© 2008 Nature Publishing Group http://www.nature.com/natureimmunology
ARTICLES
Silica crystals and aluminum salts activate the NALP3
inflammasome through phagosomal destabilization
Veit Hornung1,5, Franz Bauernfeind2,5, Annett Halle1, Eivind O Samstad1,3, Hajime Kono4, Kenneth L Rock4,
Katherine A Fitzgerald1 & Eicke Latz1,3
Inhalation of silica crystals causes inflammation in the alveolar space. Prolonged exposure to silica can lead to the development
of silicosis, an irreversible, fibrotic pulmonary disease. The mechanisms by which silica and other crystals activate immune
cells are not well understood. Here we demonstrate that silica and aluminum salt crystals activated inflammasomes formed by
the cytoplasmic receptor NALP3. NALP3 activation required phagocytosis of crystals, and this uptake subsequently led to
lysosomal damage and rupture. ‘Sterile’ lysosomal damage (without crystals) also induced NALP3 activation, and inhibition
of either phagosomal acidification or cathepsin B activity impaired NALP3 activation. Our results indicate that the NALP3
inflammasome senses lysosomal damage as an endogenous ‘danger’ signal.
Crystalline silica, also known as silicon dioxide (SiO2), is found in
nature as sand or quartz. Although ingestion of silica is harmless and
nontoxic, inhalation of small silica crystals can lead to acute lung
inflammation. Chronic, mostly occupational, inhalative exposure to
silica crystals causes the pneumoconiosis silicosis. Silicosis is an
incurable, irreversible, progressive lung fibrosis that is associated
with other diseases, including cardiopulmonary failure, mycobacterial
infection, autoimmunity and lung cancer. No effective treatment is
available for silicosis, and the mechanisms of silica crystal–induced
lung inflammation and fibrosis are not well understood1.
Inhalation of silica crystals leads to their deposition in small airways
of the lung, where mucocilial clearance is not functional. Subsequent
ingestion of silica crystals by resident macrophages elicits an inflammatory response characterized by the release of cytokines, such as
interleukin 1b (IL-1b; A003663) and tumor necrosis factor, and
factors that can induce fibrosis. Moreover, uptake of silica crystals
can initiate apoptotic cell death with concomitant release of ingested
silica particles, which are re-engulfed by other alveolar macrophages,
inducing a cycle of sustained inflammation2.
Reports have shown that other medically relevant crystals, such as
crystals of monosodium urate (MSU) or calcium pyrophosphate
dihydrate, which trigger inflammation in gout or pseudogout, respectively, can activate the cytoplasmic receptor NALP3 (also called
cryopyrin or NLRP3)3. NALP3 is a member of a family of cytoplasmic
Nod-like receptors that control the activity of inflammatory caspase-1
by forming multimolecular complexes called ‘inflammasomes’4. After
being activated, NALP3 recruits the adaptor molecule ASC, which in
turn binds to procaspase-1, leading to its autocatalytic processing and
activation5. Active caspase-1 catalyzes cleavage of the procytokines
IL-1b and IL-18, which are secreted and biologically active only in
their processed forms6. The ‘upstream’ mechanisms of activation of
the NALP3 inflammasome are poorly understood. Several ligands of
diverse physicochemical nature have been reported to activate the
NALP3 inflammasome. For example, in addition to crystaline MSU
and calcium pyrophosphate dihydrate, bacterial cell-wall components,
several bacterial toxins and high concentrations of extracellular ATP
are all reported to activate the NALP3 inflammasome4.
Here we investigate the mechanisms of crystal-induced inflammation and demonstrate that like MSU, silica crystals and aluminum salt
(alum) crystals activated the NALP3 inflammasome. We further found
that NALP3 activation by crystals required phagocytosis and that
crystals taken up by phagocytosis induced lysosomal swelling and
damage. NALP3 activation by silica crystals was dependent on
lysosomal acidification and involved the lysosomal cysteine protease
cathepsin B. Crystal-independent lysosomal damage was sufficient to
induce NALP3 inflammation. Our results suggest a common mechanism of crystal-induced activation of the NALP3 inflammasome
whereby lyosomal perturbation, not the crystal structure itself, is
being sensed.
RESULTS
Silica induces IL-1b maturation and caspase-1 activation
The NALP3 inflammasome recognizes crystalline material appearing
in joint fluids as a ‘danger’ signal3. Inhalation of silica crystals is
known to induce a strong proinflammatory response, including the
release of active IL-1b. We thus hypothesized that silica crystals could
1Department of Infectious Diseases and Immunology, University of Massachusetts Medical School, Worcester, Massachusetts 01605, USA. 2Division of Clinical
Pharmacology, Department of Internal Medicine, Ludwig-Maximilians University of Munich 80336, Germany. 3Institute of Cancer Research and Molecular Medicine,
Norwegian University of Science and Technology, N-7489 Trondheim, Norway. 4Department of Pathology, University of Massachusetts Medical School, Worcester,
Massachusetts 01605, USA. 5These authors contributed equally to this work. Correspondence should be addressed to: E.L. ([email protected]).
Received 10 April; accepted 10 June; published online 11 July 2008; doi:10.1038/ni.1631
NATURE IMMUNOLOGY
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800
8
6
4
2
200
(kDa)
17
.5
12
5
.3
62
0
.6
31
15
.5
12
5
15
0
AT
P
dA
dT
0
None
50
0
25
0
1,
00
0
50
e
Silica
(µg/ml)
.3
10
0
IB: cleaved
IL-1β
0
.6
IB: cleaved
caspase-1
25
Silica (µg/ml)
– priming
17
on
+ priming
400
(kDa)
0
IB: cleaved
IL-1β
N
0
3.
9
7.
8
15
.6
31
.3
62
.5
12
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25
0
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5
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(kDa)
17
600
62
IL-1β (ng/ml)
10
IL-1β (ng/ml)
z-YVAD-fmk
Silica (µg/ml)
MSU
(µg/ml)
Figure 1 Silica induction of the release of mature IL–1b and activated caspase-1 by human PBMCs is caspase-1 dependent. ELISA (above) and immunoblot
analysis (IB; below) of the production of IL-1b (a–c) or caspase-1 (b) by human PBMCs. (a) PBMCs primed for 3 h with LPS (25 pg/ml; + priming) or left
untreated (– priming) and then stimulated for an additional 6 h with silica crystals (Silica). Each dot above a lane represents one donor; small horizontal
lines indicate the mean. kDa, kilodaltons. Data are from two experiments with four independent donors (ELISA) and or one representative donor
(immunoblot). (b) LPS-primed PBMCs left unstimulated (None) or stimulated with silica crystals, MSU crystals (MSU) or ATP or transfected with poly(dA:dT)
(dAdT) and then incubated for 6 h. Data are from two experiments with one representative donor of three. (c) LPS-primed PBMCs stimulated with silica
crystals in the presence (z-YVAD-fmk) or absence (None) of the caspase-1 inhibitor z-YVAD (10 mM) and then incubated for 6 h. Results are the mean and
s.d. of two independent donors, presented as the ‘fold increase’ relative to only LPS priming (ELISA), or are from one representative donor (immunoblot).
Data are representative of two experiments.
848
Silica crystals activate the NALP3 inflammasome
To investigate whether silica crystals can activate the NALP3 inflammasome, we did experiments with macrophages from mice deficient
b
n=5
1.25
1.0
0.75
0.5
n=5
n=3
n=4
10
n=4
n=4
8
6
n=4
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W
T
yD
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-K
O
IL
1R
-K
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PBS
W
T
yD
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TR 8-K
IF O
-K
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1R
-K
O
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Neutrophils in lavage
(× 106)
a
T
Silica induces influx of neutrophils into the lung via IL-1b
After exposed people inhale silica crystal dust, lung-resident immune
cells subsequently engulf the crystal material by phagocytosis and
induce an inflammatory response. Large doses of silica dust lead to the
clinical syndrome of acute silicosis, characterized by the rapid influx of
immune cells into the exposed area and massive production of
chemokines and proinflammatory cytokines, including IL-1b2. To
investigate the in vivo relevance of the IL-1 response in silica-induced
lung inflammation, we transorally instilled silica crystals into wildtype and IL-1 receptor (IL-1R)-deficient mice and then monitored the
mice for acute inflammation after 16–18 h. We counted the cells in
bronchoalveolar lavage fluid and assessed their phenotype by flow
cytometry. We detected much more infiltration of neutrophils in lungs
of wild-type mice exposed to silica crystals than in those of mice
treated with the carrier fluid; this did not occur in IL-1R-deficient
mice exposed to silica crystal (Fig. 2a). Other cell types remained
mostly unchanged (data not shown).
We noted almost complete abrogation of silica-induced influx of
neutrophils in mice doubly deficient in the adaptor molecules MyD88
(A003535) and TRIF, whereas absence of these molecules had little or
no effect on zymosan-induced inflammation (Fig. 2b). Commercial
zymosan preparations are known to activate signaling pathways
dependent on Toll-like receptor 2 and dectin 1 (refs. 8,9), which
could explain the lower zymosan-induced neutrophil recruitment.
These results collectively indicate that silica crystals induce IL-1 release
and that the IL-1 signal–transduction molecules IL-1R and MyD88 are
critical in the development of acute inflammation in vivo after
exposure to silica crystal.
W
exert their inflammatory potential through the activation of a
similar pathway. We incubated human peripheral blood mononuclear
cells (PBMCs) from several donors with baked, lipopolysaccharide
(LPS)-free silica crystals. Pro-IL-1b is not constitutively expressed
and requires transcriptional induction in response to, for example, a
Toll-like receptor stimulus. Although silica crystals did not induce
cleavage and release of IL-1b in human PBMCs by themselves,
LPS-primed PBMCs strongly responded to the addition of silica
crystals in a dose-dependent way (Fig. 1a). Silica crystal–mediated
activation of human PBMCs was as potent as other known activators
of the NALP3 inflammasome, such as MSU crystals, ATP or the
NALP3-independent stimulus of transfected double-stranded
DNA7 (poly(dA:dT); Fig. 1b). IL-1b production, as measured by
enzyme-linked immunosorbent assay (ELISA), correlated strongly
with the detection of cleaved IL-1b and caspase-1 by immunoblot
analysis, which indicated that activated cells released mature IL-1b.
Inhibition of caspase-1 by the specific peptide inhibitor z-YVAD
almost completely abolished the release of IL-1b in response to
treatment with silica crystals (Fig. 1c). These data suggest that silica
crystals activate IL-1b in a caspase-1-dependent way in human
immune cells.
Neutrophils in lavage
(× 106)
© 2008 Nature Publishing Group http://www.nature.com/natureimmunology
c
12
31
b
45
40
35
30
25
20
15
10
5
0
IB: cleaved
IL-1β
IL-1β (‘fold induction’)
a
Silica
Zymosan
Figure 2 Silica-mediated neutrophil influx in a model of acute lung
inflammation is mediated by IL-1. Flow cytometry of neutrophils in lung
lavage fluid from wild-type mice (WT), mice doubly deficient in MyD88 and
TRIF (MyD88-KO TRIF-KO) or IL-1R-deficient mice (IL-1R-KO) assessed
16–18 h after orotracheal administration of PBS or silica crystals (200 mg
per mouse; a) or zymosan (50 mg per mouse; b). Numbers above bars
indicate number of mice per group. Data are from one representative
experiment of two (error bars, s.d.).
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WT (C57BL6)
NALP3-KO
Lysate
ASC-KO
1.2
1.2
1.2
1.0
1.0
0.8
0.6
0.4
0.8
0.6
0.4
0.8
0.6
0.4
Supernatants
IL-1β (ng/ml)
1.2
IL-1β (ng/ml)
1.4
1.0
Supernatants
IL-1β (ng/ml)
1.4
IL-1β (ng/ml)
1.4
Lysate
1.4
1.0
0.8
0.6
0.4
Lysate
1.4
1.4
1.2
1.2
1.0
1.0
0.8
0.6
0.4
0.8
0.6
0.4
0
0
Silica
(µg/ml)
Silica
(µg/ml)
b
NUMBER 8
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0
50
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0
50
0
AT
P
dA
dT
e
VOLUME 9
25
on
N
in NALP3 or the ‘downstream’ adaptor molecule ASC. Similar to their
response to known inflammasome activators such as MSU crystals,
ATP or transfected poly(dA:dT), macrophages from wild-type mice
produced large amounts of IL-1b after exposure to silica crystals
(Fig. 3a, left). In contrast, macrophages lacking NALP3 or ASC failed
to release cleaved IL-1b in response to silica crystals (Fig. 3a), which
indicated a requirement for NALP3 and ASC in the processing of
IL-1b after exposure to silica crystals. Consistent with published
reports3,7,10,11, responses to MSU crystals and ATP were dependent
on both NALP3 and ASC, whereas the response to transfected
poly(dA:dT) was ASC dependent yet NALP3 independent (Fig. 3a,
middle and left). As IL-1b processing and release could both
be influenced by activation by silica crystals, we next examined the
cleavage of procaspase-1 into active caspase-1. We found that like the
release of IL-1b, caspase-1 cleavage was induced in a dose-dependent
way after treatment with silica crystals, MSU, ATP or poly(dA:dT)
(Fig. 3b). This response was completely dependent on ASC for all
stimuli, as ASC-deficient macrophages failed to trigger caspase-1
cleavage in response to any of these ligands. Consistent with the
IL-1b release, caspase-1 cleavage in response to poly(dA:dT) was
independent of NALP3. A published report has suggested uric acid
released from cells is a chief trigger for IL-1b production after
stimulation of cells with other crystal preparations12. We assessed
whether uricase influenced the IL-1b response to MSU or silica
crystals and found that the response to silica was unimpaired after
incubation with uricase (Fig. 3c). We also generated immortalized
macrophage cell lines from wild-type, NALP3- and ASC-deficient
mice and examined their response to inflammasome ligands. Immortalized macrophages had responses qualitatively similar to those of
freshly isolated bone marrow–derived macrophages (Supplementary
Fig. 1 online). These results collectively suggest that silica crystals
activate the NALP3-ASC complex, leading to the activation of caspase1 and subsequent cleavage of pro-IL-1b into mature, secreted IL-1b.
10
10
10
on
Silica (250 µg/ml)
Silica (125 µg/ml)
(kDa)
Figure 3 Silica-mediated release of matured IL-1b and activated
caspase-1 is mediated by the NALP3 inflammasome. (a) ELISA
of IL-1b production by bone marrow–derived wild-type, NALP3deficient or ASC-deficient macrophages primed for 3 h with LPS
and then left unstimulated or stimulated with silica crystals,
MSU crystals or ATP or transfected with poly(dA:dT); IL-1b was
analyzed supernatants 6 h after stimulation (Supernatants) and
intracellular pro-IL-1b was assessed in lysates of primed but
Silica
MSU
unstimulated cells (Lysate). (b) Immunoblot analysis of the
(µg/ml)
(µg/ml)
cleavage of caspase-1 to its active p10 subunit in cells treated
as described in a. (c) ELISA of IL-1b production by LPS-primed
mouse macrophages stimulated with silica or MSU crystals or ATP in the presence or absence of uricase, presented
(% max). Data are from one representative experiment of two (error bars, s.d.).
NATURE IMMUNOLOGY
MSU
(µg/ml)
c
WT (C57BL6)
IB: caspase-1
p10
NALP3-KO
IB: caspase-1
p10
ASC-KO
IB: caspase-1
p10
N
0
on
25
N
e
on
N
MSU
(µg/ml)
MSU
ATP
125
IL-1β response (% max)
MSU
(µg/ml)
N
on
N
on
25
Silica
(µg/ml)
e
0
50
1, 0
00
0
25
0
50
0
AT
P
dA
dT
0
e
0
on
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00
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dT
0.2
0
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P
dA
dT
0.2
50
1, 0
00
0
25
0
0.2
e
0.2
0
0.2
N
© 2008 Nature Publishing Group http://www.nature.com/natureimmunology
IL-1β (ng/ml)
Supernatants
IL-1β (ng/ml)
a
100
75
50
25
0
0.1
1
Uricase (µg/ml)
10
as the percent of maximum production
Activation of the NALP3 inflammasome requires crystal uptake
To delineate the ‘upstream’ mechanisms involved in silica crystal–
induced activation of the NALP3 inflammasome, we first determined
if uptake of crystalline inflammasome activators influenced cell
activation. We pretreated human PBMCs with cytochalasin D, a well
characterized inhibitor of phagocytosis that impairs actin filament
assembly, and then stimulated the cells with MSU or silica crystals as
well as with two noncrystalline NALP3 activators, the imidazoquinoline derivative R-848 and ATP13. Cytochalasin D completely
abrogated IL-1b release after treatment with MSU or silica crystals,
whereas the response to R-848 or ATP was unaffected (Fig. 4a). We
obtained similar results with mouse macrophages (Supplementary
Fig. 2a online). These results indicate crystal binding and uptake by
phagocytosis are prerequisites for activation of the cytosolic
NALP3 inflammasome.
To visualize the uptake of crystalline material in living cells, we
devised a method to image crystals simultaneously with cellular
components stained with fluorescent dyes (Supplementary Fig. 3
online). We used this method to study the uptake of crystals into
mouse macrophages. After incubating cells with silica crystals for
30 min, we stained the cell surface with the membrane-binding
reagent fluorescent cholera toxin B-subunit in the presence or absence
of cytochalasin D. Macrophages rapidly engulfed crystals into intracellular compartments by phagocytosis, whereas cells treated with
cytochalasin D failed to engulf silica crystals (Fig. 4b) or MSU crystals
(Supplementary Fig. 2b) by phagocytosis. The median length of silica
crystals engulfed by phagocytosis was 1.65 mm (Fig. 4c).
It is well known that phagocytosis of particulate matter in macrophages results in the generation of reactive oxygen species. Silica
crystals trigger the production of reactive oxygen species in macrophages, events reported to be linked to disease pathology in silicainduced inflammation14. On the basis of those reports and our
own data emphasizing the importance of phagocytosis in
849
(8-chloromethyl-4,4-difluoro-1,3,5,7-tetramethyl-4-bora-3a,4a-diaza-sindacene) on DQ ovalbumin is normally quenched unless the protein
is proteolytically processed into peptides in endo-lysosomal compartments. In untreated cells, processed DQ ovalbumin was localized to
small vesicular and tubular endosomes or lysosomes, as expected. In
contrast, we detected large swollen lysosomes in most cells exposed to
silica crystals. Many cells had a cytosolic pattern of fluorescently
processed DQ ovalbumin, which indicated lysosomal rupture or
leakage of lysosomal contents into the cytosol of crystal-treated cells
(Fig. 5a). Fluorescent indicator dyes that stain acidic environments
confirmed those findings (data not shown). In addition, ingested
fluorescent dextran, which traffics into the lysosomal pathway, also
stained swollen lysosomal compartments and showed cytosolic translocation in most cells (Supplementary Fig. 4 online). Such swelling of
lysosomes was not exclusive to silica crystal–treated cells, as MSU
crystals elicited similar morphological changes and rupture of lysosomal compartments (data not shown).
We also tested if crystals could be detected in cells outside
phagosomal compartments by staining the plasma membrane and
phagosomal membranes of fixed and permeabilized cells with
silica crystal–induced NALP3 activation, we hypothesized that the
phagocyte reactive oxygen species system might be involved in this
response. To address the involvement of this pathway directly, we
examined NALP3 inflammasome responses in mice lacking gp91phox,
the 91-kilodalton subunit of the phagosomal NADPH oxidase cytochrome b. Mice deficient in this subunit lack phagocyte superoxide
production and are reported to be hypersusceptible to various pathogens15. However, macrophages from these mice responded normally
to silica crystals, MSU, ATP and poly(dA:dT) (Fig. 4d,e). These results
indicate that the phagosomal respiratory-burst oxidase system is not
essential for activation of the NALP3 inflammasome by these stimuli.
Phagocytosis of crystals leads to lysosomal destabilization
We next did imaging studies with macrophages undergoing phagocytosis of crystalline material to monitor the fate of the cargo
engulfed. We used confocal reflection microscopy combined with
fluorescence imaging to study the subcellular distribution of silica
crystals over time (Supplementary Fig. 3). DQ ovalbumin is a useful
tool for monitoring the endo-lysosomal compartment in living cells
in real time. The fluorescence of the fluorophore BODIPY-FL
a
None
b
R-848
MSU
crystals
ATP
c
Silica
crystals
20
0.16
18
Relative frequency
16
IL-1β (ng/ml)
14
12
10
+ cytochalasin D
8
6
0.12
0.08
0.04
4
2
0
0
MSU
(µg/ml)
e
0.
3
0.
6
1.
3
2.
5
5
N
on
e
0.
3
0.
6
1.
3
2.
5
5
1.0
0.5
0.5
0
0
Silica
(µg/ml)
MSU
(µg/ml)
Silica
(µg/ml)
10
10
50
1, 0
00
0
25
0
50
0
AT
P
dA
dT
1.5
1.0
e
2.0
0
1.5
2.5
(kDa)
10
on
2.0
WT (C57BL/6)
IB: caspase-1
p10
gp91phox-KO
IB: caspase-1
p10
Ipaf-KO
IB: caspase-1
p10
3.0
25
3.5
2.5
N
on
e
25
0
50
1, 0
00
0
25
0
50
0
AT
dA P
dT
0
3.0
e
on
e
25
0
50
0
1,
00
0
25
0
50
0
AT
P
dA
dT
0.5
4.0
IL-1β (ng/ml)
1.0
1
2
3
4
>5
Silica crystal length (µm)
Reflection (silica)
Cholera toxin B
Hoechst
Ipaf-KO
4.5
3.5
on
e
25
0
50
1, 0
00
0
25
0
50
0
AT
P
dA
dT
IL-1β (ng/ml)
1.5
Cytochalasin D
(µM)
gp91phox-KO
4.0
2.0
IL-1β (ng/ml)
Cytochalasin D
(µM)
N
WT (C57BL/6)
Silica
(µg/ml)
on
N
Cytochalasin D
(µM)
N
2.5
0.
6
1.
3
2.
5
5
e
3
Cytochalasin D
(µM)
d
0.
on
N
0.
6
1.
3
2.
5
5
0.
N
on
e
3
0
N
© 2008 Nature Publishing Group http://www.nature.com/natureimmunology
ARTICLES
Silica
(µg/ml)
MSU
(µg/ml)
MSU
(µg/ml)
Figure 4 Inflammasome activation requires phagosomal uptake of crystals but is independent of the phagosomal reactive oxygen species system. (a) ELISA
of the release of IL-1b by human LPS-primed PBMCs left untreated (None) or treated with increasing doses of cytochalasin D and then stimulated for 6 h
with R-848, ATP, silica crystals or MSU crystals. (b) Confocal microscopy of C57BL/6 (wild-type) macrophage cells stimulated for 2 h with silica crystals in
the presence (below) or absence (above) of cytochalasin D (2.5 mM); cell membranes were stained with fluorescent cholera toxin B-subunit (red) and nuclei
were stained with Hoechst dye (blue), followed by analysis of crystal uptake (green). Scale bars, 5 mm. (c) Length of silica crystals taken up by phagocytosis
in C57BL/6 (wild-type) macrophages (n ¼ 10) stimulated for 2 h with silica crystals. (d,e) ELISA of IL-1b in supernatants (d) and immunoblot analysis of
activated caspase-1 in lysates (e) of bone marrow–derived wild-type macrophages (WT), gp91phox-deficient macrophages (gp91phox-KO) or gp91phoxsufficient, Ipaf-deficient macrophages (Ipaf-KO; mixed background control) primed for 3 h with LPS and then left unstimulated or stimulated for 6 h
with silica crystals, MSU crystals or ATP or transfected with poly(dA:dT). Data (error bars, s.d.) are from one representative experiment of two (a,c–e)
or three (b).
850
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ARTICLES
Silica
Reflection (silica)
Cholera toxin B
DQ ovalbumin
2.3
3.8
d
5.8
14.8
No crystals
Cells
NALP3-KO
Silica
2.7
2
4.2
3
4
5
2
7.2
3
4
5
0 10 10 10 10
0 10 10 10 10
No crystals
Silica
(125 µg/ml)
Reflection (silica)
Hoechst
Cholera toxin B
2
15.8
3
4
5
0 10 10 10 10
2
3
4
5
0 10 10 10 10
Silica
(250 µg/ml)
LysoSensor
45
40
35
30
25
20
15
10
5
0
None 0
Silica
(500 µg/ml)
5 50 250
Bafilomycin (nM)
Lysosomal acridine orange fluorescence
e
Fluorescence intensity (×102)
+ control
+ bafilomycin
45
40
f
800
None
Silica
(250 µg/ml)
ATP
700
Silica
(125 µg/ml)
None
N
on
e
50
25 nM
0
nM
N
on
e
50
n
25 M
0
nM
acridine orange in lysosomes directly correlates with the amount of
acidic lysosomes in cells. We took advantage of this property and
monitored the lysosomal content of macrophages before and after
treatment with silica crystals. Increasing amounts of silica crystals
resulted in loss of lysosomes, as indicated by less red fluorescence of
None
Cath B inhibitor
2.0
b
(kDa)
- 35
- 27
WT (C57BL6)
1.6
IB: Cath B
1.2
NALP3-KO
IB: Cath B
0.8
- 35
- 27
on
e
25
0
50
1, 0
00
0
25
0
50
AT 0
dA P
dT
a
0.4
N
fluorescent cholera toxin B-subunit. Most crystals were located in
phagosomes that had distinct membrane staining; however, some
crystals were in regions were not associated with membranes and
thus were translocated to the cytosol (Fig. 5b). We obtained similar
data when we examined MSU crystals (data not shown). To quantify
the degree of phagosomal rupture after crystal treatment, we used
acridine orange, a dye used to measure lysosomal integrity. Acridine
orange fluoresces green in its monomeric state and binds to nuclear
and cytosolic DNA and RNA. Because of its cationic nature, acridine
orange becomes highly concentrated in acidic compartments, resulting
in the formation of dimers and the appearance of red fluorescence
(Supplementary Fig. 5 online). The amount of red fluorescence of
N
on
e
50
25 nM
0
nM
N
on
e
50
25 nM
0
nM
IL-1β (pg/ml)
35
600
Figure 5 Phagocytosis of crystals leads to lysosomal
Bafilomycin
30
destabilization. (a,b) Confocal microscopy of wild-type
500
25
(C57BL/6) macrophages incubated for 60 min with DQ
400
20
ovalbumin (10 mg/ml; red) alone or together with silica crystals
300
15
(green), then stained with fluorescent cholera toxin B-subunit
200
10
(blue; a); or left untreated or incubated for 60 min with silica
100
5
crystals (green), then fixed, made permeable (0.01% (vol/vol)
0
0
saponin) and stained with fluorescent cholera toxin B-subunit
0 0.6 2.5 10 40
(red) and Hoechst dye (blue; b). Original magnification, 180
DQ ovalbumin (µg/ml)
(main images) with enlargement of 2 (a) or 1.8 (b) at far
right for boxed areas in adjacent main images. (c) Flow
cytometry of wild-type and NALP3-deficient macrophages stained with acridine orange and then treated for 3 h with silica crystals. Numbers above bracketed
lines indicate percent cells with loss of lysosomal staining with acridine orange (excitation, 488 nm; emission, 650–690 nm). (d,e) Flow cytometry of wildtype macrophages treated with bafilomycin and left unstained (None) or stained with 1 mM LysoSensor Green immediately before flow cytometry (d), or
incubated for 60 min with DQ ovalbumin in the presence (+ bafilomycin) or absence (+ control (dimethyl sulfoxide)) of 250 nM bafilomycin (e). (f) ELISA of
the release of IL-1b from LPS-primed wild-type macrophages treated with bafilomycin or left untreated and then stimulated for 6 h with ATP or silica
crystals. Data are from one representative experiment of three (a,b) or two (c–f; error bars, s.d.).
IL-1β (ng/ml)
© 2008 Nature Publishing Group http://www.nature.com/natureimmunology
b
WT
c
Fluorescence intensity (×102)
No crystals
Cells
a
Figure 6 Silica-mediated IL-1b production is partially dependent on
cathepsin B. (a) ELISA of IL-1b in LPS-primed wild-type macrophage cells
left untreated (None) or treated with the cathepsin B (Cath B) inhibitor
CA-074-Me (10 mM) and then left unstimulated (None) or stimulated with
silica crystals or ATP or transfected with poly(dA:dT). (b) Immunoblot
analysis of cathepsin B in supernatants of wild-type or NALP3-deficient
bone marrow–derived macrophages primed for 3 h with LPS and then left
unstimulated or stimulated for 6 h with silica crystals, MSU crystals or
ATP or transfected with poly(dA:dT). (c) Confocal microscopy of wild-type
macrophages left untreated (left) or incubated for 3 h with silica crystals
(pink; right), then stained for 1 h with fluorescent probes for activated
caspase-1 (green) and activated cathepsin B (red) and then surface-stained
with fluorescent cholera toxin B-subunit (blue). Boxed areas in main images
at right are enlarged along the far right margin (2, top; 1.5, bottom).
Scale bars, 5 mm. Data are from one representative experiment of two (a
(error bars, s.d.), b) or three (c).
NATURE IMMUNOLOGY
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c
None
125 250 500
Silica (µg/ml)
No crystals
Cholera toxin B
Caspase-1
Cathepsin B
Reflection
Silica MSU
(µg/ml) (µg/ml)
dAdT ATP
Silica
Cholera toxin B
Caspase-1
Cathepsin B
Reflection (silica crystals)
851
acridine orange (Fig. 5c, top). Furthermore, we found lysosomal
rupture due to silica phagocytosis to a similar extent in cells deficient
in NALP3 and wild-type cells, which indicated that lysosomal damage
was independent of NALP3 (Fig. 5c, bottom). Collectively, these data
show that phagocytosis of crystalline material leads to active swelling
of phagosomes, followed by phagosomal destabilization and rupture,
thus resulting in the release of phagosomal contents, which then gain
access to the cytosolic compartment.
Lysosomes contain a plethora of proteolytic enzymes, many
of which are activated by acidification of lysosomal pathways.
To assess the function of lysosomal acidification in silica-mediated
NALP3 activation, we used bafilomycin A to block the vacuolar
H+ ATPase system, which is required for the acidification of lysosomal compartments. As expected, bafilomycin A blocked the formation of acidic lysosomes in macrophages, as assessed with the
pH-sensitive dye LysoSensor Green, which fluoresces only after
accumulation in acidic environments (Fig. 5d). In addition, bafilomycin A suppressed the rapid, dose-dependent appearance of fluorescent DQ ovalbumin (Fig. 5e), which indicated lower lysosomal
proteolytic function. Notably, bafilomyin A completely blocked silicamediated IL-1b release but had no effect on ATP (Fig. 5f), which
suggested a pivotal function for lysosomes in crystal-mediated
NALP3 activation.
Alum (500 µg/ml)
1.0
0.8
0.6
0.4
0.2
Alum
f
Alum
DQ ovalbumin
Cholera toxin B
8
6
4
2
T
-K
O
W
R
-K
O
-K
O
C
DQ ovalbumin
Cholera toxin B
10
AS
T
W
P3
Alum
g
1.0
Alum
(500 µg/ml)
Alum
(250 µg/ml)
Alum
IL-1β (ng/ml)
0.8
0.6
0.4
0.2
120
100
80
60
40
20
0
0
B No
in n
Ba hib e
fil ito
om r
yc
in
C
at
h
B No
in n
Ba hib e
fil ito
om r
yc
in
Caspase-1
Cathepsin B
0.1
1
10
Uricase (µg/ml)
h
Caspase-1
Cathepsin B
Cholera toxin B
Alum
at
Acridine orange
DNA and RNA
Lysosomes
Alum
C
Acridine orange
DNA and RNA
Lysosomes
MSU (125 µg/ml)
MSU (250 µg/ml)
Alum (125 µg/ml)
Alum (250 µg/ml)
h
IL-1β response (% max)
None
None
N
– priming
Alum (µg/ml)
e
d
0
0
7.
8
15
.6
31
.3
62
.5
12
5
25
0
50
0
12
5
25
0
50
0
0
9
3.
+ priming
Alum
(100 µg)
PBS
-1
IL-1β (ng/ml)
1.2
(kDa)
-17
12
Neutrophils (× 106)
1.4
c
IL
1.6
T
b
AL
9
8
7
6
5
4
3
2
1
0
IB: cleaved
IL-1β
Lysosomal destabilization triggers inflammasome activation
Because one important function of lysosomal acidification is the
pH-dependent activation of proenzymes to further degrade lysosomal
contents, we hypothesized that activation of lysosomal proenzymes
could be a critical event in silica-mediated inflammasome activation.
In particular, the cathepsin family of proenzymes was a likely
candidate. To address their function in silica-mediated inflammasome
activation, we tested several cathepsin inhibitors for their ability to
affect silica-mediated IL-1b release. Among the inhibitors tested,
CA-074-Me, a cathepsin B–specific inhibitor16, led to much less
caspase-1 activation after silica crystal treatment (Fig. 6a). We also
detected mature cathepsin B in supernatants of crystal-stimulated cells
independently of NALP3 (Fig. 6b). To assess whether release of
cathepsin B from lysosomes was temporally associated with caspase-1
activation, we incubated mouse macrophages with silica crystals and
added fluorescent peptides that indicate cathepsin B activity by an
increase in red fluorescence and caspase-1 activity by green fluorescence. When we incubated resting cells with those two reporter
peptides, we found red fluorescence in lysosomal compartments,
indicative of cathepsin B activity, whereas resting cells did not acquire
green fluorescence because they lacked caspase-1 activity (Fig. 6c, left).
Cells treated with silica crystals acquired either red fluorescence
because of lysosomal containment of the cathepsin B–indicator
W
a
IL-1β (ng/ml)
© 2008 Nature Publishing Group http://www.nature.com/natureimmunology
ARTICLES
Figure 7 Alum activates the NALP3 inflammasome through lysosomal destabilization. (a) ELISA (above; supernatants) and immunoblot analysis (below)
of IL-1b production by human PBMCs left untreated or primed for 3 h with LPS (25 pg/ml) and then stimulated for 6 h with increasing doses of alum.
Each dot above a lane represents one donor; small horizontal lines indicate the mean. Results are from four donors (ELISA) or one representative donor
(immunoblot). (b) ELISA of IL-1b in supernatants of wild-type, NALP3-deficient or ASC-deficient bone marrow–derived macrophages primed for 3 h with LPS
and then stimulated with alum (500 mg/ml). (c) Flow cytometry of neutrophils in peritoneal lavage fluid from wild-type mice (n ¼ 5) and IL1R-deficient mice
(n ¼ 5) given peritoneal injection of alum (100 ug) or PBS 16–18 h before. (d–f) Confocal microscopy of wild-type macrophages incubated for 60 min with
DQ ovalbumin (10 mg/ml; red) alone or together with alum (green) and then stained with fluorescent cholera toxin B-subunit (blue; d); or stained with
acridine orange and then treated with alum (blue; e); or left untreated (right) or incubated for 3 h with alum (pink; left), then stained for 1 h with fluorescent
probes for activated caspase-1 (green) and activated cathepsin B (red) and then surface-stained with fluorescent cholera toxin B-subunit (blue; f). Scale
bars, 5 mm. (g) ELISA of the release of IL-1b by LPS-primed bone marrow–derived macrophages left untreated or treated with the cathepsin B inhibitor
CA-074-Me (10 mM) or bafilomycin (250 nM) and then stimulated with alum. (h) ELISA of IL-1b production by LPS-primed bone marrow–derived
macrophages treated for 6 h with alum or MSU crystals in the presence of increasing doses of uricase, presented as the percent of the maximum (100%)
obtained in the absence of uricase. Data (error bars, s.d.) are from one representative experiment of two (c,d,f–h) or three (b,e).
852
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peptide or green cytoplasmic fluorescence indicative of caspase-1
activity (Fig. 6c, right). Activated macrophages obtained from caspase-1-deficient mice did not stain with the caspase-1–indicator
fluorescent peptide, which demonstrated the specificity of the reagent
(data not shown). This notable difference between staining of either
lysosomal cathepsin B or activated caspase-1 suggested that cells that
lose lysosomal cathepsin B after lysosomal rupture (loss of red
lysosomal fluorescence) rapidly activate caspase-1. These results collectively suggest that the NALP3 inflammasome senses lysosomal
contents released into the cytosol of phagocytic macrophages after
crystal-induced lysosomal damage.
Alum triggers NALP3 activation through lysosomal destabilization
Aluminum salts (alum) are the most commonly used vaccine adjuvants, and all alum preparations contain crystals. Aluminum
hydroxide has been reported to induce the cleavage of IL-1b and
IL-18 in a caspase-1-dependent way17. To determine whether alum
induces inflammation by a mechanism similar to that of silica crystals,
we did experiments with a mixture of aluminum hydroxide and
magnesium hydroxide (Imject alum). Alum induced IL-1b maturation
and release by human PBMCs (Fig. 7a) in a caspase-1-dependent way
(data not shown). In mouse macrophages, the alum-induced release of
a
IL-1b was dependent on NALP3 and ASC (Fig. 7b), which indicated
that alum triggers inflammation through activation of the NALP3
inflammasome. Additionally, the influx of neutrophils into the peritoneum after intraperitoneal administration of alum was dependent
on IL-1 activity (Fig. 7c). Of note, alum added to cells induced
considerable morphological changes and led to lysosomal rupture, as
shown by the cytosolic translocation of DQ ovalbumin (Fig. 7d) and
the lysosomal staining pattern of acridine orange (Fig. 7e). As noted
with silica crystals, cells incubated with alum stained positively with a
cathepsin B indicator or the caspase-1 indicator (Fig. 7f), which
demonstrated close correlation of lysosomal loss and caspase-1 activation. In agreement with that idea, alum-induced release of IL-1b was
partially dependent on lysosomal acidification and cathepsin B activity
(Fig. 7g). Uricase added to crystal- or ATP-stimulated cells inhibited
the MSU crystal–elicited release of IL-1b in a dose-dependent way but
did not change the response to alum (Fig. 7h).
NALP3 activation by crystal-independent lysosomal damage
To determine if lysosomal rupture alone was sufficient to activate the
inflammasome, we turned to a model with which we could assess the
function of lysosomal rupture without the requirement of the addition
of known inflammasome activators. We loaded mouse macrophages
b
c
None
Leu-Leu-OMe
None
Leu-Leu-OMe
Dextran
Cath B
inhibitor
(µM)
Dextran
NALP3-KO ASC-KO
Leu-LeuOMe (µM)
1.6
WT
2.5
1.4
NALP3-KO
2.0
1.2
IL-1β (ng/ml)
IL-1β (ng/ml)
1.0
0.8
0.6
0.4
1.5
1.0
0.5
0.2
0
Leu-Leu- Leu-LeuOMe (µM) OMe (µM)
on
e
62
.5
12
5
25
0
dA
dT
N
0
dT
dA
5
25
12
5
62
.
on
e
0
N
1.8
1.6
1.4
1.2
1.0
0.8
0.6
0.4
0.2
0
on
e
50
1, 0
00
0
N
on
e
5
1, 00
00
0
P
AT
0
00
1,
N
Leu-LeuOMe (mM)
0
e
IL-1β (ng/ml)
WT
on
lic
a
AT
dA P
dT
Si
1
2
N
on
e
IL-1β (ng/ml)
1.4
1.2
1.0
0.8
0.6
0.4
0.2
0
1.8
1.6
1.4
1.2
1.0
0.8
0.6
0.4
0.2
0
50
e
Cath B
inhibitor
(µM)
N
Lysosome
disruption
Acridine orange
DNA and RNA
Lysosomes
2
AT
dA P
d
N T
on
e
Dextran
d
NALP3-KO
Lysosome
disruption
10
2
AT
dA P
dT
N
on
e
10
WT
Lysosome
disruption
None
IL-1β (ng/ml)
Silica (µg/ml)
Silica (µg/ml)
1.6
ASC-KO
None
Cath B inhibitor
Bafilomycin
1.4
Figure 8 Rupture of ‘sterile’ lysosomes activates the NALP3 inflammasome. (a) Microscopy of wild-type
1.2
macrophage cells incubated for 30 min in the presence of fluorescent dextran (red) and then left untreated or
1.0
treated with hypertonic and hypotonic solutions to induce lysosomal rupture; left, fluorescent image; right,
0.8
transmitted light image. (b) Immunoblot analysis of activated caspase-1 in supernatants of wild-type or NALP30.6
deficient bone marrow–derived macrophages treated as described in a for 5 h with or without the cathepsin B
0.4
inhibitor CA-074-Me (10 mM or 2 mM); the addition of ATP or poly(dA:dT) serves as a control. (c) Confocal
0.2
microscopy of wild-type macrophages labeled with acridine orange (top row) or incubated for 3 h in the presence of
0
fluorescent dextran (red; bottom row) and incubated with Leu-Leu-OMe (1,000 mM). (d) ELISA of the release of
IL-1b by LPS-primed wild-type macrophages incubated for 6 h with Leu-Leu-OMe (1 or 2 nM), silica crystals
(250 mg/ml), ATP or poly(dA:dT). (e) ELISA of the release of IL-1b by wild-type, NALP3-deficient or ASC-deficient
Silica (µg/ml)
bone marrow–derived macrophages primed with LPS and then stimulated with Leu-Leu-OMe (500 or 1,000 mM) or
ATP (above) or stimulated with silica crystals or poly(dA:dT) (below) in the presence or absence of the cathepsin B inhibitor CA-074-Me (10 mM) or bafilomycin
(250 nM). Original magnification, 160 (a) or 180 (c). Data (error bars, s.d.) are from one representative experiment of two (a,b,e) or three (c,d).
N
on
e
62
.5
12
5
25
0
dA
dT
IL-1β (ng/ml)
© 2008 Nature Publishing Group http://www.nature.com/natureimmunology
ARTICLES
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© 2008 Nature Publishing Group http://www.nature.com/natureimmunology
ARTICLES
with a hypertonic solution and then placed them in hypotonic media,
which resulted in the rupture of endo-lysosomal compartments. This
strategy successfully translocated endo-lysosomal fluorescent dextran
into the cytosol, as shown by diffuse cytosolic staining of cells after the
treatment (Fig. 8a). After this crystal-independent disruption of
lysosomes, there was caspase-1 cleavage (Fig. 8b, left) that was
partially inhibited by a cathepsin B inhibitor. Notably, this crystalindependent lysosomal rupture was completely dependent on NALP3
(Fig. 8b, right). These results collectively indicate that rupture of
lysosomal compartments and leakage of lysosomal contents into the
cytosol even in the absence of crystal material was sufficient to trigger
activation of the NALP3 inflammasome in a partially cathepsin
B–dependent way. Additionally, we used THP-1 human monocytes
differentiated in the presence of phorbol 12-myristate 13-acetate
(PMA) in this lysosomal rupture assay; this represents a model
system that does not rely on microbial stimuli for pro-IL-1b priming.
Similar to the findings with mouse macrophages, THP-1 cells were
also activated to release cleaved IL-1b after lysosomal rupture in a
partially cathepsin B–dependent way (Supplementary Fig. 6 online).
To confirm those findings, we used an alternative method to disrupt
lysosomes with the reagent Leu-Leu-OMe (L-leucyl-L-leucine methyl
ester), which is known to induce lysosomal damage18,19. Leu-Leu-OMe
is a ‘functionalized’ dipeptide that is converted into a membrane-lysing
compound by the lysosomal enzyme dipeptidyl peptidase I. We
incubated mouse macrophages with Leu-Leu-OMe at the reported
effective doses and noted considerable swelling and rupture of lysosomes in most cells, as indicated by lysosomal morphology, loss of red
fluorescence of acridine orange and diffuse cytoplasmic staining by
fluorescent dextran (Fig. 8c). Inhibition of lysosomal acidification by
bafilomycin resulted in much less altered lysosomal morphology and
rupture (data not shown). We next assessed whether Leu-Leu-OMe
activated Il-1b maturation. We detected large quantities of IL-1b in
cellular supernatants in response to Leu-Leu-OMe, similar to the
concentrations elicited by known inflammasome activators (Fig. 8d).
To test whether the Leu-Leu-OMe-induced lysosomal damage induced
NALP3 activation, we analyzed macrophages from mice deficient in
NALP3 and ASC. As noted with silica crystals, cells deficient in NALP3
and ASC failed to respond to Leu-Leu-OMe-induced lysosomal
damage and released only small quantities of IL-1b (Fig. 8e, left).
Finally, inhibition of changes in lysosomal pH by bafilomycin or
inhibition of cathepsin B also inhibited the release of IL-1b in response
to Leu-Leu-OMe (Fig. 8e, right).
DISCUSSION
Chemically and structurally diverse stimuli, ranging from the small
molecules ATP and imidazoquinoline derivatives (such as R-848 and
R-837) to bacterial toxins and various crystals, can activate the NALP3
inflammasome4. It has been reported that asbestos and silica crystals
can activate the NALP3 inflammasome20 and that alum can activate
the NALP3 inflammasome21. Undoubtedly, the list of NALP3 inflammasome activators will grow, yet the mechanisms by which the NALP3
inflammasome can sense different activators are not yet fully understood. One possibility is that NALP3 acts as a pattern-recognition
receptor that can directly bind and respond to various ligands. Indeed,
reports suggest that NALP3 activators could gain access to the cytoplasmic space22,23. A precedent for such a mechanism is the large
spectrum of ligands that can be sensed by certain Toll-like receptors24.
Another possibility is that the NALP3 inflammasome detects an
intermediate molecule generated by a common mechanism elicited by
many different ligands. It has been shown that potassium efflux
is required for NALP3 activation after various inflammasome
854
stimuli25,26. However, it is unclear whether potassium efflux alone
represents a NALP3-inflammasome stimulus itself or whether low
intracellular potassium acts as an additional requirement for inflammasome assembly after the appearance of an as-yet-undefined primary
stimulus. An example of this potential mechanism is found in
the formation of the closely related apoptosome27,28. The loss of
intracellular potassium during apoptosis is an early, essential step for
successful assembly of the apoptosome, which forms after cytochrome
c is released from mitochondria in stress situations. It is therefore
possible that formation of the NALP3 inflammasome is highly favored
at low intracellular potassium concentrations, which may be required
yet not sufficient for activation.
We shave shown here that activation of the NALP3 inflammasome
by crystals or alternatively induced lysosomal damage required the
uptake and acidification of lysosomes and that the ingested crystals led
to lysosomal swelling and leakage. Notably, inhibition of a single
lysosomal protease, cathepsin B, led to a substantial yet incomplete
decrease in activation of the NALP3 inflammasome by lysosomal
damage. In this context it is noteworthy that the NALP3 stimulus
nigericin, thought to activate solely by potassium efflux, induces
lysosomal leakage and subsequent caspase-1 activation through the
function of cathepsin B29. As it has also been shown that pannexin 1,
which helps in establishing larger lysosomal channels, is required
for nigericin-mediated NALP3 activation30,31, it is conceivable that
pannexin 1 not only allows access of bacterial ligands to the
cytoplasm22,23 but could also initiate lysosomal destabilization.
Other studies have also suggested involvement of cathepsin B in the
function of the NALP3 inflammasome, yet those data have indicated
involvement of cathepsin B ‘downstream’ of NALP332,33 rather than
‘upstream’ of NALP3, as we have demonstrated here. It will be useful
to define molecular targets of cathepsin B and potentially other
pH-activated proteases, as this may lead to the identification of
common ‘upstream’ NALP3-activating factors. It has been shown
that the related cathepsin K is also involved in innate immune
recognition. Cathepsin K acts ‘upstream’ of the innate signaling
receptor Toll-like receptor 9 (ref. 34), which signals from within
endo-lysosomal compartments35,36.
Collectively, our data suggest the hypothesis that lysosomal damage
or leakage is perceived by the immune system as an endogenous
‘danger’ signal. The NALP3 inflammasome can respond to internal
membrane perturbations and thereby respond to different stimuli,
which all have in common the ability to induce lysosomal destabilization. Phagocytes that ingest endogenous or exogenous crystallized
material or possibly also aggregated proteins or peptides are thus able
to sense lysosomal damage induced by the cargo taken up by
phagocytosis. After the NALP3 inflammasome is activated, highly
inflammatory cytokines are generated, which leads to the recruitment
of other immune cells to the affected tissue and to the induction of
inflammatory mechanisms that assist in clearing the crystallized
material. However, it is also possible that NALP3 activation by this
mechanism can lead to sustained inflammation and resulting tissue
damage. For example, chronic inhalative exposure to silica or asbestos
can lead to chronic inflammation, tissue damage and carcinogenesis.
NALP3 activation by particulate matter can also intentionally be
exploited therapeutically. Aluminum salt preparations, commonly used
as adjuvants, contain small crystalline materials. We have shown here
that alum activated immune cells through the NALP3 inflammasome
by a mechanism involving lysosomal destabilization. It is likely that
other vaccine candidates that are of particulate nature or that can
perturb lysosomal membranes act by a similar NALP3-activation
mechanism. Our findings should thus aid in the development of new
VOLUME 9
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ARTICLES
vaccines and can lead to new therapeutic ideas for the treatment of
pathologies related to the NALP3 inflammasome.
© 2008 Nature Publishing Group http://www.nature.com/natureimmunology
METHODS
Mice. NALP3-deficient (Nlrp3–/–) mice13, ASC-deficient (Pycard–/–) mice13 and
mice deficient in the protease-activating factor Ipaf (Nlrc4–/–)37 were provided
by Millennium Pharmaceuticals. C57BL/6 mice, 129/Sv mice, C57BL/6 129
F1 mice, IL-1R-deficient (Il1r1–/–) mice and gp91phox-deficient (Cybb–/–) mice
were from Jackson Laboratories. Mice doubly deficient in MyD88 and TRIF
were generated from MyD88-deficient (Myd88–/–) mice and TRIF-deficient
(Ticam1–/–) mice (provided by S. Akira). Mice 7–9 weeks of age were used in all
experiments. All mouse strains were bred and maintained in specific pathogen–
free conditions in the animal facilities at the University of Massachusetts
Medical School. All experiments involving live animals were in accordance
with guidelines set forth by the University of Massachusetts Medical School
Department of Animal Medicine and the Institutional Animal Care and
Use Committee.
Reagents. Acridine orange, ATP, bafilomycin A1, cytochalasin D, LPS, PMA,
poly(dA:dT) sodium salt, sucrose and zymosan were from Sigma-Aldrich.
CA-074-Me and polyethylene glycol 1000 were from Calbiochem. DQ ovalbumin, Alexa Fluor 647–conjugated dextran, Alexa Fluor 647–conjugated cholera
toxin B-subunit, LysoSensor Green and Hoechst stain were from Molecular
Probes–Invitrogen. Alum (Imject alum adjuvant; a mixture of aluminum
hydroxide and magnesium hydroxide) was from Pierce. Leu-Leu-OMeHCl
was from Chem-Impex International. Uricase (Elitek) was from Sanofi-Aventis.
Silica crystals (MIN-U-SIL-15) were provided by US Silica. A ‘poly-dispersed’
preparation of silica crystals of up to 15 mm in length was used in all
experiments. MSU crystals were prepared as described38.
In vivo silica model. Mice were exposed by direct orotracheal instillation of
40-ml aqueous suspensions of 200 mg silica crystals (MIN-U-SIL-15) in PBS or
50 mg crude zymosan; control mice received PBS. Mice were killed 16–18 h after
instillation and bronchoalveolar lavage fluid was obtained by three consecutive
instillations and withdrawals of 1 ml of 1% (vol/vol) BSA in PBS. Recovered
fluid was pelleted by centrifugation and cells were counted. Subsequently, cells
were stained for surface markers and neutrophils were identified as cells double
positive for MCA771B (7/4; Serotec) and Ly6G (1A8; BD Biosciences) by flow
cytometry. For alum experiments, mice were injected intraperitoneally with
100 mg of a mixture of aluminum hydroxide and magnesium hydroxide
(Pierce) in 200 ml PBS. After challenge (16–18 h), mice were killed and their
peritoneal cavities were washed with 6 ml PBS containing 3 mM EDTA and
10 U/ml of heparin. Total peritoneal exudate cells were counted with a
hematocytometer and neutrophils were counted as described above.
Cell isolation and culture. Bone marrow–derived macrophages were generated
as described39. Human PBMCs were isolated by from whole blood of healthy
volunteers by density-gradient centrifugation (Institutional Review Board
protocol H-12497). Red blood cells were lysed with red blood cell lysis buffer
(Sigma). PBMCs and macrophages were used at a density of 2 106 cells per
ml. All primary cells and cell lines except THP-1 cells were cultured in DMEM
supplemented with L-glutamine, ciprofloxacin (Cellgro) and 10% (vol/vol) FCS
(Hyclone). THP-1 cells were cultured in RPMI medium supplemented with
10% (vol/vol) FCS (Hyclone), L-glutamine, sodium pyruvate (Cellgro) and
ciprofloxacin. At 1 d before stimulation, THP-1 cells were differentiated for 3 h
with 0.5 mM PMA, were washed three times and were plated for stimulation.
All experiments for immunoblot analysis used serum-free DMEM medium.
Where appropriate, cells were stimulated with 5 mM ATP at 1 h before
supernatants were collected.
Immortalized macrophage cell lines. Immortalized macrophage cell lines were
generated with a published J2 recombinant retrovirus (carrying v-myc and
v-raf(mil) oncogenes)40. Primary bone marrow cells were incubated in L929
mouse fibroblast–conditioned medium for 3-4 d for the induction of macrophage differentiation. Subsequently, cells were infected with J2 recombinant
retrovirus. Cells were maintained in culture for 3–6 months and were slowly
‘weaned off’ L929 supernatant until they were growing in the absence
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AUGUST 2008
of conditioned medium. Macrophage phenotype was verified by surface
expression of the markers CD11b (M1/70; BD Pharmingen) and F4/80
(BM8; eBiosciences) as well as a range of functional parameters, including
responsiveness to Toll-like receptor ligands and bacterial uptake. Macrophage
cell lines were generated from wild-type (C57BL/6), NALP3-deficient and
ASC-deficient mice.
Flow cytometry. For evaluation of lysosomal rupture, cells were incubated for
15 min with acridine orange (1 mg/ml), were washed three times and then were
stimulated. Lysosomal rupture was assessed by flow cytometry as loss of
emission at 600–650 nm. An LSRII (BD Biosciences) was used for all flow
cytometry. Data were acquired by DIVA (BD Biosciences) and were analyzed
with FlowJo software (Tree Star).
Confocal microscopy. Confocal reflection microscopy was combined with
fluorescence microscopy on a Leica SP2 AOBS confocal laser-scanning microscope. Reflection was captured by placement of the detector channel directly
over the wavelength of the selected laser channel for reflection light capture and
the AOBS microscope was set to allow 5–15% of laser light into the collection
channel. Fluorescence was simultaneously captured by standard confocal
imaging techniques.
ELISA. Cell culture supernatants were assayed for IL-1b with ELISA kits from
BD Biosciences according to the manufacturer’s instructions. For measurement
of intracellular IL-1b, cells were washed and subjected to three cycles of freezing
and thawing in assay diluent.
Lysosomal rupture in THP–1 cells. THP-1 cells differentiated with PMA were
incubated for 10 min at 37 1C in hypertonic DMEM medium containing 10%
(vol/vol) polyethylene glycol 1000, 1.4 M sucrose, 20 mM HEPES, pH 7.2, and
5% (vol/vol) FCS. Cells were subsequently washed and were incubated for
2 min in hypotonic DMEM medium (DMEM/H2O, 3:2) for induction of
lysosomal rupture. Cells were then incubated for an additional 4 h in serumfree DMEM.
Staining of cathepsin B and caspase-1. After treatments, cells were incubated
for 30 min at 37 1C with the fluorescent cathepsin B substrate Magic Red
(cresyl violet ‘bisubstituted’ by amide linkage to the dipeptide argininearginine) together with the fluorochrome inhibitor of caspase-1, green fluorescent peptide FAM-YVAD-fmk (5-carboxyfluorescein–Tyr-Val-Ala-Asp–
fluoromethyl ketone), according to the manufacturer’s recommendations
(Immunochemistry Technologies). After being washed three times in PBS,
cells were visualized by confocal microscopy.
Immunoblot analysis. Cell culture supernatants were precipitated by the
addition of an equal volume of methanol and 0.25 volumes of chloroform,
then were vortexed and centrifuged for 10 min at 20,000g. The upper phase was
discarded and 500 ml methanol was added to the interphase. This mixture was
centrifuged for 10 min at 20,000g and the protein pellet was dried at 55 1C,
resuspended in Laemmli buffer and boiled for 5 min at 99 1C. Samples were
separated by 15% SDS-PAGE and were transferred onto nitrocellulose
membranes. Blots were incubated with rabbit polyclonal antibody to mouse
caspase-1 p10 (sc-514; Santa Cruz Biotechnology), rabbit polyclonal antibody
to the human caspase-1 subunit p10 (sc-515; 2021 Santa Cruz Biotechnology),
rabbit polyclonal antibody to human cleaved IL-1b (Asp116; Cell Signaling) or
rabbit polyclonal antibody to mouse cathepsin B (AF965; R&D Systems).
Accession codes. UCSD-Nature Signaling Gateway (http://www.signaling-gate
way.org): A003663 and A003535.
Note: Supplementary information is available on the Nature Immunology website.
ACKNOWLEDGMENTS
MyD88-deficient and TRIF-deficient mice were provided by S. Akira (Osaka
University). We thank A. Cerny and Joseph Boulanger for animal husbandry
and genotyping; D. Kalvakolanu (University of Maryland School of Medicine)
for providing J2 recombinant retroviruses; and J. Lee and H. Kornfeld for
help with the lung inflammation model. Supported by the Deutsche
Forschungsgemeinschaft (Ho2783/2-1 to V.H. and GK1202 to F.B.) and the
855
ARTICLES
US National Institutes of Health (R01 AI-065483 to E.L., RO1 AI-067497 to
K.A.F. and RO1 AI043543 to K.L.R.).
© 2008 Nature Publishing Group http://www.nature.com/natureimmunology
Published online at http://www.nature.com/natureimmunology/
Reprints and permissions information is available online at http://npg.nature.com/
reprintsandpermissions/
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