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doi:10.1182/blood-2008-03-146720
Prepublished online Dec 22, 2008;
Robert J. Mason, Bart Jan Kullberg, Anna Rubartelli, Jos W.M. Van der Meer and Charles A. Dinarello
van der Meer, Frank L. van de Veerdonk, Gerben Ferwerda, Bas Heinhuis, Isabel Devesa, C. Joel Funk,
Mihai G. Netea, Claudia A Nold-Petry, Marcel F. Nold, Leo A.B. Joosten, Bastian Opitz, Jonathan H.M.
processing and release of IL-1{beta} in monocytes and macrophages
Differential requirement for the activation of the inflammasome for
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. Hematology; all rights reservedCopyright 2007 by The American Society of
DC 20036.
by the American Society of Hematology, 1900 M St, NW, Suite 200, Washington
Blood (print ISSN 0006-4971, online ISSN 1528-0020), is published semimonthly
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Differential requirement for the activation of the inflammasome for
processing and release of IL-1β in monocytes and macrophages
Running title: IL-1β release in monocytes vs. macrophages
Mihai G. Netea1,2,4,#, Claudia A. Nold-Petry1,*, Marcel F. Nold1,*, Leo A.B. Joosten2,4, Bastian
Opitz6, Jonathan H.M. van der Meer1, Frank L. van de Veerdonk2,4, Gerben Ferwerda2, Bas
Heinhuis3, Isabel Devesa3, C. Joel Funk5, Robert J. Mason5, Bart Jan Kullberg2,4, Anna
Rubartelli7, Jos W. M. Van der Meer2,4, Charles A. Dinarello1
1Division of Infectious Diseases, University of Colorado Health Sciences Center, Denver,
Colorado
Departments of 2Medicine, and 3
Rheumatology, Radboud University Nijmegen Medical
Center, Nijmegen, The Netherlands
4Nijmegen Institute for Infection, Inflammation and Immunity (N4i), Nijmegen, The
Netherlands
5Department of Medicine, National Jewish Medical and Research Center, Denver, Colorado
6Department of Internal Medicine/Infectious Diseases and Pulmonary Medicine, Charité -
Universitätsmedizin Berlin, Germany
7Department of Experimental Oncology E (Cell Biology), Istituto Nazionale per la Ricerca sul
Cancro, Italy
*These two authors contributed equally to this study
Blood First Edition Paper, prepublished online December 22, 2008; DOI 10.1182/blood-2008-03-146720
Copyright © 2008 American Society of Hematology
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#To whom correspondence should be addressed :
Mihai G. Netea
Department of Medicine
Radboud University Nijmegen Medical Center
Nijmegen, The Netherlands
Tel: +31-24-3618819 Fax: +31-24-3541734 E-mail: m.netea@aig.umcn.nl
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Abstract
The processing of pro-IL-1β depends on activation of caspase-1. Controversy has arisen
whether TLR ligands alone can activate caspase-1 for release of IL-1β. Here we demonstrate
that human blood monocytes release processed IL-1β after a one-time stimulation with either
TLR2 or TLR4 ligands, due to constitutively activated caspase-1 and release of endogenous
ATP. The constitutive activation of caspase-1 depends on the inflammasome components
ASC and NALP3, but in monocytes caspase-1 activation is uncoupled from PAMP
recognition. In contrast, macrophages are unable to process and release IL-1β solely by TLR
ligands, and require a second ATP stimulation. We conclude that IL-1β production is
differentially regulated in monocytes and macrophages, and this reflects their separate
functions in host defense and inflammation.
Keywords: Cytokines, Caspase-1, Inflammation, Host Defense
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Introduction
Much interest has been generated regarding processing and release of bioactive interleukin-1β
(IL-1β), since the discovery that autoinflammatory disorders specifically respond to blockade
of IL-1 receptor with the IL-1 receptor antagonist (IL-1Ra), or neutralization of IL-1β by the
monoclonal anti-IL-1β antibodies. These syndromes include familial Mediteranean fever 1,
familial cold auto-inflammatory syndrome 2, Muckle-Wells syndrome 3, neonatal onset
multisystem inflammatory disease 4, hyperimmunoglobulin D syndrome 5, and adult-onset
Still’s disease 6. Blood monocytes from patients with some of these disorders, especially
cryopyrinopathies, readily release more IL-1β than monocytes from unaffected controls,
revealing a loss of the tight control regulating processing and release of active IL-1β.
Several mechanisms control the production and activity of IL-1β, incuding the
processing of the 31-kDa inactive IL-1β precursor form into the bioactive 17-kDa IL-1β 7, and
the release from secretory lysosomes through K+-dependent mechanisms 8,9. In addition,
control over IL-1 activity is exerted by the IL-1 receptor antagonist (IL-1Ra) or the type II
decoy receptors 10. Processing of bioactive IL-1β depends on activation of caspase-1 by the
protein complex termed the inflammasome 11. Several protein platforms/inflammasomes have
been described for the activation of caspase-1, each of them include members of the NOD-
like receptor (NLR) family of proteins 12. The most intensely studied have been the
inflammasomes formed by the NLR family members NALP3 and NALP1, that also include
the adapter protein ASC for the activation of caspase-1. Mutations in NALP3 exits in familial
cold-induced autoinflammatory syndrome (FCAS) 2, Muckle-Wells Syndrome (MWS), and
neonatal onset multisystem inflammatory disease (NOMID), whereas specific NALP-1
polymorphisms have been associated with vitiligo and autoimmune diseases 13. In addition to
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the NALPs, another NLR member, IPAF, forms an inflammasome that activates caspase-1 in
response to intracellular flagellin in an ASC-independent manner 14,15.
However, controversy surrounds the capacity of TLR ligands such as LPS to activate
caspase-1 resulting in the release of active IL-1β. By using transfected cell lines and/or
NALP3 knock-out mice, a broad panel of stimuli have been proposed to activate the NALP3
inflammasome, including bacterial products such as peptidoglycans and muramyl dipeptide
(MDP) 16, bacterial toxins 17, but also endogenous products such as uric acid 18 or ATP 17.
Based on responses in the leukemic cell line THP-1, a concept has arisen that IL-1β
production induced by the often-employed monocyte stimulant lipopolysacchardie (LPS) is
due to contamination with non-LPS ligands such as peptidoglycans 16, while LPS by itself is
ineffective as a stimulator of IL-1β release. A second signal, such as muramyldipeptide or
ATP, is required, and this would induce activation of caspase-1 followed by IL-1β processing
and release 19. This model is derived from data in THP-1 cells 16 and in primary mouse
macrophages 20; however, it is inconsistent with many studies showing abundant production
and release of IL-1β from blood monocytes by purified LPS, lipopeptides, lipoteichoic acid as
well as cytokines such as TNFα and IL-1 itself 21,22. In addition, several studies report that
synthetic products, which exclude contamination with NALP1 or NALP3 ligands, stimulate
IL-1β release 23,24.
In the present study we demonstrate a remarkable difference in the level of
constitutive caspase-1 activation and subsequent IL-1β release between freshly-obtained
blood monocytes and macrophages. We conclude that there is no basis for the concept that
LPS is unable to induce processing and release of IL-1β from blood monocytes. We also
conclude that unlike the blood monocytes, the macrophages require not only LPS but also a
second signal for the activation of the inflammasome and release of processed IL-1β.
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Materials and methods
Reagents. LPS (E. coli serotype 055:B5) and staphylococcal peptidoglycan (PepG)
were purchased from Sigma (St. Louis, MO). LPS was re-purified as previously described 25.
Synthetic Pam3Cys was purchased from EMC Microcollections (Tubingen, Germany).
Synthetic MDP was obtained from Sigma. The reversible caspase-1 inhibitor (termed ICE-i)
Ac-Tyr-Val-Ala-Asp-2,6-dimethylbezoyloxymethylketone (YVAD) was purchased from
Alexis Biochemicals (San Diego, CA) and solubilized in dimethyl sulfoxide (DMSO) at
10 mg/ml. The ICE-i was diluted to the desired concentration in RPMI. Anti-human caspase-1
p10 (sc515), anti-caspase-1 p45 (sc-622), and anti-human ASC (sc30153) antibodies were
purchased from Santa Cruz Biotechnologies (Santa Cruz, CA). Anti-human IL-1β antibodies
were purchased from Cell Signaling (Danvers, MA). Recombinant G-CSF, GM-CSF and IL-4
were purchased from R&D (Minneapolis, MN). The TLR4 antagonist B. quintana LPS was
prepared as previously described 26. oxATP was purchased from Sigma.
Chinese Hamster Ovary Cells. The Chinese Hamster Ovary (CHO)/CD14 cell line
(clone 3E10) was transfected with plasmids expressing either human TLR4 or human TLR2
surface proteins, kindly provided by Douglas Golenbock (University of Massachussetts,
Worcetser, MA). Upon engagement of TLR4 or TLR2, a nuclear factor kB (NF-kB)-
dependent reporter plasmid drives the expression of surface CD25, as a result of NF-kB
translocation. Surface CD25 was assessed by flow cytometry after stimulation of CHO/TLR2
or CHO/TLR4 cells with 1 μg/ml LPS. In addition, peritoneal macrophages of TLR2 and
TLR4 knock-out mice were stimulated with LPS (1 μg/ml) for 24h at 37°C, and TNF was
measured by specific ELISA.
Isolation of mononuclear cells and stimulation of cytokine production. These
studies were approved by the Colorado Multiple Institutional Review Board. After obtaining
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informed consent in accordance with the Declaration of Helsinki, the PBMC fraction was
obtained by differential centrifugation over Ficoll-Paque (Sigma). Cells adjusted to 5x106
cells/ml were suspended in culture medium (RPMI 1640) supplemented with gentamicin 10
μg/ml, L-glutamine 10 mM and pyruvate 10 mM. 100 μl cells were incubated with either 100 μl
of culture medium (negative control), or purified LPS (various concentrations as described in
the figure legends), Pam3Cys (10 μg/ml), heat-killed S. epidermidis (106 microroganisms/ml),
MDP (10 μg/ml), or combinations of MDP and LPS. In separate experiments, inhibitors
(YVAD caspase-1 inhibitor, 20 µM) or IL-1Ra (R & D, Minneapolis, MN) were added. After
24 hours, the supernatants were collected and stored at -70°C until assayed. Intracellular were
assessed after adding 200 μl RPMI to the adherent cells and cell lysis by two cycles of freeze-
thaw.
In order to investigate the role of monocytes for the production of IL-1β, the
monocytes were purified using a separation assay with magnetic beads coated with anti-CD14
antibodies, as described by the manufacturer (Miltenyi Biotec, Bergische Gladbach,
Germany).
To investigate the effect of LPS followed by the second stimulus of ATP on cytokine
production, PBMC or purified monocytes were initially stimulated for 4 hours with LPS.
After 4 hours, the supernatants were collected and RPMI containing 1mM ATP was added to
the cells for another 15 min. The LPS-dependent IL-1β production during the first 4 hours and
the ATP-dependent IL-1β secretion after the additional 15 minutes was assessed in the
supernatant. The role of the endogenous ATP release for the stimulation of IL-1β was
investigated by blocking P2X7 receptors with oxATP (300 μM) during the stimulation of
cells for 4h with LPS 27.
Cytokine and ATP determinations. Cytokine concentrations were determined by
electrochemiluminescence assays. The IL-1β precursor and the mature IL-1β were measured
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by specific ELISA from R&D Systems. ATP concentrations in the supernatants were assessed
using a firefly luciferase assay (ATP determination kit, Invitrogen, Carlsbad, CA).
Macrophage and dendritic cell differentiation. The adherent monocytes were
obtained after incubation of PBMC for 2h at 37°C, after which the non-adherent lymphocytes
were discarded. The monocytes were incubated for 5 days with either 10% pooled human
plasma (macrophage differentiation), 50 ng/ml G-CSF (macrophage differentiation), or a
combination of GM-CSF (50 ng/ml) and IL-4 (20 ng/ml) (dendritic cell differentiation). On
day 3, medium was refreshed. After 5 days the supernatant was removed and cells were
stimulated with the various stimuli. In addition, alveolar macrophages collected from healthy
volunteers by bronchoalveaolar lavage were suspended to a concentration of 5x106 cells/ml
and stimulated for cytokine production.
Immunoblotting. For immunoblotting 10x10 6 cells were lysed in 100 μl lysis buffer
(50 mM Tris, pH 7.4, 150 mM NaCl, 2 mM EDTA, 2 mM EGTA, 10% glycerol, 1% Triton
X-100, 40 mM α-glycerophosphate, 50 mM sodium fluoride, 200 μM sodium vanadate, 10
μg/ml leupeptin, 10 μg/ml aprotinin, 1 μM pepstatin A, and 1 mM phenylmethylsulfonyl
fluoride). The homogenate was frozen, thawed then centrifuged at 4 for 10 min at 14,000
rpm, and the supernatant was taken for Western blotting. Equal amounts of protein were
subjected to SDS-PAGE using 10% and 15% polyacrylamide gels at a constant voltage of
100V. After SDS-PAGE, proteins were transferred to nitrocellulose membrane (0.2 μm).
The membrane was blocked with 3% (w/v) milk powder in PBS for 1 hour at room
temperature followed by incubation over night at 4°C with the primary antibody in 5%
BSA/TBST (5% bovine serum albumin / Tris-buffered saline/Tween 20). After over night
incubation the blots were washed three times with TBST and incubated with HRP-conjugated
goat anti-rabbit antibody at a dilution of 1:10 000 in 3% (w/v) milk powder in PBS for 1h at
room temperature. After washing the blots three times with TBST the blots where developed
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with ECL according to manufacture’s instructions. The quantification of protein expression
was performed by densitometry (GS-670, Biorad, Veenendaal, Netherlands) and signal
analysis using Molecular AnalystTM software. The ratio between the intensity of the protein of
interest and β–actin was calculated. The activation of caspase-1 was assessed by calculating
the ratio between the p10 and p45 fragments.
siRNA experiments. For siRNA experiments specific cell line and primary monocyte
protocols for electroporation in the Amaxa chamber were used, according to the instructions of
the manufacturer. Specific sets of siRNA for ASC and NALP3 as well as control, non-silencing
siRNA were obtained from Dharmacon (Boulder, CO) or Ambion (Huntingdon, UK). After
counting with trypan blue, PBMC were centrifuged at 200 g for 10 min and resuspended in 100
μl prewarmed Human Monocyte Nucleofector solution (Amaxa) per transfer condition. Without
delay, siRNA was added and electroporation was performed using program Y-001. After
electroporation, the primary monocytes were allowed to recover overnight in polypropylene
tubes, in order to avoid adhesion. 2 µg siRNA were used (control-sense 5´-
UUCUCCGAACGUGUCACGUtt-3`; control-antisense 5´-
ACGUGACACGUUCGGAGAAtt-3´; ASC-sense 5´-GAUGCGGAAGCUCUUCAGUtt-3´;
ASC-antisense 5´-ACUGAAGAGCUUCCGCAUCtt-3´; Nalp3-sense 5´-
GGUGUUGGAAUUAGACAACtt-3´; Nalp3-antisense GUUGUCUAAUUCCAACACCtg-
3´) per 106 cells. On the next day, in case of THP1 cells, they were treated with PMA for 24 h,
washed and incubated for additional 24 h in culture medium. The following day the cells were
stimulated with LPS or LPS/ATP as described above. Control experiments to check the
inhibition of ASC or NALP3 expression were performed by RT-PCR and immunoblotting.
RT-PCR. Ten million freshly isolated PBMC were incubated with the various stimuli.
After 4 hours of incubation at 37°C, total RNA was extracted in 1 ml of TRIzol reagent.
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Isolated RNA was treated with DNase before being reverse transcribed into complementary
DNA using oligo(dT) primers and MMLV reverse transcriptase. PCR was performed using a
Peltier Thermal Cycler-200 (Watertown, MA). Primer sequences for human IL-1β are: sense:
5'-GGA-TAT-GGA-GCA-ACA-AGT-GG-3' and antisense: 5'-ATG-TAC-CAG-TTG-
GGG-AAC-TG-3', and for TNFα: sense 5’-ACA-AGC-CTG-TAG-CCC-ATG-TT-3’ and
antisense 5’-AAA-GTA-GAC-CTG-CCC-AGA-CT-3’ GAPDH was used as a reference gene,
for which the primers were: 5-GGC-AAA-TTC-AAC-GGC-ACA-3 (forward) and 5-GTT-
AGT-GGG-GTC-TCG-CTC-TG-3 (reverse). PCR conditions were as follows: 2 minutes at
50°C and 10 minutes at 95°C, followed by 30 cycles of PCR reaction at 94°C for 45 seconds,
70°C for 2 minutes, and 59°C for 1 minute. The PCR products were run on 1% agarose gels
stained with ethidium bromide.
Statistical analysis. The cytokine induction experiments were performed in triplicate
wells, and the data are presented as cumulative results. The differences were analyzed by
paired t-test. The data are given as means ± SEM.
Results
Purified LPS stimulates IL-1β synthesis and release by freshly obtained blood
monocytes. It has been suggested that LPS induces the release of active IL-1β only due to
contamination with bacterial cell wall products such as peptidoglycans and MDP 16. To
exclude the presence of non-LPS microbial products, we performed a double purification of
the standard commercial preparation of E. coli O55:B5 LPS 25. We tested the properties of
the preparation in several ways: purified LPS stimulated CD14/TLR4 but not CD14/TLR2
transfected CHO-cells (Fig. 1A and 1C); TNFα production by the purified preparation was
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completely reversed in the presence of the specific TLR4 antagonist Bartonella quintana LPS
26 (Fig.1B) or in TLR4-/- mice (not shown); moreover, there was no reduction in LPS-induced
TNFα production from peripheral blood mononuclear cells (PBMC) isolated from individuals
carrying the frame shift mutation in the MDP-receptor NOD2 (homozygous for the 3020insC
mutation) (Fig. 1D). In addition, we did not observe a reduction in production induced by the
purified LPS in TLR2 deficient mice (data not shown).
When human PBMC were stimulated with this purified LPS, the levels of both proIL-
1β precursor and the mature IL-1β, were significantly increased (Fig. 2C). To demonstrate the
presence of the mature processed form of the IL-1β, Western blots of supernatants of cells
stimulated with either control medium, MDP or LPS were performed. As shown in the insert
to Figure 2C, LPS stimulates the release of the active 17-kDa form of IL-1β.
The secretion of IL-1β by LPS from monocytes was significantly inhibited by a
caspase-1 inhibitor (Fig. 2D). We employed two strategies to assess the bioactivity of
endogenous IL-1β stimulated by LPS. On the one hand, the release of the mature form of IL-
1β was inhibited in the presence of a caspase-1 inhibitor, and that in turn reduced significantly
the production of IL-1α and IL-6, as shown in Figure 2E and F. Moreover, the activity of IL-
1β can be blocked at the level of the receptor by the competitive binding of IL-1Ra. Blockade
of IL-1 receptors by adding IL-1Ra to the medium reduced the production and release of IL-6
and IL-1α (Fig. 2E and F).
Different capacity of monocytes, macrophages and dendritic cells to produce and
release IL-1β. In order to compare the capacity of monocytes and macrophages to stimulate
IL-1β synthesis and release, freshly-isolated monocytes and monocyte-derived macrophages
were stimulated with either purified TLR4 (LPS) or TLR2 (Pam3Cys) ligands, or with whole
heat-killed Staphylococcus epidermidis, an inducer of IL-1β 28. As shown in Fig. 3A,
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monocytes released large amounts of IL-1β in the supernatant after 24h stimulation, whereas
no IL-1β was present in the supernatant of stimulated macrophages. Macrophages that were
differentiated in the presence of 10% pooled human plasma did not release IL-1β with each of
the three stimulants. Similarly, there was no production of IL-1β by macrophages
differentiated for 5 days with 50 ng/ml G-CSF (data not shown).
In addition to monocyte-derived macrophages, differentiation of monocytes into
dendritic cells also resulted in a loss in the capacity to release mature IL-1β (Fig.3B). In
contrast, both monocytes and monocyte-derived macrophages released comparable amounts
of TNFα (Fig.3C) or IL-1α (Fig.3D) in the same cultures. IL-1α concentrations were lower in
macrophages than in monocytes, most likely due to the absent autocrine stimulatory loop
induced by endogenous IL-1β.
Differential activation of caspase-1 and IL-1β excretion in monocytes and
macrophages. Despite the absence of IL-1β in the supernatants of macrophages stimulated
with microbial stimuli, LPS induced abundant amounts of precursor IL-1β mRNA in both the
monocytes and macrophages (Fig. 4A). Moreover, significant amounts of the IL-1β precursor
were present intracellularly in the macrophages (Fig.4B), demonstrating that a post-
translational defect is the mechanism of defective release of IL-1β in macrophages.
In order to test the hypothesis that a processing defect of the IL-1β precursor accounts
for the differences between the monocyte and macrophage, Western-blots of the inactive p45
caspase-1 and the active p10 caspase-1 form were performed in both cell types. As shown in
Fig.4C, after 2 hours of incubation, monocytes display clear activation of caspase-1 regardless
of LPS stimulation. The constitutive presence of both p45 and the active p10 caspase-1 can be
observed in freshly isolated mononuclear cells that, in order to avoid stimulation, were
directly lysed after isolation at 4°C (Fig.4D). In contrast to monocytes, macrophages have
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significant amounts of the inactive p45 caspase-1 form, but less of the active p10 caspase-1
fragment, even after stimulation with LPS (Fig.4C).
THP-1 cells exhibit no activation of caspase-1 (Fig.4C), and there was no release of
active IL-1β when stimulated with LPS alone (data not shown). However, when primed for 3
days with 10 μg/ml PMA, THP-1 cells released significant amounts of IL-1β into the
supernatant after stimulation for 24h with 100 ng/ml LPS (1.4 ± 0.4 ng/ml, n=5). This was
due to a moderate activation of caspase-1 after PMA priming, as well as the spontaneous
release of endogenous ATP (1.4 + 0.4 nM vs. <0.2 nM in non-primed cells), followed by the
production of IL-1β upon stimulation with LPS (see above).
Despite the relatively ineffective activation of caspase-1 by LPS in macrophages,
Western blots of cell lysates reveal the intracellular presence of the precursor as well as the
mature forms of IL-1β (Fig.4E). These findings suggest that the defect in the release of
mature IL-1β is partially at the level of inflammasome activation and partially at the level of
IL-1β secretion.
The role of the inflammasome components for the stimulation and release of IL-
1β in primary blood monocytes. In order to assess the role of the inflammasome
components ASC and NALP3 for the activation of caspase-1 and IL-1β release, we
transfected PMA-primed THP-1 cells with siRNA against either ASC or NALP3. The
transfection of PMA-primed THP-1 cells with siRNA decreased the steady state levels of
ASC by 48 + 13% (Fig.5A), and of NALP3 by 67 + 19% (Fig.5B). These reductions were
accompanied by a significant inhibition of IL-1β release that was induced by LPS (Fig.5C).
No effects on IL-8 production were apparent (Fig.5D). Similarly, freshly isolated PBMC
were transfected with siRNA specific for ASC as well as a control of scrambled siRNA. As
depicted in Figure 5E, decreasing ASC expression in primary monocytes was associated with
a decrease in IL-1β release, which was induced after a 24h-exposure to LPS (single
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stimulation) (Fig.5E). A similar reduction was observed after a short 4 hour exposure to LPS
followed by a second stimulation with ATP (double stimulation) (Fig.5F). There was no
effect on the production of the caspase-1-independent cytokine TNFα in these cultures
(Fig.5E). Moreover, inhibition of ASC expression by siRNA (Fig.5G) in primary cells
resulted in a significantly decreased activation of caspase-1: the p10/p45 ratio was decreased
from 0.37 + 0.19 to 0.14 + 0.09, p<0.05 (Fig.5H).
Double stimulation is necessary for the release of IL-1β by macrophages. We
investigated whether inflammasome stimuli can reverse the defect of IL-1β release in
macrophages and increase release of mature IL-1β from monocytes. The costimulation of
macrophages with LPS plus MDP did not result in the release of IL-1β, demonstrating that
activation of the inflammasome alone is not sufficient for the release of IL-1β (Fig.6A). In
contrast, the co-stimulation of LPS-primed cells with ATP (that induces both caspase-1
activation and IL-1β secretion) induced the release of IL-1β from both monocytes and
macrophages (Fig.6B). As shown in Figure 6C, stimulation of monocytes with LPS plus ATP
induced a significant decrease in intracellular caspase-1 p10 and p45, consistent with a release
of the inflammasome components into the supernatant (Fig.6C).
A pioneering study published by Ferrari and colleagues has demonstrated that human
monocytes can release endogenous ATP 29. We compared the release of ATP into the
supernatant of PBMC or macrophages. As shown in Figure 6D, monocytes from the PBMC,
but not macrophages, release endogenous ATP. Similarly, purified monocytes had an
identical effect on ATP release (1.8 + 0.4 nM). Since monocytes cultured with LPS for 4
hours spontaneously release ATP, we blocked the ATP interaction with P2X7 receptor with
oxidized ATP (oATP) 27. As shown in Figure 6E, inhibition of endogenous ATP binding to
the P2X7 receptor in the presence of oATP resulted in near total reduction in the release of
IL-1β release.
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IFNγ stimulation or prevention of adherence affects IL-1β release. We assessed
the effect of the macrophage-activating cytokine IFNγ on macrophages differentiated from
blood monocytes. Incubation of macrophages differentiated with IFNγ resulted in substantial
IL-1β release following stimulation with TLR ligands or heat-killed Staphylococcus
epidermidis for an additional 24h (Fig.6F). In addition, the differentiation of macrophages in
the presence of IFNγ led to the activation of caspase-1 (Fig.6H) and the release of endogenous
ATP (Fig.6I). This suggests that IFNγ-activated macrophages have a more proinflammatory
phenotype, capable of releasing active IL-1β.
We also exploited a simple but effective method to prevent the adherence of
monocytes to the polystyrene plate during the differentiation process. By slowly rotating
PBMC in polypropylene tubes, one prevents adherence, which also partially reversed the near
total lack of IL-1β release from macrophages adhering to the polystyrene plates during
differentiation (Fig.6G).
Alveolar macrophages also require double stimulation for release of IL-1β. We
sought to assess whether naturally occurring macrophages are also deficient in the release of
IL-1β, and are similar to the in-vitro differentiated macrophages in requiring two stimuli for
production of IL-1β. Although in alveolar macrophages LPS, Pam3Cys or heat-killed S.
epidermidis stimulated high levels of TNFα, there was no release of IL-1β after 24h of
incubation with these stimuli alone (Fig.7A). There were, however, substantial amounts of
intracellular precursor IL-1β (884 + 219 pg/ml) in alveolar macrophages after LPS. While
LPS did not induce IL-1β release from the alveolar macrophages, the addition of ATP
induced partial release of the IL-1β into the extracellular compartment (223 + 45 pg/ml),
similar to its effects in monocyte-derived macrophages (Fig.7B).
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Discussion
The Controversy. The well-known capacity of human blood monocytes to release IL-1β
after a single stimulation with TLR ligands such as LPS has been recently challenged by
studies using the THP-1 leukemic cell line 16,30 or mouse peritoneal macrophages 17,20. In
human cell lines and mouse macrophages, LPS fails to activate caspase-1 and release mature
IL-1β. Those reports propose that release of IL-1β by LPS requires an additional stimulation
of the inflammasome with MDP or ATP 19,31. It has been claimed that the large number of
published research on LPS stimulation of the synthesis and release of IL-1β is based on
contamination of LPS with bacterial peptidoglycans 16. However, this claim contradicts a
large body of evidence that human blood monocytes release bioactive IL-1β upon stimulation
by purified LPS or other (sometimes synthetic) TLR ligands 32-34.
If human blood monocytes were indeed unable to respond to LPS with release of
IL-1β, a large body of clinical studies using freshly obtained human blood and their
conclusions would be brought into question. Therefore, the purpose of the present study was
to assess whether a sole TLR ligand can induce the synthesis, processing and release of IL-1β
from blood monocytes and whether primary cells differ from THP-1 cells. We demonstrate
that synthesis and release of IL-1β differ between human monocytes and macrophages.
Monocytes have constitutively activated caspase-1, leading to release of active IL-1β after a
single stimulation event with bacterial ligands such as LPS, whereas macrophages need two
distinct stimuli: one stimulus induces transcription and translation, and a second stimulus is
needed for caspase-1 activation with subsequent IL-1β processing and secretion.
The major differences between the various studies that assessed IL-1β production and
release are represented by the type of cell studied and by the stimulation model employed.
Practically all studies challenging the capacity of TLR ligands alone to induce IL-1β release
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17
have used either cell-lines (especially THP-1) or mouse macrophages stimulated for short
intervals (2 to 4h) with LPS followed by ATP. In contrast, the “classical” IL-1β stimulation
is performed in primary human monocytes in which release of the processed cytokine takes
place over 24 hours. The first aim of our study was to verify the validity of classical 24 hour
IL-1β stimulation. Using several approaches, we demonstrate that the purified LPS used in
the present study is a TLR4 agonist not contaminated with peptidoglycans or bacterial
lipopeptides. This highly purified LPS preparation induced the release of mature IL-1β from
human monocytes in the routine 24h stimulation assay.
The Inflammasome Needs a Second Signal in Macrophages and Dendritic Cells. A
strikingly different response occurred when monocytes were differentiated into macrophages
or dendritic cells. When stimulated with either LPS, the TLR2 ligand Pam3Cys, or even
whole S. epidermidis microorganisms, macrophages or dendritic cells did not release IL-1β.
The lack of IL-1β release was observed not only for the in vitro differentiated monocyte-
derived macrophages, but also for alveolar macrophages isolated from healthy volunteers, the
latter observation being consistent with earlier studies 35,36. Nevertheless, macrophages and
monocytes produced comparable amounts of the caspase-1-independent TNFα.
To investigate the molecular mechanisms responsible for these differences, we
dissected the steps of IL-1β production and release. LPS induced marked increases in steady-
state levels of IL-1β mRNA as well as of intracellular levels of the IL-1β precursor, clearly
demonstrating that the defect in IL-1β release is post-translational. Thereafter, we performed
Western blots of the inactive and cleaved caspase-1 fragments in unstimulated or stimulated
monocytes and macrophages. Unexpectedly, freshly-isolated primary blood monocytes
contained the activated form of caspase-1, even when lysed immediately after the isolation
procedure without adherence to plastic. Moreover, stimulation with LPS had little additional
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effect (Fig.4). In contrast, macrophage expression of caspase-1 was much lower, and
activation by IFNγ was required for the induction of active p10 fragments. The presence of
both the IL-1β precursor and mature IL-1β in the macrophage lysate is consistent with the
lack of IL-1β release despite LPS stimulation, and only a second stimulation event with ATP
was able to induce IL-1β secretion. In addition, ATP also induces a decrease in intracellular
caspase-1 components 8.
Why are the monocytes capable of IL-1β secretion, while macrophages are not ? An
earlier study has reported that monocytes can release endogenous ATP 29. In the present study
we confirm the importance of endogenous ATP for IL-1β secretion by monocytes, and show
that macrophages are not capable of ATP release (see Fig.6). It is therefore the defective
activation of caspase-1, coupled with the lack of endogenous ATP release, that render human
macrophages incapable of secretion of bioactive IL-1β.
IL-1β Release from Monocytes of Patients with Autoinflammatory Diseases.
Although the human monocytic leukemia cell-line THP-1 is studied to investigate
inflammasome activation, we believe these cells are inappropriate for extrapolating to
responses of primary cells. In contrast to primary monocytes, THP-1 cells did not express
constitutively activated caspase-1, and responded poorly to LPS-induced IL-1β synthesis and
release. In order to release IL-1β, THP-1 cells requires either double stimulation with LPS
and ATP 16, or priming with PMA leading to constitutive release of endogenous ATP.
Although they are highly responsive to TLR stimulation and they release IL-1β due to
constitutively active caspase-1, blood monocytes also respond to ATP challenge, leading to a
greater release of IL-1β 8. This is highly relevant to the role of NALP-3 in monocytes isolated
from patients with autoinflammatory diseases. Monocytes from patients with Muckle-Wells
syndrome and NALP3 mutations did not require additional ATP for the release of IL-1β 37.
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Similarly, 24-hour stimulation with LPS of monocytes isolated from patients with NOMID 38
or hyper IgD syndrome 39 also exhibit increased release of IL-1β compared to unaffected
volunteers. Thus, the NALP3 mutations result in an inflammasome which is already
maximally stimulated without the need for a second signal from ATP 37. Having a maximally
activated inflammasome that efficiently processes IL-1β even after minimal stimulation may
explain the inflammatory attacks in these syndromes, which are induced by most trivial of
stimuli 2,40.
Alveolar Macrophages. Consistent with the failure of in vitro differentiated
macrophages to release IL-1β are the alveolar macrophages. Wewers and colleagues obtained
similar results and proposed a post-transcriptional defect in freshly obtained alveolar
macrophages 41. Recently, they reported differences in pyrin expression between monocytes
and macrophages, and suggested that pyrin induces IL-1β release 42. However, it is unclear
how pyrin modulates the inflammasome, as other studies propose inhibitory effects of pyrin
on the inflammasome 1,43. Monocytes from patients with FMF and mutations in the carboxy
terminal domain of pyrin release more IL-1β than cells from control subjects, suggesting a
failure to suppress the activation of caspase-1 1.
Importance of ASC in Monocytes and Macrophages. It is clear from the studies in
mice deficient in ASC and NALP-3 that the inflammasome contributes to LPS responses in
vivo 44,45. However, we are not aware of data showing the importance of inflammasome
components for the release of IL-1β following LPS stimulation in primary cells from healthy
subjects or mouse monocytes. We showed here that when ASC or NALP3 expression was
inhibited in primed THP-1 cells or primary monocytes by specific siRNA, caspase-1
activation and IL-1β release were significantly reduced, even after stimulation with LPS
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alone. Moreover, as expected, these inhibitory effects by specific siRNAs were observed in
cells stimulated with LPS for 4 hours followed by ATP. We also observed a similar reduction
in caspase-1 activation and release of IL-1β in primary monocytes stimulated only with LPS
for 24 hours. Our data in primary human cells are consistent with in vivo LPS responses in
mice deficient in ASC and NALP-3 44,45, which support a role of ASC and Nalp3 in the
responses to TLR ligands through their capacity to control the constitutive caspase-1
activation and IL-1β processing. On the other hand, the caspase-1 activation in unstimulated
cells is also consistent with data showing that activation of the inflammasome is independent
of TLRs 20, but rather dependent on the hemichannel protein pannexin 20.
These data imply a paradigm shift in our understanding of the inflammasome. The
demonstration of a role of ASC and Nalp3 in the constitutive activation of caspase-1,
independent of stimulation by TLRs or inflammasome ligands, uncouples caspase-1 activation
from PAMP recognition in human primary monocytes. This new model, in which the
inflammasome components ASC and Nalp3 form a protein platform responsible for the
constitutive activation of caspase-1, explains why the IL-1β induced in monocytes by a very
diverse panel of stimuli (including TLR ligands) is caspase-1 dependent, as well as the
resistance to experimental endotoxemia in ASC-/- and Nalp-3-/- mice 44,45, and the cytokine
production in patients with autoinflammatory disorders. The rate-limiting step is represented
in macrophages by the presence of danger signals such as ATP, that induce both an increase
in IL-1β processing, but especially secretion. A common pathway in IL-1β secretion is
represented by decreased intracellular potassium concentrations 46, induced in monocytes by
P2X7 engagement by endogenous ATP release, and in macrophages by exogenous ATP
released from tissue damage.
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Relevance for the Role of IL-1β in Host Responses to Exogenous Challenges. The
single (TLR only) stimulation in monocytes compared with the double (TLR/ATP)
stimulation in macrophages (see schematic diagram in Fig.7C) likely represents an adaptation
of each cell type to their respective environments. Circulating monocytes function in the
surveillance of an essentially pathogen-free environment, so they must respond promptly to
any danger signal (especially of microbial origin). Our experiments showing the importance
of adhesion for the inhibitory effects on IL-1β production suggest that the migration and
homing of monocyte into the tissues is essential for the phenotypic loss of IL-1β production
capacity. On the other hand macrophages are confined to an environment (e.g. alveolar space,
mucosal surfaces) in which they are constantly exposed to microbial stimuli but also to
carcinogenic substances. A sensitive response in macrophages to such stimuli for the release
of IL-1β for each encounter with such exogenous stimuli would result in recurring and
deleterious inflammatory reactions. Thus, repeated bouts of inflammation are likely reduced
by the requirement of a second stimulus for the activation of the inflammasome and release of
active IL-1β. Such second stimuli would be available at sites of infection, trauma or necrosis
where ATP levels are elevated and can trigger the P2X7 receptor 47. In addition, second
signals can come from the cathelicin-derived peptide LL37 from infiltrating neutrophils 48,
IFNγ from a lymphocytic infiltrate (Fig. 6), or the release of bacterial toxins 17.
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Acknowledgements
This work was supported by the NIH grants AI-15614 and CA-046934 to C.A.D.
M.G.N. was supported by a Vidi Grant of the Netherlands Organization for Scientific
Research.
Author contribution
M.G.N. designed and performed the research, analysed the data, wrote the manuscript, C.N-P,
M.F.N, B.O., F.vd V, B.H., J.H.M vdM., L.J., ID, G.F., and J.F performed research and
corrected the manuscript, R. J. M., B-J K, A. R., J.W.M. vdM. designed the research and
corrected the manuscript, C.A.D. designed the experiments and wrote the manuscript.
The authors have no financial conflicts of interest.
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Figure legends
Figure 1. Double purified LPS is a specific TLR4 agonist. A. Effect of LPS (1 µg/ml),
Staphylococcus peptidoglycan (PepG) (10 μg/ml), and Pam3Cys (10 µg/ml) on surface
expression of CD25 in CD14/TLR4 transfected CHO cells (relative flow cytometry units are
shown). The results of one representative experiment of three are presented. B. Mean (±
SEM) TNFα levels in supernatants from LPS (10 ng/ml)-stimulated PBMC in the absence or
absence of the TLR4 antagonist B.quintana (100 ng/ml) indicated by aTLR4. N=5,
p<0.01(compared to LPS stimulation). C. Effect of LPS (1 µg/ml), Staphylococcus
peptidoglycan (PepG) (10 μg/ml), and Pam3Cys (10 µg/ml) on surface expression of CD25
in CD14/TLR2 transfected CHO cells (relative flow cytometry units are shown). The results
of one representative experiment of three are presented. D. Mean ± SEM TNFα levels in 24
hour supernatants of LPS-stimulated PBMC from 5 volunteers bearing wild-type NOD2 allele
(NOD2 WT) and 4 individuals with a frame-shift mutation in NOS2 (NOD2fs).
Figure 2. LPS induces bioactive IL-1β in primary monocytes. A. Human PBMC, purified
CD14+ monocytes, or the CD14- lymphocytes were incubated with 10 ng/ml LPS and the IL-
1β concentrations were measured in the supernatant by specific ELISA after 24h incubation.
B. CD14+ monocytes were stimulated with various stimuli for the induction of IL-1β. C.
Human PBMC were incubated with 10 ng/ml LPS and the levels of mature and precursor IL-
1β or were measured in the supernatant by specific ELISA after 24h incubation. Insert of C.
Western blot of IL-1β and pro-IL-1β the supernatant derived from PBMC stimulated with
LPS (100 ng/ml) or MDP (10 μg/ml). D. Dose-response of caspase-1 inhibitor ZVAD-fmk on
the 24 hour levels of LPS-induced IL-1β from PBMC. E and F. The inhibitory effects of the
caspase-1 inhibitor (20 μM) and of IL-1Ra (10 μg/ml) on the induction of IL-6 (E) and
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intracellular IL-1α measured in cell-lysates (F) by 10 ng/ml LPS. Data from all four panels
are presented as means + SEM of cells harvested from 6 volunteers. *p<0.05 compared to
LPS stimulation alone.
Figure 3. Differential production of cytokines by monocytes, macrophages and dendritic
cells. Freshly isolated human PBMC, macrophages differentiated after 5 days incubation with
10% human plasma, or dendritic cells differentiated after 5 days GM-CSF (50 ng/ml) + IL-4
(20 ng/ml), were stimulated with culture medium, LPS (100 ng/ml), Pam3Cys (10 μg/ml), or
S. epidermidis (106 organisms/ml). IL-1β (A and B), TNF (C) or IL-1α (D) were measured in
the supernantant after 24h incubation. Data are presented as means + SEM of cells harvested
from 6 volunteers. *p<0.01 compared to the stimulation in monocytes.
Figure 4. mRNA levels and processing of IL-1β in monocytes vs. macrophages. A.
Steady-state levels of IL-1β and TNFα in freshly isolated PBMC or monocyte-derived
macrophages after 4 hours of incubation with, LPS (100 ng/ml), Pam3Cys (10 μg/ml), or S.
epidermidis (106 organisms/ml). B. Intracellular levels of precursor IL-1β in monocyte-
derived macrophages stimulated for 24h with the various stimuli as in A. C. Western blot of
caspase-1 peptides in monocytes (Mo), macrophages (Mf) or THP-1 cells after two hours with
and without LPS (100 ng/ml). D. Western blot of the active p10 fragment of caspase-1 (sc515
antibody from Santa-Cruz) in freshly isolated monocytes (column 1) and in monocytes that
have adhered for 2h in polystyrene plates (column 2). E. IL-1β and proIL-1β Western blots of
lysates of monocyte-derived macrophages before and after stimulation for 24 hours with 100
ng/ml LPS.
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Figure 5. The role of ASC and NALP3 in the production and release of IL-1β. THP-1
cells were transfected with siRNA against ASC or NALP3 or control scramble (siC). RT-PCR
of lysates after overnight incubation for ASC (A) or NALP3 (B). Cells were transfected with
scrambled or target siRNA, then primed for 24h with PMA (100 ng/ml), and thereafter
stimulated for 24h with LPS (1 μg/ml). After 24 hoursIL-1β (C) or IL-8 (D) were assessed in
the supernatants (*p<0.05 compared to the stimulation with LPS in cells treated with scramble
siRNA). E. Levels of supernatant IL-1β and TNFα in primary PBMC following transfection
with scramble or siRNA for ASC and subsequent stimulation with LPS for 24h. (n=5, mean
±SEM, *p<0.05 compared to LPS/scramble) F. Levels of supernatant IL-1β in PBMC
stimulated for 3h with LPS (100 ng/ml), after which ATP (1 mM) or a combination of LPS
and ATP was added for an additional 15min. PBMC were stimulated which were transfected
with either scramble or siRNA against ASC. (n=5, means + SEM, *p<0.05 compared to
LPS/ATP in the cells transfected with scramble siRNA). G. Western blot of ASC in PBMC
transfected with either scramble or siRNA against ASC. H. Western blot of the p10 and p45
caspase-1 in PBMC transfected with either scramble (column 1) or siRNA (column 2) against
ASC.
Figure 6. ATP induces IL-1β secretion in both monocytes and macrophages. A. Human
monocytes and macrophages were incubated with RPMI, LPS (1 μg/ml), or a combination of
LPS and MDP (10 μg/ml) for 24h. Intracellular pro-IL-1β or secreted mature IL-1β were
measured by specific ELISA kits. (n=6, mean ±SEM). B. Monocytes or macrophages were
incubated with RPMI or LPS for 4h, followed by ATP (1 mM) for 15 minutes (RPMI/ATP or
LPS/ATP) . Mean levels of mature IL-1β secreted in the supernatant were measured by
ELISA. (n=6, mean ±SEM). C. Western-blots of caspase-1 in lysates after stimulation of
monocytes with RPMI, LPS (1 μg/ml), ATP (1 mM), or a combination of LPS and ATP. D.
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Monocytes and macrophages were incubated with RPMI or LPS (1 μg/ml) for 4h and ATP
was measured in the supernatant using a luciferase assay (n=6, mean ±SEM, *p<0.05
compared to monocytes). E. Human monocytes and macrophages were incubated with RPMI
or LPS (1 μg/ml) for 4h, and P2X7 receptors were blocked by adding oxidized ATP (oATP,
300 μM). Concentrations of mature IL-1β were measured in the supernatants by ELISA (n=6,
mean ±SEM, *p<0.05 compared with RPMI). F. Macrophages (Mf) differentiated in the
presence of 10 ng/ml IFNγ were stimulated for 24h with LPS (1 μg/ml), MDP (10 μg/ml) or
heat-killed S. epidermidis (106 organisms/ml). IL-1β in the supernatant was measured by
ELISA. (n=6, mean ±SEM). G. Macrophages differentiated for 5 days in RPMI with 10%
plasma while in rotating (4 rpm) in 50 ml polypropylene tubes were stimulated for 24h with
LPS, MDP or heat-killed S. epidermidis. IL-1β in the supernatant was measured by ELISA.
(n=6, mean ± SEM). H. Western blot of the active caspase-1 p10 fragment in macrophages
differentiated in the absence (RPMI) or presence (IFNγ) of IFNγ. Data from two volunteers (1
and 2) are presented. I. ATP release from macrophages differentiated in the absence or
presence of IFNγ (n=5, mean + SEM, *p<0.05 compared to macrophages without IFNγ).
Figure 7. Alveolar macrophages require the second stimulation by ATP to release
mature IL-1β after LPS. A. Human alveolar macrophages were stimulated for 24h with
RPMI, LPS (100 ng/ml), Pam3Cys (10 μg/ml), or S. epidermidis (106 mo/ml). B. Alveolar
macrophages were stimulated with LPS (1 μg/ml) or a combination of LPS and ATP (1mM).
Intracellular proIL-1β after LPS stimulation after 4h, and the extracellular IL-1β release after
stimulation with LPS or LPS/ATP, was assessed by ELISA. The stimulation experiments
were performed in cells harvested from 5 volunteers (means + SEM, *p<0.05 compared to
LPS stimulation alone).
For personal use only. at CANCRO RESEARCH INST on January 9, 2009. www.bloodjournal.orgFrom
30
C. Diagram representing the IL-1β activation pathways in monocytes and macrophages.
Caspase-1 is constitutively activated in monocytes, and these cells release mature IL-1β after
single stimulation with TLR ligands. IL-1β secretion is induced by endogenously-released
ATP. In contrast, macrophages need a double stimulation: one stimulus (TLR-ligands)
induces transcription, and a second stimulus (ATP) induces IL-1β secretion.
For personal use only. at CANCRO RESEARCH INST on January 9, 2009. www.bloodjournal.orgFrom
