7
Golgi-recycling endosomes
TNF
Macrophages
11
Golgin p230
Golgi-recycling endosomes
TNF
Macrophages
18
Phosphatidylinositol 3-kinase ␦ (PI3K␦)
Golgi-recycling endosomes
TNF
Macrophages
19
Adaptor AP-1
Recycling endosomes, plasma membrane
TNF
Macrophages
20
SNARE regulator SCAMP5
Recycling endosomes, plasma membrane
TNF
Macrophages
21
SNARE regulator Munc18c
Recycling endosomes, plasma membrane
TNF
Macrophages
17,22
GTPase Rab11a
Recycling endosomes, plasma membrane
TNF
Macrophages
11
Recycling endosomes, plasma membrane
IFN␥, TNF
NK cells
12
10,11
Q-SNAREs
SNAP-23
15
16
11,14,17
15
Others
Examples of specific SNAREs (R-SNAREs and Q-SNAREs) identified as part of the membrane fusion machinery required for vesicles or granules transporting cytokines
from one membrane compartment to fuse with the next in classical secretory pathways are listed. Examples of “other” trafficking machinery components that have been
implicated in cytokine trafficking are also listed.
SCAMP5 indicates secretory carrier membrane protein 5.
final event necessary for the surface delivery and release of
cytokines. A Q-SNARE complex consisting of syntaxin-4 and
synaptosomal-associated protein 23 (SNAP-23) on the plasma
membrane of many innate immune cells commonly performs the
final fusion step by pairing with different VAMPs on approaching
granules or vesicles (Table 1).28 Identifying the specific cognate
pairs of R- and Q-SNAREs for intracellular transport steps has
been a useful experimental approach to help chart the trafficking
routes of cytokines through cells.28 SNARE-binding proteins, such
as Munc proteins, can regulate Q-SNARE complexes, and Rab
GTPases work with SNAREs to align docking and fusion of
vesicles at the target membranes for IFN␥ and TNF trafficking.29
Whereas the trafficking machinery in cells was previously
thought of as having a passive housekeeping role, in fact, many
trafficking molecules are tightly coupled to cell activation and
respond to the need for cytokine secretion through being recruited
to membranes or through up-regulation of their expression. Thus,
in macrophages, LPS and IFN␥ up-regulate the expression of
specific SNAREs such as VAMP-3 and induce recruitment of
regulators such as PI3K␦ to TGN membranes.19,28 LPS also acutely
Figure 2. Binding model for SNAREs in cytokine
secretion. Ligand/receptor binding elicits signaling that
initiates trafficking in classic secretory pathways. At each
step of transport (eg, a secretory granule fusing with the
plasma membrane), a specific R-SNARE (VAMP) in the
secretory organelle membrane partners with a Q-SNARE
complex (eg, composed of SNAP-23 and syntaxin-4) in
the target membrane. SNARE binding involves the formation of a 4-helix coiled-coil structure that is modeled to
winch membranes together to allow membrane fusion
and extrusion of cytokines from the granule interior.
increases the budding of a subpopulation of TGN carriers. Taken
together, these LPS-induced changes enhance the activated cell’s
ability to rapidly traffic and secrete proinflammatory cytokines.
Regulated and constitutive exocytosis of
cytokines in granule-containing cells
Although all innate immune cells have the capacity for constitutive
exocytosis, in some cell types, notably granulocytes, cytokines or
other proteins may be loaded into secretory granules, lysosomerelated organelles, or secretory lysosomes for storage and later
release at the cell surface via ⱖ 1 of several mechanisms. Such
pathways are the “regulated” or “granule-mediated” pathways
for exocytosis or secretion which can use a combination of
granules and smaller secretory vesicles to actually release stored
cytokines. The storage of preformed cytokines in granules
allows cells to orchestrate their immediate release into the local
microenvironment within minutes of receptor stimulation. This
12
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BLOOD, 7 JULY 2011 VOLUME 118, NUMBER 1
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Figure 3. Piecemeal degranulation from eosinophils. (A) Piecemeal release of crystalloid granules with a “hollowed out” appearance, shown by electron microscopic
analysis of human cultured eosinophils. Arrows show lucent vesicles budding into granule interior, suggestive of vesicular trafficking to and from crystalloid granules. Reprinted
from Am J Pathol. 1991;138:69-82 with permission from the American Society for Investigative Pathology.56 (B) Time course of IFN␥-induced CCL5/RANTES release (green)
by piecemeal degranulation from major basic protein-positive crystalloid granules (red) in eosinophils. This research was originally published in Blood.57 Copyright The
American Society of Hematology. (C) Immunogold labeling of IL-4 (arrowheads) present in small vesicles budding from a crystalloid granule (Gr), indicating piecemeal
degranulation as a pathway for cytokine release. Reproduced with permission from Spencer et al.58 (Cytokine receptor-mediated trafficking of preformed IL-4 in eosinophils
identifies an innate immune mechanism of cytokine secretion. Proc Natl Acad Sci U S A. 2006;103(9):3333-3338. Copyright 2006 National Academy of Sciences, USA.)
(D) Meshed model of an eosinophil granule with small vesicles budding from it, using contours generated by automated electron tomography. A protrusion can be seen
progressively emerging from the granule, in blue (arrowheads). In the center panel, a vesicle is shown in yellow to indicate the way in which vesicles dissociate from the
granule. In the right panel, the arrow indicates the same vesicle from a different perspective near the granule (Gr). This model was generated by meshes of contours arising
from serial sections of the same granule in a single cell. Cells were fixed and processed for conventional transmission electron microscopy after stimulation with eotaxin.
Images were reproduced from Melo et al.59 Reprinted with permission from Traffic. 2005;6:1047-1057, published by John Wiley and Sons.
rapid response to injury or infection provides granulecontaining cells with a unique ability to spatiotemporally
modulate the inflammatory response.
Neutrophils are a prominent granulocyte at the first line of
defense in innate immunity and possess ⱖ 4 different types of
secretory organelles: azurophilic granules, specific granules, tertiary granules, and secretory vesicles.30 However, surprisingly little
is known about the way in which these organelles contribute to
cytokine trafficking and release. Neutrophils produce a wide range
of cytokines and chemokines, and their precise intracellular
locations are not known. Immunohistologic examination of mature
peripheral blood neutrophils suggests that TGF␣, TNF, IL-6, IL-12,
and CXCL2/IL-8 are stored within peroxidase-negative organelles,31-33 which may be secondary or tertiary granules, or endosomal secretory vesicles. SNAREs, including VAMP-2, VAMP-7,
syntaxin-4, syntaxin-6, and SNAP-23, are expressed in neutrophils
and regulate the trafficking and release of a number of these
secretory organelles.34-37 However, the dependency of neutrophil
cytokine release on Rab GTPases, SNAREs, and other trafficking
machinery has not been characterized. Thus, evidence for neutrophil cytokine production and trafficking is scant, and much more
work is needed to understand the contribution of each of the
neutrophil secretory organelles to cytokine release. The basophil is
another granulocyte that is important in the generation of cytokines, particularly IL-4 in allergic inflammation,38 although the
trafficking pathway for release of this cytokine is also not known.
Mast cells, which are important innate immune cells in allergy
and inflammatory diseases that reside in tissues, secrete numerous
cytokines and chemokines, many of which are stored as preformed
mediators in their secretory granules.39 Classical degranulation of
secretory granules from mast cells involves simultaneous fusion of
a large number of granules with the cell surface for bulk release of
histamine and other mediators, determined by capacitance measurements and electron microscopic analysis.40,41 Cytokines are released during classical degranulation, shown by the rapid secretion
of IL-4 and TNF during receptor-mediated exocytosis by crosslinking cell surface complexes of IgE and Ag.42-45 However,
cytokine release from mast cells may involve trafficking of a
distinct population of secretory vesicles independently of the
granules, in a process similar to piecemeal degranulation or
constitutive exocytosis (Figure 1). Piecemeal degranulation is a
type of regulated exocytosis that is considered to be a general
secretory pathway used by immune cells and other cell types,
including enteroendocrine cells of the gastrointestinal tract, adrenal
chromaffin cells, and chief cells of the parathyroid gland.46 This
form of vesicular trafficking is characterized by small vesicles
budding from large secretory granules, which then perform the
final step of transporting cytokines to the cell surface for release.47-49
Piecemeal degranulation or constitutive exocytosis may occur
in cytokine secretion from mast cells, whereby IL-1–induced IL-6
trafficking to the cell surface occurs in the absence of secretory
granule release.50 Cytokine trafficking that takes place independently of secretory granule exocytosis is further supported by
studies on the SNARE protein VAMP-8 in granule release with the
use of BM mast cells from VAMP-8 knockout mice.51 Degranulation measured as ␤-hexosaminidase and histamine release in
response to Fc⑀RI cross-linking was defective in VAMP-8 knockout mast cells, whereas the release of cytokines TNF, IL-6, and
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CCL3 occurred normally in these cells.51 Similar findings were
found in another study showing that VAMP-8 was necessary for
granule protein release in response to Fc⑀RI-mediated secretion but
not for TNF release.52 Thus, despite mast cells having a prominent,
characteristic capacity for VAMP-8–mediated degranulation, cytokine secretion largely occurs through a distinct and probable
constitutive pathway. Similarly, stimulation by distinct TLR2
activators in mast cells leads to differential cytokine release of
GM-CSF and IL-1␤ independently of degranulation.53 These
studies highlight the presence of multiple classic secretory pathways for granule proteins and cytokines in mast cells.
Another major effector cell in allergy and asthma is the
eosinophil, which can release ⱕ 35 different cytokines, chemokines, and growth factors.54,55 Many of these cytokines are stored as
preformed mediators in the eosinophil’s unique crystalloid granules
for later release in response to receptor stimulation (Figure 3).54
Eosinophils have been shown to use piecemeal degranulation as a
trafficking mechanism for cytokines. Piecemeal degranulation in
eosinophils is characterized by “hollowed out” crystalloid granules
along with the appearance of numerous tubulovesicular vesicles
(carriers) (Figure 3A). A structure that closely resembles piecemeal
degranulation is frequently observed by electron microscopy in
tissue eosinophils from allergic subjects, with ⱕ 67% of all nasal
polyp eosinophils from subjects with allergic rhinitis exhibiting
this type of exocytosis (Figure 3A).60
For example, eosinophils synthesize and store CCL5/RANTES,
a CC chemokine that acts as a potent chemoattractant, in their
crystalloid granules (Figure 3B).57,61 On stimulation by IFN␥,
eosinophils release CCL5/RANTES in a piecemeal manner by
shuttling this chemokine through a pool of small secretory vesicles
from the crystalloid granule to the cell surface (Figure 3B).57 These
small secretory vesicles are selectively released and located quite
apart from main basic protein-containing crystalloid granules, and
their presence was confirmed by subcellular fractionation of
homogenized eosinophils separated by linear density gradients.57,58
Eosinophils were shown to traffic Th1 (IFN␥, IL-12) and Th2
cytokines (IL-4, IL-13), as well as TNF, IL-6, and IL-10, through a
tubulovesicular system and small secretory vesicles that bud from
the crystalloid granules, and that serve to shuttle cytokines from the
granules to the cell membrane.62,63 Immunoelectron microscopy
confirms that IL-4 is synthesized and stored in crystalloid granules.58,59,63 After stimulation by CCL11/eotaxin, a potent chemokine that triggers chemotaxis in eosinophils, IL-4 is transported
through the tubulovesicular system and small secretory vesicles
(Figure 3C).58 These secretory vesicles bud off the crystalloid
granules to form the extensive tubulovesicular network found in
the cytoplasm of eosinophils (Figure 3D). Intriguingly, the IL-4
receptor actually participates in trafficking and release of the
cytokine. This led to the proposal that IL-4 is linked to its receptor
on the luminal side of the secretory vesicle membrane to allow its
selective packaging, trafficking, and release.58
The use of cytokine receptors to transport cytokines presages
what may be a mechanism used more widely for the release of
cytokines in other pathways and different cell types. Indeed, other
innate immune cells also express cytokine receptors in their
intracellular granules. In particular, neutrophils secrete IL-1064 and
express IL-10 receptor in association with their specific granules.65
Moreover, mast cell granules contain chemokine receptors CCR3
and CXCR2,66,67 and human mast cells secrete the ligands for these
receptors: eotaxin, CCL5/RANTES, and IL-8. Finally, cytokine
receptor-mediated trafficking has also been described for constitutive trafficking and secretion of the proinflammatory cytokine
13
IL-15 from DCs. In these cells, IL-15 is colocalized with the IL-15
receptor ␣ chain as preassembled complexes in the ER and Golgi
complex.68
Some of the trafficking machinery governing piecemeal degranulation of CCL5/RANTES in eosinophils has been characterized,
using a pathway that depends on the membrane fusion R-SNARE
protein, VAMP-2 (synaptobrevin-2), and the Q-SNAREs
syntaxin-4 and SNAP-23 (Figure 4).9,15 Human eosinophils express
VAMP-2 in small secretory vesicles containing CCL5/RANTES,
which translocate to the cell surface on stimulation by IFN␥
(Figure 4). Syntaxin-4 and SNAP-23 localize to the cell surface
where they function as putative cognate intracellular receptors for
VAMP-2, allowing the vesicles to fuse with the cell surface.
Eosinophils also express VAMP-7 and VAMP-8 on their crystalloid
granules, where VAMP-7 is required for membrane fusion during
crystalloid granule exocytosis (Figure 4).37
NK cells are cytotoxic cells of the innate immune system.
NK cells are armed with lytic granules that release proteins such as
perforin and granzyme to kill virally infected or tumorigenic target
cells.69 Similar to mast cells and eosinophils, cytokine release in
NK cells is mediated independently of lytic granules. However, the
carriers for NK cell–generated cytokines differ from granulocytes.
TNF and IFN␥ produced in response to NK-cell activation are
sorted away from the contents of lytic granules at the TGN and are
instead packaged together into dynamic carriers for constitutive
transport and release.12 Although lytic granules are targeted to the
immunologic synapse for docking and release, cytokine carriers are
released from all points of the NK-cell surface for nonpolarized
release of cytokines. TNF is delivered both to the immunologic
synapse and to the rest of the cell surface for dissemination.12 Thus,
despite having secretory granules for the release of specialized
contents, often in a polarized fashion, NK cells also maintain the
ability to widely broadcast cytokines by releasing them via separate
carriers and constitutive pathways.
Taken together, a major route for cytokine release from mast
cells and NK cells is through separate pathways from secretory
granules. In the case of eosinophils, cytokine trafficking occurs
through piecemeal degranulation relying on a tubulovesicular
network that buds from crystalloid granules and provides a readily
available pool of cytokines for immunoregulatory processes in
inflammation and infection. This allows cells to modulate or
fine-tune the immune response through separate intracellular
machinery apart from granule exocytosis.
Constitutive exocytosis of inflammatory
cytokines in macrophages
Constitutive exocytosis is the predominant mechanism for cytokine
release from macrophages and DCs, which do not have typical
secretory granules.70 The trafficking pathway and molecular machinery for TNF in activated macrophages is currently one of the most
comprehensively studied pathways for cytokine release. TNF is a
potent proinflammatory cytokine secreted by many innate immune
cells, particularly activated macrophages, but also neutrophils,
mast cells, eosinophils, DCs, and NK cells.31,32,43,71,72 The exocytic
pathways responsible for TNF release vary between these cell
types, and in macrophages TNF is released by constitutive exocytosis only after being synthesized in response to inflammatory
stimuli.71 Macrophages also produce and secrete a cascade of other
proinflammatory and anti-inflammatory cytokines, such as IL-6,
IL-10, IL-12, and a host of chemokines that are trafficked and
14
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Figure 4. Regulated and constitutive exocytosis of cytokines. Representative pathways are shown for SNARE-mediated regulated (eosinophil, left) and constitutive
(macrophage, right) release of CCL5/RANTES and TNF, respectively. In regulated exocytosis, IFN␥-stimulated eosinophils release preformed CCL5/RANTES by piecemeal
degranulation from their crystalloid granules via small secretory vesicles that express the Q-SNARE VAMP-2 (red) for binding to cognate R-SNAREs, SNAP-23 (green), and
syntaxin-4 (blue). Constitutive exocytosis occurs during stimulation by LPS, which leads to continuous trafficking of newly synthesized pro-TNF from the ER through the Golgi
to recycling endosomes, which use the SNARE complex Vti1b/syntaxin-6/syntaxin-7 to transport pro-TNF to recycling endosomes. From the recycling endosome to the cell
membrane, the R-SNARE VAMP-3 (red) on recycling endosomes binds to Q-SNAREs SNAP-23 (green) and syntaxin-4 (blue) in the cell membrane to fuse the recycling
endosomes for TNF cleavage and release at the cell exterior. Rab11a (purple) is involved in directing SNARE binding at the cell membrane.
secreted by constitutive exocytosis, moving, sometimes simultaneously, through the ER and Golgi complex.70,71,73
Macrophages have a pool of constitutively transcribed TNF
mRNA, which, on TLR4 signaling, is stabilized and rapidly
translated, giving rise to a transmembrane TNF precursor,74 which
is trafficked from the ER to the Golgi complex, where it can be
detected by immunostaining within 20 minutes of LPS activation.71
By imaging live cells, GFP-tagged TNF can be seen exiting the
TGN in highly dynamic, tubular carriers that stretch out and break
off, undergoing fission, to release TNF-loaded carriers.10,18 Fluorescently tagged IL-6 can also be visualized in live cells being loaded,
as a soluble cargo, into tubules at the TGN. The extension of TGN
membranes into tubules helps to load and sort the cargo proteins.
Members of the coiled-coil GRIP golgin family of tethering
proteins, golgin97 and golgin-245 (p230), label separate TGN
tubular carriers,75 and live cell imaging shows that TNF is
specifically transported in p230-labeled tubules. LPS up-regulates
the number of p230 tubules forming at the Golgi in activated
macrophages, generating a greater capacity for cytokine trafficking. RNAi knockdown of p230 in macrophage cell lines or in
macrophages from retrogenic p230-deficient mice inhibits the
formation of TNF carriers, halting TNF at the Golgi and blocking
its secretion.18
Membrane lipids also have a major role in the formation and
function of these tubules and carriers, undergoing rearrangements
to allow curvature, extension, and fission of membranes and also
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binding the cytoplasmic proteins needed to mediate these processes.76 Choline cytidylyltransferase mediates synthesis of phosphatidylcholine at the TGN in macrophages to allow the exit and
subsequent secretion of TNF and IL-6.77 Although PI3Ks were
previously thought to regulate trafficking predominantly at endosomes and at the cell surface, recent studies show that one isoform,
PI3K␦, functions at the TGN to produce carriers for TNF exit and
secretion. The p110 subunit of PI3K␦ is recruited to Golgi
membranes in LPS-activated macrophages, where it, in turn,
recruits dynamin II for fission of the tubules.19 Pharmacologic or
genetic inactivation of PI3K␦ reduces TNF secretion from macrophages as a result of impaired fission of p230 Golgi-derived
carriers for TNF.19 PI3K␦ probably variably regulates the secretion
of different cytokines, depending on which tubules they use for exit
from the TGN. The differential exit of cytokines from the TGN is
emerging as a definitive point in the constitutive secretory pathway,
and one that may be amenable to pharmacologic intervention.
Previous dogma held that constitutively secreted proteins were
trafficked directly from the TGN to the cell surface for release.
However, with the advent of live cell imaging, it became apparent
that many newly synthesized proteins leaving the TGN are next
delivered to intervening endosomes, before being transported in a
subsequent step to the plasma membrane for release (Figure 4).
These are tubulovesicular “recycling endosomes,” based on the
presence of markers such as Rab11 and VAMP-3 and their
recycling cargo of transferrin,78 and in many cell types they are
now recognized as compartments in both endocytic and exocytic
pathways.79,80 Most cell types, both immune and nonimmune cells,
contain recycling endosomes and, although these organelles have
been studied in NK cells, mast cells, and macrophages, they are yet
to be characterized in neutrophils or eosinophils. Cytokine trafficking through recycling endosomes is not unique to macrophages.
Microglial cells represent yet another innate immune cell type in
which TNF secretion occurs by recycling endosomes.81 In NK cells,
the constitutive, nonpolarized release of TNF and IFN␥ also
requires involvement of recycling endosomes.12
SNARE complexes and SNARE-interacting proteins mediate
membrane fusion at successive transport steps from the Golgi to the
cell membrane to facilitate the secretion of cytokines in macrophages.28 Carriers exiting the Golgi with TNF as cargo display a
distinct Q-SNARE complex composed of syntaxin-6/syntaxin-7/
Vti1b.14 This complex engages with its cognate R-SNARE,
VAMP-3, on recycling endosomes11 to deliver TNF to these
endosomal/exocytic compartments in the cell periphery. VAMP-3,
or in some cells VAMP-2, is the principal R-SNARE typically
found on recycling endosomes and on the cell surface, where it
mediates constitutive exocytosis at the cell surface (Figure 4). By
high-resolution imaging, TNF colocalizes with VAMP-3 in the
membrane of recycling endosomes, and these 2 proteins migrate
together to the cell surface where VAMP-3 engages with the
Q-SNARE complex of syntaxin-4 and SNAP-23 for fusion, finally
delivering TNF to the cell surface.10,11,17
Additional machinery interacts with the SNAREs either directly
or indirectly to sort and load cargo and affect its movement. For
example, the sec/munc protein Munc-18c also participates in this
SNARE-mediated fusion at the macrophage cell surface (Table
1),17 although its potential role remains controversial. Secretory
carrier membrane proteins are transmembrane proteins associated
with secretory organelles, and in macrophages secretory carrier
membrane protein (SCAMP) 5 on recycling endosomes interacts
with the relevant SNARE proteins to regulate calcium-triggered
15
traffic and secretion of TNF and CCL5/RANTES in response to
ionomycin or TLR4 signaling.21
Thus, although many individual proteins of the macrophage
trafficking machinery have been implicated in cytokine secretion,
as yet we do not have a unified picture of how they all coordinately
work to transport newly synthesized cytokines step-wise through
the cell and regulate their release during immune responses.
Polarized release of cytokines
Polarized cytokine delivery has been functionally associated with
microtubule organizing center polarization at the immunologic
synapse in T cells and NK cells.69 A recent study has shown that
DCs also secrete the Th1 cytokine IL-12 in a polarized manner
toward the immunologic synapse and that this polarization of IL-12
release depends on Cdc42 GTPase.82 Recycling endosomes have a
key role in the selective delivery of cytokines to polarized domains
of the cell surface. In macrophages, the transmembrane precursor
of TNF moves in a polarized fashion from recycling endosomes to
the filopodia and nascent phagocytic cups at the cell surface.11 In
the recycling endosomes, TNF destined for phagocytic cups is
sorted away from other cargo, including recycling transferrin or
soluble cytokine IL-6, which are delivered to other points on the
cell surface.10 The clustering and sorting of TNF at the recycling
endosome is reportedly mediated by the adaptor complex AP-1 in
combination with clathrin, after which cleaved subunits of AP-1
shepherd the TNF toward the phagosome.20,83 Recycling endosomes labeled for GFP-VAMP-3 and Rab11a contribute extra
membrane needed to form phagocytic cups as they extend out from
the cell surface during the initial stages of phagocytosis.83-89 By
comigrating with this recycling endosome membrane TNF is
conjointly, and efficiently, delivered to the cell surface.11,20,22,83
TNF is delivered to raft domains in the cholesterol-rich membranes
of phagocytic cups and filopodia. Finally, the TNF cleaving
enzyme (TACE, or ADAM17) is also concentrated at the phagocytic cup, ready to cleave the transmembrane TNF for extracellular
release before the phagocytic cup closes over and internalizes.11,22
Thus, cytokine trafficking through classic secretory pathways,
whether through regulated or constitutive exocytic routes, is
regulated both temporally and spatially to orchestrate immune
responses. The mode of release is often customized to enhance the
rapidity or efficiency of cytokine release, to direct cytokines to a
target, or to maximize their dispersal.
Nonclassical secretory pathways
Cytokines such as IL-1␤, IL-15, IL-18, and macrophage inhibitory
factor lack the N-terminal signal sequence required for ER entry
and are thus synthesized in the cytoplasm and thereafter released
from cells by so called “nonclassical” pathways.90 A number of
possible, and quite different, routes have been proposed for these
cytokines to cross the plasma membrane and achieve exit from
the cell.
The proinflammatory cytokine IL-1␤ is one of the most crucial
mediators of inflammation and host responses to infection,91
although its mechanism of release remains among the least
understood of all cytokines. IL-1␤ is first generated as pro-IL-1␤,
which is biologically inactive, and is synthesized directly in the cell
cytoplasm.91 This precursor is processed into mature, biologically
active IL-1␤ by caspase-1and then released directly into the
16
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BLOOD, 7 JULY 2011 VOLUME 118, NUMBER 1
䡠
LACY and STOW
extracellular milieu. How it exits the cell, by crossing the plasma
membrane directly or by entering or associating with intracellular
membranes, is still controversial. Several different mechanisms
have been proposed for IL-1␤ release, and these have been
reviewed in detail previously.91 Purported nonclassical mechanisms for release include (1) export of IL-1␤ through ABC
transporters in the plasma membrane,92 (2) release of IL-1␤–
containing exosomes from multivesicular bodies, and (3) shedding
of plasma membrane microvesicles.91 There is also evidence that a
significant proportion of IL-1␤ production may occur through
necrosis and cell lysis (Figure 1).
Similarly, IL-18, a potent inducer of IFN␥,93 is made in the
cytoplasm from where it is translocated, by unknown mechanisms,
into secretory lysosomes for classic secretion in DCs.94 These
secretory lysosomes are restricted to the immunologic synapse in a
calcium-dependent manner to release IL-18 at this site.95 The
polarization and constrained release of IL-18 allows activation of
NK cells to up-regulate cytotoxicity without spreading this cytokine nonspecifically to collateral tissues.
In addition, a cytolytic pathway of nonclassical granule release
has been described in eosinophils that generate “cell-free” granules
that can be detected in tissues of persons with allergies.60 Cytolysis
is responsible for ⬃ 33% of the degranulation events seen in vivo
during allergic reactions, aside from the predominant form of
release through piecemeal degranulation. Although controversial,
cell-free granules were shown to function as extracellular secretory
organelles for IL-4 and IL-6 release in response to IFN␥.96 This
intriguing observation deserves further exploration to understand
how cell-free granules may function in immunomodulation.
Looking ahead: future directions
Cytokines are currently at the forefront of medicine, both as
culprits in the pathogenesis of a large number of diseases associated with inflammation and as increasingly popular targets for
therapeutic antibodies.97,98 Both issues compel us to develop a
comprehensive understanding of cytokine trafficking and release,
first to explain how individual cytokines are differentially handled
and regulated in different cell types and second to identify the
trafficking machinery involved. Such knowledge may then help us
to refine the use of therapeutic antibodies, or it may show new
targets or new strategies for selectively reducing or controlling
cytokine release in disease.
A plethora of cytokines are also released by cells of the
adaptive immune system. Thanks to the use of approaches such
as live-cell imaging, some of the pathways and molecules regulating cytokine release in helper and cytotoxic T-cell populations have
been elucidated, and these are described elsewhere.99 Eventually,
the combined knowledge of cytokine release in both innate and
adaptive immune systems will instruct our understanding of cell
communication and effectors across the spectrum of immunity and
disease.
Finally, studies into cytokine trafficking will benefit greatly
from systems biology100 and “omic” approaches to trafficking and
from sophisticated bioinformatics being used to predict, test, and
visualize molecular networks.101-103 With high-throughput imaging and RNAi approaches it is now eminently possible to study
and compare all members of molecular families, such as Rabs,
SNAREs, adaptors, lipid kinases, and phosphatases, in trafficking pathways. Such family-wide studies have yet to be applied
in earnest to studies of trafficking in cells of the innate immune
system. Cytokine secretion from single cells can be measured
with great sensitivity,104 and the behavior of cytokine-secreting
cells can be visualized with increasing sensitivity with the use of
in vitro live-cell imaging, alongside 2-photon imaging in vivo.
Finally, super-resolution fluorescence imaging and ultrastructural approaches are anticipated to define the membrane microdomains and molecular interactions that underpin secretion in
innate immune cells.105
Acknowledgments
We thank Amanda Stanley and Adam Wall for many helpful
suggestions in the preparation of this manuscript.
This work was supported by grants from the Alberta Heritage
Foundation for Medical Research (Visiting Speaker from Alberta
Award), the Australian Research Council International Discovery
Grant, and the National Health and Medical Research Council of
Australia.
Authorship
Contribution: P.L. and J.L.S. contributed equally to this paper.
Conflict-of-interest disclosure: The authors declare no competing financial interests.
Correspondence: Paige Lacy, Pulmonary Research Group, 559
HMRC, Department of Medicine, University of Alberta, Edmonton, AB T6G 2S2, Canada; e-mail: [email protected].
References
1. Iwasaki A, Medzhitov R. Regulation of adaptive
immunity by the innate immune system. Science.
2010;327(5963):291-295.
2. McGettrick AF, O’Neill LA. Toll-like receptors: key
activators of leucocytes and regulator of haematopoiesis. Br J Haematol. 2007;139(2):185-193.
3. Hu X, Ivashkiv LB. Cross-regulation of signaling
pathways by interferon-␥: implications for immune responses and autoimmune diseases. Immunity. 2009;31(4):539-550.
4. Giepmans BN, Adams SR, Ellisman MH, Tsien RY.
The fluorescent toolbox for assessing protein location and function. Science. 2006;312(5771):217-224.
5. Lippincott-Schwartz J. Dynamics of secretory
membrane trafficking. Ann N Y Acad Sci. 2004;
1038(1):115-124.
6. Hughson FM, Reinisch KM. Structure and
mechanism in membrane trafficking. Curr Opin
Cell Biol. 2010;22(4):454-460.
7. De Matteis MA, Luini A. Exiting the Golgi complex. Nat Rev Mol Cell Biol. 2008;9(4):273-284.
8. Stenmark H. Rab GTPases as coordinators of
vesicle traffic. Nat Rev Mol Cell Biol. 2009;10(8):
513-525.
9. Lacy P, Logan MR, Bablitz B, Moqbel R. Fusion
protein vesicle-associated membrane protein 2 is
implicated in IFN-␥-induced piecemeal degranulation in human eosinophils from atopic individuals. J Allergy Clin Immunol. 2001;107(4):671-678.
10. Manderson AP, Kay JG, Hammond LA, Brown DL,
Stow JL. Subcompartments of the macrophage
recycling endosome direct the differential secretion of IL-6 and TNF␣. J Cell Biol. 2007;178(1):
57-69.
11. Murray RZ, Kay JG, Sangermani DG, Stow JL. A
role for the phagosome in cytokine secretion. Science. 2005;310(5753):1492-1495.
12. Reefman E, Kay JG, Wood SM, et al. Cytokine
secretion is distinct from secretion of cytotoxic
granules in NK cells. J Immunol. 2010;184(9):
4852-4862.
13. Pushparaj PN, Tay HK, Wang CC, Hong W,
Melendez AJ. VAMP8 is essential in anaphylatoxin-induced degranulation, TNF-␣ secretion,
peritonitis, and systemic inflammation. J Immunol. 2009;183(2):1413-1418.
14. Murray RZ, Wylie FG, Khromykh T, Hume DA,
Stow JL. Syntaxin 6 and Vti1b form a novel
SNARE complex, which is up-regulated in activated macrophages to facilitate exocytosis of tumor necrosis factor-␣. J Biol Chem. 2005;
280(11):10478-10483.
From bloodjournal.hematologylibrary.org at UNIVERSITY OF ALBERTA LIBRARY on July 7, 2011. For personal
use only.
CYTOKINE RELEASE FROM INNATE IMMUNE CELLS
BLOOD, 7 JULY 2011 䡠 VOLUME 118, NUMBER 1
15. Logan MR, Lacy P, Bablitz B, Moqbel R. Expression of eosinophil target SNAREs as potential
cognate receptors for vesicle-associated membrane protein-2 in exocytosis. J Allergy Clin Immunol. 2002;109(2):299-306.
16. Suzuki K, Verma IM. Phosphorylation of
SNAP-23 by I␬B kinase 2 regulates mast cell degranulation. Cell. 2008;134(3):485-495.
17. Pagan JK, Wylie FG, Joseph S, et al. The
t-SNARE syntaxin 4 is regulated during macrophage activation to function in membrane traffic
and cytokine secretion. Curr Biol. 2003;13(2):
156-160.
18. Lieu ZZ, Lock JG, Hammond LA, La Gruta NL,
Stow JL, Gleeson PA. A trans-Golgi network golgin is required for the regulated secretion of TNF
in activated macrophages in vivo. Proc Natl Acad
Sci U S A. 2008;105(9):3351-3356.
19. Low PC, Misaki R, Schroder K, et al. Phosphoinositide 3-kinase ␦ regulates membrane fission of
Golgi carriers for selective cytokine secretion.
J Cell Biol. 2010;190(6):1053-1065.
20. Braun V, Deschamps C, Raposo G, et al. AP-1
and ARF1 control endosomal dynamics at sites of
FcR mediated phagocytosis. Mol Biol Cell. 2007;
18(12):4921-4931.
21. Han C, Chen T, Yang M, Li N, Liu H, Cao X. Human SCAMP5, a novel secretory carrier membrane protein, facilitates calcium-triggered cytokine secretion by interaction with SNARE
machinery. J Immunol. 2009;182(5):2986-2996.
22. Kay JG, Murray RZ, Pagan JK, Stow JL. Cytokine
secretion via cholesterol-rich lipid raft-associated
SNAREs at the phagocytic cup. J Biol Chem.
2006;281(17):11949-11954.
23. Spang A, Shiba Y, Randazzo PA. Arf GAPs: gatekeepers of vesicle generation. FEBS Lett. 2010;
584(12):2646-2651.
24. Verhey KJ, Hammond JW. Traffic control: regulation of kinesin motors. Nat Rev Mol Cell Biol.
2009;10(11):765-777.
25. Loubery S, Coudrier E. Myosins in the secretory
pathway: tethers or transporters? Cell Mol Life
Sci. 2008;65(18):2790-2800.
26. Jahn R, Scheller RH. SNAREs–engines for membrane fusion. Nat Rev Mol Cell Biol. 2006;7(9):
631-643.
27. Fasshauer D, Sutton RB, Brunger AT, Jahn R.
Conserved structural features of the synaptic fusion complex: SNARE proteins reclassified as Qand R-SNAREs. Proc Natl Acad Sci U S A. 1998;
95(26):15781-15786.
28. Stow JL, Manderson AP, Murray RZ. SNAREing
immunity: the role of SNAREs in the immune system. Nat Rev Immunol. 2006;6(12):919-929.
29. Wickner W. Membrane fusion: five lipids, four
SNAREs, three chaperones, two nucleotides, and
a Rab, all dancing in a ring on yeast vacuoles.
Annu Rev Cell Dev Biol. 2010;26:115-136.
30. Borregaard N, Sorensen OE, Theilgaard-Monch
K. Neutrophil granules: a library of innate immunity proteins. Trends Immunol. 2007;28(8):340345.
31. Beil WJ, Weller PF, Peppercorn MA, Galli SJ,
Dvorak AM. Ultrastructural immunogold localization of subcellular sites of TNF-␣ in colonic
Crohn’s disease. J Leukoc Biol. 1995;58(3):284298.
32. Calafat J, Janssen H, Stahle-Backdahl M,
Zuurbier AE, Knol EF, Egesten A. Human monocytes and neutrophils store transforming growth
factor-␣ in a subpopulation of cytoplasmic granules. Blood. 1997;90(3):1255-1266.
33. Denkers E, Del Rio L, Bennouna S. Neutrophil
production of IL-12 and other cytokines during
microbial infection. In: Cassatella MA, ed. The
Neutrophil: An Emerging Regulator of Inflammatory and Immune Response. Basel, Switzerland:
Karger;2003:95-114.
34. Martin-Martin B, Nabokina SM, Blasi J, Lazo PA,
Mollinedo F. Involvement of SNAP-23 and syntaxin 6 in human neutrophil exocytosis. Blood.
2000;96(7):2574-2583.
35. Mollinedo F, Martin-Martin B, Calafat J, Nabokina SM,
Lazo PA. Role of vesicle-associated membrane protein-2, through Q-soluble N-ethylmaleimide-sensitive
factor attachment protein receptor/R-soluble Nethylmaleimide-sensitive factor attachment protein receptor interaction, in the exocytosis of specific and tertiary granules of human neutrophils.
J Immunol. 2003;170(2):1034-1042.
36. Mollinedo F, Calafat J, Janssen H, et al. Combinatorial SNARE complexes modulate the secretion of cytoplasmic granules in human neutrophils. J Immunol. 2006;177(5):2831-2841.
37. Logan MR, Lacy P, Odemuyiwa SO, et al. A critical role for vesicle-associated membrane protein-7 in exocytosis from human eosinophils and
neutrophils. Allergy. 2006;61(6):777-784.
38. Yoshimoto T, Yasuda K, Tanaka H, et al. Basophils contribute to T(H)2-IgE responses in vivo via
IL-4 production and presentation of peptide-MHC
class II complexes to CD4⫹ T cells. Nat Immunol.
2009;10(7):706-712.
39. Marshall JS. Mast-cell responses to pathogens.
Nat Rev Immunol. 2004;4(10):787-799.
40. Fernandez JM, Neher E, Gomperts BD. Capacitance measurements reveal stepwise fusion
events in degranulating mast cells. Nature. 1984;
312(5993):453-455.
41. Gomperts BD. GE: a GTP-binding protein mediating exocytosis. Annu Rev Physiol. 1990;52:591606.
42. Wilson SJ, Shute JK, Holgate ST, Howarth PH,
Bradding P. Localization of interleukin (IL)-4 but
not IL-5 to human mast cell secretory granules by
immunoelectron microscopy. Clin Exp Allergy.
2000;30(4):493-500.
43. Gordon JR, Galli SJ. Mast cells as a source of
both preformed and immunologically inducible
TNF-␣/cachectin. Nature. 1990;346(6281):274276.
44. Olszewski MB, Groot AJ, Dastych J, Knol EF.
TNF trafficking to human mast cell granules: mature chain-dependent endocytosis. J Immunol.
2007;178(9):5701-5709.
45. Olszewski MB, Trzaska D, Knol EF, Adamczewska V,
Dastych J. Efficient sorting of TNF-␣ to rodent
mast cell granules is dependent on N-linked glycosylation. Eur J Immunol. 2006;36(4):997-1008.
46. Crivellato E, Nico B, Mallardi F, Beltrami CA,
Ribatti D. Piecemeal degranulation as a general
secretory mechanism? Anat Rec A Discov Mol
Cell Evol Biol. 2003;274(1):778-784.
47. Dvorak AM. Basophils and mast cells: piecemeal
degranulation in situ and ex vivo: a possible
mechanism for cytokine-induced function in disease. Immunol Ser. 1992;57:169-271.
48. Dvorak AM, Ackerman SJ, Furitsu T, Estrella P,
Letourneau L, Ishizaka T. Mature eosinophils
stimulated to develop in human-cord blood mononuclear cell cultures supplemented with recombinant human interleukin-5, II: vesicular transport of
specific granule matrix peroxidase, a mechanism
for effecting piecemeal degranulation. Am J
Pathol. 1992;140(4):795-807.
49. Crivellato E, Candussio L, Mallardi F, Ribatti D.
Recombinant human ␣-2a interferon promotes an
atypical process of mast cell secretion with ultrastructural features suggestive for piecemeal degranulation. J Anat. 2002;201(6):507-512.
50. Kandere-Grzybowska K, Letourneau R, Kempuraj D,
et al. IL-1 induces vesicular secretion of IL-6 without
degranulation from human mast cells. J Immunol.
2003;171(9):4830-4836.
51. Tiwari N, Wang CC, Brochetta C, et al. VAMP-8
segregates mast cell-preformed mediator exocytosis from cytokine trafficking pathways. Blood.
2008;111(7):3665-3674.
52. Puri N, Roche PA. Mast cells possess distinct secretory granule subsets whose exocytosis is
17
regulated by different SNARE isoforms. Proc Natl
Acad Sci U S A. 2008;105(7):2580-2585.
53. McCurdy JD, Olynych TJ, Maher LH, Marshall JS.
Cutting edge: distinct Toll-like receptor 2 activators selectively induce different classes of mediator production from human mast cells. J Immunol.
2003;170(4):1625-1629.
54. Lacy P, Moqbel R. Eosinophil cytokines. Chem
Immunol. 2000;76:134-155.
55. Hogan SP, Rosenberg HF, Moqbel R, et al. Eosinophils: biological properties and role in health and
disease. Clin Exp Allergy. 2008;38(5):709-750.
56. Dvorak AM, Furitsu T, Letourneau L, Ishizaka T,
Ackerman SJ. Mature eosinophils stimulated to
develop in human cord blood mononuclear cell
cultures supplemented with recombinant human
interleukin-5. Part I. Piecemeal degranulation of
specific granules and distribution of CharcotLeyden crystal protein. Am J Pathol. 1991;138(1):
69-82.
57. Lacy P, Mahmudi-Azer S, Bablitz B, et al. Rapid
mobilization of intracellularly stored RANTES in
response to interferon-␥ in human eosinophils.
Blood. 1999;94(1):23-32.
58. Spencer LA, Melo RC, Perez SA, Bafford SP,
Dvorak AM, Weller PF. Cytokine receptormediated trafficking of preformed IL-4 in eosinophils identifies an innate immune mechanism of
cytokine secretion. Proc Natl Acad Sci U S A.
2006;103(9):3333-3338.
59. Melo RC, Spencer LA, Perez SA, Ghiran I,
Dvorak AM, Weller PF. Human eosinophils secrete preformed, granule-stored interleukin-4
through distinct vesicular compartments. Traffic.
2005;6(11):1047-1057.
60. Erjefalt JS, Greiff L, Andersson M, et al. Allergeninduced eosinophil cytolysis is a primary mechanism for granule protein release in human upper
airways. Am J Respir Crit Care Med. 1999;
160(1):304-312.
61. Ying S, Meng Q, Taborda-Barata L, et al. Human
eosinophils express messenger RNA encoding
RANTES and store and release biologically active RANTES protein. Eur J Immunol. 1996;26(1):
70-76.
62. Melo RC, Perez SA, Spencer LA, Dvorak AM,
Weller PF. Intragranular vesiculotubular compartments are involved in piecemeal degranulation by
activated human eosinophils. Traffic. 2005;6(10):
866-879.
63. Spencer LA, Szela CT, Perez SA, et al. Human
eosinophils constitutively express multiple Th1,
Th2, and immunoregulatory cytokines that are
secreted rapidly and differentially. J Leukoc Biol.
2009;85(1):117-123.
64. Piskin G, Bos JD, Teunissen MB. Neutrophils infiltrating ultraviolet B-irradiated normal human skin
display high IL-10 expression. Arch Dermatol
Res. 2005;296(7):339-342.
65. Elbim C, Reglier H, Fay M, et al. Intracellular pool
of IL-10 receptors in specific granules of human
neutrophils: differential mobilization by proinflammatory mediators. J Immunol. 2001;166(8):52015207.
66. Price KS, Friend DS, Mellor EA, De Jesus N,
Watts GF, Boyce JA. CC chemokine receptor 3
mobilizes to the surface of human mast cells and
potentiates immunoglobulin E-dependent generation of interleukin 13. Am J Respir Cell Mol Biol.
2003;28(4):420-427.
67. Lippert U, Artuc M, Grutzkau A, et al. Expression
and functional activity of the IL-8 receptor type
CXCR1 and CXCR2 on human mast cells. J Immunol. 1998;161(5):2600-2608.
68. Mortier E, Woo T, Advincula R, Gozalo S, Ma A.
IL-15R␣ chaperones IL-15 to stable dendritic cell
membrane complexes that activate NK cells via
trans presentation. J Exp Med. 2008;205(5):
1213-1225.
69. Stinchcombe JC, Griffiths GM. Secretory mechanisms in cell-mediated cytotoxicity. Annu Rev Cell
Dev Biol. 2007;23:495-517.
18
From bloodjournal.hematologylibrary.org at UNIVERSITY OF ALBERTA LIBRARY on July 7, 2011. For personal
use only.
BLOOD, 7 JULY 2011 VOLUME 118, NUMBER 1
䡠
LACY and STOW
70. Stow JL, Ching Low P, Offenhauser C, Sangermani D.
Cytokine secretion in macrophages and other cells:
pathways and mediators. Immunobiology. 2009;
214(7):601-612.
71. Shurety W, Merino-Trigo A, Brown D, Hume DA,
Stow JL. Localization and post-Golgi trafficking of
tumor necrosis factor-␣ in macrophages. J Interferon Cytokine Res. 2000;20(4):427-438.
72. Beil WJ, Weller PF, Tzizik DM, Galli SJ, Dvorak AM.
Ultrastructural immunogold localization of tumor necrosis factor-␣ to the matrix compartment of eosinophil secondary granules in patients with idiopathic
hypereosinophilic syndrome. J Histochem Cytochem. 1993;41(11):1611-1615.
73. Gordon S. The macrophage: past, present and
future. Eur J Immunol. 2007;37(S1):S9-S17.
74. Anderson P, Phillips K, Stoecklin G, Kedersha N.
Post-transcriptional regulation of proinflammatory
proteins. J Leukoc Biol. 2004;76(1):42-47.
75. Goud B, Gleeson PA. TGN golgins, Rabs and
cytoskeleton: regulating the Golgi trafficking highways. Trends Cell Biol. 2010;20(6):329-336.
76. Bard F, Malhotra V. The formation of TGN-toplasma-membrane transport carriers. Annu Rev
Cell Dev Biol. 2006;22:439-455.
77. Tian Y, Pate C, Andreolotti A, et al. Cytokine secretion requires phosphatidylcholine synthesis.
J Cell Biol. 2008;181(6):945-957.
78. van Ijzendoorn SC. Recycling endosomes. J Cell
Sci. 2006;119(pt 9):1679-1681.
79. Lock JG, Stow JL. Rab11 in recycling endosomes
regulates the sorting and basolateral transport of
E-cadherin. Mol Biol Cell. 2005;16(4):1744-1755.
80. Ang AL, Taguchi T, Francis S, et al. Recycling endosomes can serve as intermediates during
transport from the Golgi to the plasma membrane
of MDCK cells. J Cell Biol. 2004;167(3):531-543.
81. Hulse RE, Swenson WG, Kunkler PE, White DM,
Kraig RP. Monomeric IgG is neuroprotective via
enhancing microglial recycling endocytosis and
TNF-␣. J Neurosci. 2008;28(47):12199-12211.
82. Pulecio J, Petrovic J, Prete F, et al. Cdc42mediated MTOC polarization in dendritic cells
controls targeted delivery of cytokines at the immune synapse. J Exp Med. 2010;207(12):27192732.
83. Mazzolini J, Herit F, Bouchet J, Benmerah A,
Benichou S, Niedergang F. Inhibition of phagocytosis in HIV-1-infected macrophages relies on
Nef-dependent alteration of focal delivery of recycling compartments. Blood. 2010;115(21):42264236.
84. Huynh KK, Kay JG, Stow JL, Grinstein S. Fusion,
fission, and secretion during phagocytosis. Physiology. 2007;22:366-372.
85. Zhang Q, Cox D, Tseng C-C, Donaldson JG,
Greenberg S. A requirement for ARF6 in Fc␥ receptor-mediated phagocytosis in macrophages.
J Biol Chem. 1998;273(32):19977-19981.
86. Cox D, Lee DJ, Dale BM, Calafat J, Greenberg S.
A Rab11-containing rapidly recycling compartment in macrophages that promotes phagocytosis. Proc Natl Acad Sci U S A. 2000;97(2):680685.
87. Bajno L, Peng XR, Schreiber AD, Moore HP,
Trimble WS, Grinstein S. Focal exocytosis of
VAMP3-containing vesicles at sites of phagosome formation. J Cell Biol. 2000;149(3):697706.
88. Niedergang F, Colucci-Guyon E, Dubois T,
Raposo G, Chavrier P. ADP ribosylation factor 6
is activated and controls membrane delivery during phagocytosis in macrophages. J Cell Biol.
2003;161(6):1143-1150.
89. Allen LA, Yang C, Pessin JE. Rate and extent of
phagocytosis in macrophages lacking vamp3.
J Leukoc Biol. 2002;72(1):217-221.
90. Nickel W. The mystery of nonclassical protein secretion. A current view on cargo proteins and potential export routes. Eur J Biochem. 2003;
270(10):2109-2119.
91. Eder C. Mechanisms of interleukin-1␤ release.
Immunobiology. 2009;214(7):543-553.
92. Flieger O, Engling A, Bucala R, Lue H, Nickel W,
Bernhagen J. Regulated secretion of macrophage migration inhibitory factor is mediated by a
non-classical pathway involving an ABC transporter. FEBS Lett. 2003;551(1-3):78-86.
93. Nakamura K, Okamura H, Wada M, Nagata K,
Tamura T. Endotoxin-induced serum factor that
stimulates gamma interferon production. Infect
Immun. 1989;57(2):590-595.
94. Blott EJ, Griffiths GM. Secretory lysosomes. Nat
Rev Mol Cell Biol. 2002;3(2):122-131.
95. Semino C, Angelini G, Poggi A, Rubartelli A.
NK/iDC interaction results in IL-18 secretion by
DCs at the synaptic cleft followed by NK cell activation and release of the DC maturation factor
HMGB1. Blood. 2005;106(2):609-616.
96. Neves JS, Perez SA, Spencer LA, et al. Eosinophil granules function extracellularly as receptormediated secretory organelles. Proc Natl Acad
Sci U S A. 2008;105(47):18478-18483.
97. Apostolaki M, Armaka M, Victoratos P, Kollias G.
Cellular mechanisms of TNF function in models of
inflammation and autoimmunity. Curr Dir Autoimmun. 2010;11:1-26.
98. Taylor PC, Feldmann M. Anti-TNF biologic agents:
still the therapy of choice for rheumatoid arthritis. Nat
Rev Rheumatol. 2009;5(10):578-582.
99. Huse M, Quann EJ, Davis MM. Shouts, whispers
and the kiss of death: directional secretion in
T cells. Nat Immunol. 2008;9(10):1105-1111.
100. Collinet C, Stoter M, Bradshaw CR, et al. Systems survey of endocytosis by multiparametric
image analysis. Nature. 2010;464(7286):243249.
101. Sprenger J, Lynn Fink J, Karunaratne S, Hanson K,
Hamilton NA, Teasdale RD. LOCATE: a mammalian
protein subcellular localization database. Nucleic
Acids Res. 2008;36(Database issue):D230-D233.
102. Zielinska DF, Gnad F, Wisniewski JR, Mann M.
Precision mapping of an in vivo N-glycoproteome
reveals rigid topological and sequence constraints. Cell. 2010;141(5):897-907.
103. Walter T, Shattuck DW, Baldock R, et al. Visualization of image data from cells to organisms. Nat
Methods. 2010;7(3 Suppl):S26-S41.
104. Han Q, Bradshaw EM, Nilsson B, Hafler DA,
Love JC. Multidimensional analysis of the frequencies and rates of cytokine secretion from
single cells by quantitative microengraving. Lab
Chip. 2010;10(11):1391-1400.
105. Simons K, Gerl MJ. Revitalizing membrane rafts:
new tools and insights. Nat Rev Mol Cell Biol.
2010;11(10):688-699.