1914 Johanneke Kleinnijenhuis et al. DOI 10.1002/eji.200839115 Eur. J. Immunol. 2009.

39: 1914–1922

Transcriptional and inflammasome-mediated

pathways for the induction of IL-1b production by
Mycobacterium tuberculosis
Johanneke Kleinnijenhuis1,2, Leo A. B. Joosten1,2, Frank L.
van de Veerdonk1,2, Nigel Savage3, Reinout van Crevel1,2,
Bart Jan Kullberg1,2, Andre van der Ven1,2, Tom H. M. Ottenhoff3,
Charles A. Dinarello4, Jos W. M. van der Meer1,2 and Mihai G. Netea1,2

1
Department of Medicine, Radboud University Nijmegen Medical Center, Nijmegen,
The Netherlands
2
Nijmegen Institute for Infection, Inflammation, and Immunity (N4i), Nijmegen,
The Netherlands
3
Department of Immunohematology and Blood Transfusion, Leiden University Medical Center,
Leiden, The Netherlands
4
Division of Infectious Diseases, University of Colorado Health Sciences Center, Denver,
CO, USA

Proinflammatory cytokines of the IL-1 family play an important role for the anti-myco-
bacterial host defense mechanisms. In the present study we have deciphered the path-
ways leading from recognition of Mycobacterium tuberculosis to the production and release
of IL-1b, the most important member of the IL-1 family. By stimulating cells defective in
various pattern recognition receptors, we could demonstrate that IL-1b production is
induced by M. tuberculosis through pathways involving TLR2/TLR6 and NOD2 receptors. In
contrast, TLR4, TLR9 and TLR1 receptors are not involved in IL-1b induction. Recognition
of M. tuberculosis by TLR and NOD2 leads to transcription of proIL-1b through mechanisms
involving ERK, p38 and Rip2, but not JNK. Interestingly, although caspase-1 is necessary
for the processing of proIL-1b, activation of caspase-1 is not dependent on the stimulation
of cells by M. tuberculosis. Monocytes expressed constitutively active caspase-1. The
secretion of IL-1b is dependent on the activation of P2X7-induced pathways by endogen-
ously released ATP. In conclusion, we have dissected the molecular mechanisms
responsible for IL-1b production by M. tuberculosis, and that may contribute to a deeper
knowledge of the mechanisms of cell activation by M. tuberculosis.

Key words: Cytokines . Infections – bacterial . Inflammation

Introduction
It is estimated that approximately one-third of the world
population is infected with Mycobacterium tuberculosis, the agent
causing tuberculosis (TB), although only 5% of these individuals
Correspondence: Dr. Mihai G. Netea
e-mail: m.netea@aig.umcn.nl
will eventually develop clinical disease. The WHO estimated that
1.6 million deaths resulted from TB in 2005, equaling 4400
deaths a day [1]. The highest rates of infection and especially the
mortality are seen in the developing countries, and rates continue
to climb in countries where the prevalence of HIV infection is
high, despite the implementation of TB control programs [2].
Furthermore, the increased prevalence of multidrug-resistant
strains of M. tuberculosis represents a challenge for treatment
programmes [3].

& 2009 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.eji-journal.eu
Eur. J. Immunol. 2009. 39: 1914–1922
The first line of defense against M. tuberculosis is formed by
alveolar macrophages, which ingest and sequester the bacilli
within granulomatous structures. The control and resolution of the
infection involve the activation of macrophages, in which activated
T lymphocytes play a crucial role [4]. The release of
proinflammatory cytokines such as IL-1b, TNF, IL-12 or IL-18 from
monocytes and monocyte-derived macrophages induces the
production of T-cell-derived cytokines, most importantly IFN-g,
which in turn will activate macrophages for the killing and elim-
ination of the microorganisms [5, 6]. Patients with defects in
receptors for IL-12 and IFN have an increased susceptibility to
mycobacterial infections [7–9], while the association between low
CD4 counts and TB is well-known in HIV-infected patients [2].
While most of the research has focused on the mechanisms
responsible for the IFN-g induction, relatively little is known
about the mechanisms leading to the production and release of
the first wave of cytokines that are needed for the activation of
T cells. While TNF-a is known to be important for the integrity of
the granuloma, two of the cytokines of the IL-1 family, IL-1b and
IL-18, are considered to be crucial for the induction of IFN-g.
IL-1-mediated signals have been recently demonstrated to be an
essential component of the MyD88-dependent innate response to
M. tuberculosis infection, while IL-18-dependent pathways
seemed to be dispensable [10].
In contrast to other proinflammatory cytokines, IL-1b lacks a
signal peptide [11]. After transcription and translation, pro-IL-1b
is processed by the cysteine protease caspase-1, followed by
secretion from the cell [12]. Activation of caspase-1 requires
generation of a protein complex called the inflammasome, which
recognizes specific ligands such as muramyl dipeptide, ATP or
uric acid leading to caspase-1 activation [13]. Detection of the
PAMP or danger signals in the inflammasome is achieved by
proteins of the NOD-like receptor family such as NALP3, NALP1
or IPAF, leading to a conformational change and caspase-1 acti-
vation [14]. Caspase-1 activity is essential in host defenses
against infection with Francisella tularensis [15], Legionella
pneumophila [16], Shigella [17] and Pseudomonas aeruginosa
[18]. The fact that IL-1b and IFN-g play an important role during
infection with M. tuberculosis and are regulated by caspase-1
activity suggests that inflammasome activation may be important
for anti-mycobacterial defense mechanisms. In the present study,
we investigate the mechanisms responsible for IL-1b activation by
M. tuberculosis in human monocytes and alveolar macrophages,
including the receptor pathways leading to proIL-1b mRNA
transcription and the mechanisms of caspase-1 activation.
Results
M. tuberculosis induces IL-1b production through
caspase-1-dependent mechanisms
PBMC produced significant amounts of IL-1b when stimulated
with TLR2 (Pam3Cys) and TLR4 (LPS) stimuli, as well as with
M. tuberculosis (Fig. 1A). Both heat-killed and live M. tuberculosis
& 2009 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
Innate immunity 1915
stimulated comparable amounts of IL-1b. Primary alveolar
macrophages isolated from healthy volunteers also released
IL-1b after stimulation with M. tuberculosis (Fig. 1B). The
transcription of proIL-1b mRNA was stimulated by mycobacteria
(Fig. 1C), and this resulted in the release of active IL-1b in the
supernatant (Fig. 1D). The release of the mature form of IL-1b
from the cell was dependent on caspase-1 (Fig. 1E).
Both TLR2/TLR6 and NOD2 pathways mediate
induction of IL-1b by M. tuberculosis
When murine macrophages isolated from the various mouse
knock-out strains were stimulated with M. tuberculosis, cells from
TLR2/ and TLR6/ mice released significantly less IL-1b
compared with wild-type controls (Fig. 2A and B), similar to
stimulation induced by the specific TLR2 agonist Pam3Cys (not
shown). In contrast, macrophages from TLR4/ and TLR1/
mice released normal amounts of IL-1b (Fig. 2C and D). TLR9
had no role in the stimulation of IL-1b after challenge with
M. tuberculosis (Fig. 2E). LPS and CpG induced cytokine
production only in wild-type macrophages, but not in cells
isolated from TLR4/ or TLR9/ mice, respectively. Interest-
ingly, PBMC from individuals defective in NOD2 function
(Crohn’s disease patients homozygous for the 3020insC NOD2
mutation) [19] stimulated with M. tuberculosis released signifi-
cantly less IL-1b than either healthy volunteers or Crohn’s disease
patients homozygous for the wild-type NOD2 allele (Fig. 2F).
The intracellular pathways leading to IL-1b mRNA
transcription involve MyD88 and ERK/p38 kinases
To assess the role of the intracellular pathways involved in the
induction of IL-1b by M. tuberculosis, cells from MyD88/ and
TRIF-defective mice were stimulated with the microorganisms for
24 h. IL-1b production was strongly dependent on MyD88, but
not on TRIF (Fig. 3). In addition, stimulation of IL-1b in PBMC by
M. tuberculosis was mediated by the ERK, as well as p38 and/or
Rip2 kinases, but not by JNK (Fig. 4A). Quantitative PCR analysis
demonstrated that p38 and ERK inhibitors decreased IL-1b mRNA
expression (Fig. 4B). In line with these data, pro-IL-1b
intracellular concentrations were decreased by 55–70% by
addition of p38 and/or ERK inhibitors (not shown). Similar
effects were observed for the induction of IL-6 production. In
contrast, the induction of TNF-a was regulated through ERK and
JNK-dependent pathways, while blockade of p38 had no effect
(Fig. 4A).
Posttranslational mechanisms involved in the
processing and secretion of IL-1b
Active caspase-1 is important for the processing and release of
IL-1b. Surprisingly, unstimulated monocytes expressed both the
www.eji-journal.eu
1916 Johanneke Kleinnijenhuis et al. Eur. J. Immunol. 2009. 39: 1914–1922

A 5 B 1.2
*
1
4

IL-1β ng/ml

IL-1β ng/ml
0.8
3
0.6
2 0.4
1 0.2
0 0
RPMI MTB

ys

ve
I
PM

LP

-H

-li
3C
R

TB

TB
m

M
Pa

M
C RPMI MTB E RPMI MTB MTB+casp1 inh

β2m 8

6
IL-1β

ng/ml
4
D RPMI MTB 2
*
pro-IL-1β 0
IL-1 beta IL-6

IL-1β

Figure 1. M. tuberculosis induces IL-1b production through caspase-1-dependent mechanisms. (A) Human PBMC were stimulated with Pam3Cys
(1 mg/mL), LPS (10 ng/mL), heat-killed, or live M. tuberculosis (1  105 mo/mL) for 24 h at 371C. IL-1b in supernatant was measured by ELISA. (B) Human
primary alveolar macrophages isolated from healthy volunteers were stimulated with M. tuberculosis (1  105 mo/mL) for 24 h at 371C. IL-1b in
supernatant was measured by ELISA. (C) Human PBMC were stimulated with M. tuberculosis (1  105 mo/mL) for 4 h at 371C. The transcription of
proIL-1b mRNA was assessed by RT-PCR. (D) Western blot for pro-IL-1b and IL-1b of the supernatants of PBMC stimulated for 24 h with either RPMI
or M. tuberculosis (1  105 mo/mL). (E) Human PBMC were stimulated with M. tuberculosis (1  105 mo/mL), in the presence or absence of a caspase-1
inhibitor (10 mM) for 24 h at 371C. IL-1b and IL-6 in the supernatant were measured by ELISA (n 5 10, mean7SD, po0.05 by Wilcoxon ranked paired
test).

inactive (p45) and the active forms of caspase-1 (p10) even
before stimulation, while LPS and M. tuberculosis had little
additional activating effects (Fig. 5A). These data were supported
using a functional colorimetric assay that showed that in contrast
to control medium alone (1173 conventional units/mL), lysates
of freshly isolated PBMC contained 91715 units/mL caspase-1.
The secretion of IL-1b has been reported to be mediated by
the interaction between endogenous ATP and P2X7 receptor
[20]. This was also the case for the induction of IL-1b by
M. tuberculosis, as intracellular IL-1b was present in high
concentrations in PBMC stimulated with mycobacteria
(27807765 versus 20710 pg/mL in unstimulated cells,
po0.01), while blockade of P2X7 with oxATP significantly
inhibited the IL-1b release (Fig. 5B). This suggested that
endogenous ATP was present in the supernatants: indeed,
endogenous ATP was released in the supernatants by primary
human monocytes (3.570.7 nM).
Discussion
Cytokines of the IL-1 family play an important role for the anti-
mycobacterial host defense mechanisms. In the present study we
have deciphered the pathways leading from recognition of
M. tuberculosis to the production and release of IL-1b, one of
the most important proinflammatory cytokines. By stimulating
cells defective in various pattern recognition receptors, we
demonstrate that IL-1b production is induced by M. tuberculosis
through pathways involving TLR2/TLR6 and NOD2 receptors.
This leads to transcription of IL-1b through mechanisms involving
ERK, p38 and Rip2. Interestingly, although an active caspase-1
is required for the release of active IL-1b, its activation was
only moderately dependent on the stimulation of cells by
M. tuberculosis, due to the presence of constitutively active
caspase-1 in human primary monocytes.
The importance of understanding the mechanisms responsible
for IL-1b production by M. tuberculosis is underlined by the fact
that an intact IL-1-mediated signal is an essential component of
the host defense to mycobacteria [10, 21, 22]. Aerogenic infec-
tion with M. tuberculosis proved fatal for IL-1RI/ mice, result-
ing in a mycobacterial load increased by 2 logs in the lungs and
necrotizing pneumonia [21, 23]. Defective granuloma formation
was seen, and splenocytes of IL-1RI/ mice produced less IFN-g
[21]. These data are in contradiction with a recent study by
Master et al., suggesting that M. tuberculosis prevents inflamma-
some activation and IL-1b secretion [24]: if mycobacteria would

& 2009 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.eji-journal.eu
Eur. J. Immunol. 2009. 39: 1914–1922 Innate immunity 1917

A 1 B 0.8
control control
TLR2-/- TLR6-/-

ng/ml

ng/ml
0.5 0.4
*
* *
0 0
IL-1b TNFa IL-1b TNFa

C control D 1
control
1
TLR4-/- TLR1-/-

ng/ml
0.5

0 0
IL-1b TNFa IL-1b TNFa

E F 6000

1 control
TLR9-/- 4000
pg/ml

*
ng/ml

0.5
2000
*
0 0
IL-1b TNFa IL-1b TNFa

Figure 2. The role of TLR and NOD2 pathways for the induction of IL-1b by M. tuberculosis. Murine macrophages isolated from TLR2/ (A),
TLR6/ (B), TLR4/ (C), TLR1/ (D) and TLR9/ (E) mice were stimulated for 24 h at 371C with 1  105 mo/mL M. tuberculosis. The experiments were
performed in twice with ten mice/group. IL-1b and TNF-a in the supernatant were measured by ELISA. (F) PBMC from individuals defective in NOD2
function (n 5 4, NOD2def, white bars), Crohn’s disease patients with wild-type NOD2 (n 5 4, NOD2wt, gray bars) and healthy volunteers (n 5 10, black
bars) were stimulated for 24 h at 371C with 1  105 mo/mL M. tuberculosis (mean7SD, po0.05 by Wilcoxon rank test).

not induce IL-1b secretion and this pathway would be absent
during the anti-mycobacterial host defense, IL-1-deficient mice
could not be more susceptible to TB [21]. Our study also clearly
demonstrates the capacity of M. tuberculosis, either alive or heat-
killed, to stimulate IL-1b synthesis in human and murine primary
monocytes or macrophages. In contrast, the study of Master et al.
has shown defective IL-1b production only in the murine
macrophage cell-lines RAW 264.7 and J774A [24]. This demon-
strates the importance of using primary cells when the inflam-
masome activation and IL-1b induction is investigated.
Production and release of IL-1b are regulated at several levels:
the transcription of the gene, processing from the inactive 31 kDa
proIL-1b into the active 17 kDa IL-1b form, and secretion from
the cells [25]. The transcription of the genes encoding proin-
flammatory cytokines is under control of signals induced by
pattern recognition receptors on the immune cells. TLR have
been shown to be pivotal in the recognition of M. tuberculosis and
the induction of proinflammatory cytokines [26]. In our study,
the induction of IL-1b by M. tuberculosis is mediated by pathways
involving TLR2 and TLR6. These data are in line with the studies
involving TLR2 for the recognition of mycobacteria [27, 28], and
the increased susceptibility to M. tuberculosis infection of
TLR2/ mice [29]. TLR2 is a receptor for bacterial lipopeptides,
which are recognized by TLR2/TLR1 or TLR2/TLR6 heterodimers
[30, 31]. Cooperation of TLR2 and TLR6 has been described to be
involved in recognition of mycobacterial components [32]. In line
with this, IL-1b production induced by M. tuberculosis was
dependent on TLR2 and TLR6, but not TLR1 (Fig. 2). Moreover,
TLR4 also does not seem to mediate the induction of IL-1b by
M. tuberculosis, although it is involved in the general recognition
of mycobacteria. Differences in the transcription mechanisms
responsible for the induction of TNF and IL-1b are well known,
and this may be one of the factors explaining why TLR4 does not
modulate induction of IL-1b, while influencing other proin-
flammatory cytokines [27, 33]. Alternatively, other pathways
apart of TLR4 (e.g. TLR2/TLR6) may be sufficient for redundant
induction of IL-1b by M. tuberculosis. TLR9 was not significantly
engaged in the induction of IL-1b. TLR9/ mice have shown
only minor defects in pathogen clearance, while enhancing the
effects of TLR2 deficiency [34].
Interesting new information is the involvement of NOD2 as a
receptor mediating IL-1b production by M. tuberculosis. NOD2 is

& 2009 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.eji-journal.eu
1918 Johanneke Kleinnijenhuis et al.
A 0.8
control
MyD88-/-
ng/ml
0.4
* *
*
0
IL-1b TNFa
B 0.8 control
TRIF-/-
ng/ml
0.4
0
IL-1b TNFa
Figure 3. The role of MyD88 and TRIF pathways for the induction
of IL-1b by M. tuberculosis. Murine macrophages isolated from
MyD88/ (A) and TRIF/ (B) mice were stimulated for 24 h at 371C
with 1  105 mo/mL M. tuberculosis. IL-1b and TNF-a in the supernatant
were measured by ELISA (n 5 10, means7SD, po0.05 by Wilcoxon
rank test).
a receptor of bacterial peptidoglycans [35] and only recently we
demonstrated its role in the recognition of mycobacteria [36, 37].
It has also been recently shown that common polymorphisms of
NOD2 are strongly associated with TB in African-American
patients [38]. These data argue that TLR and NOD2 collaborate
for an effective recognition of M. tuberculosis and induction of
IL-1b production.
We further investigated the mechanism of IL-1b production by
identifying the signaling pathways leading to transcription of the
IL-1b gene. TLR2 induces intracellular signals through Mal/
MyD88, while TLR4 is able to activate a secondary TRIF/TRAM-
dependent pathway. In line with the data obtained in the various
TLR knock-out mice, IL-1b induction was dependent on MyD88,
but not on TRIF. MyD88/ mice are also known to be more
susceptible to mycobacterial infection [39, 40]. The distal intra-
cellular pathways induced by TLR involve the MAPK ERK, JNK
and p38. Recently, it has been shown that the blockade of LPS-
induced p38 kinase activation inhibits the IL-1b production in rat
pulmonary interstitial macrophages [41], but no data on the role
of MAPK on IL-1b induction by mycobacteria are available. Our
data show that both the ERK and p38 and/or Rip2 pathways are
part of the intracellular pathway of IL-1b gene activation induced
by M. tuberculosis. The pharmacological inhibitors available
cannot discern between the TLR/p38 pathways and the NOD2/
Rip2-mediated pathways, and future experiments are needed to
& 2009 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
Eur. J. Immunol. 2009. 39: 1914–1922
A 10 control
MTB
8 MTB/p38i
MTB/ERKi
MTB/JNKi
6
ng/ml
*
4
2 *
*
* *
0
IL-1beta IL-6 TNF
B 120
IL-1beta mRNA
100
80
60
40
20
0
MTB p38i ERKi
Figure 4. The role of intracellular kinase pathways for the induction of
IL-1b by M. tuberculosis. (A) PBMC were preincubated for 1 h with the
inhibitors for the kinases p38 (20 nM), Erk (10 nM), or JNK (25 M), before
the stimulation for 24 h at 371C with M. tuberculosis (1  105 mo/mL).
IL-1b, IL-6 and TNF-a in the supernatant were measured by ELISA
(n 5 6, means7SD, po0.05 by Wilcoxon rank test). (B) Inhibitors of p38
and ERK kinases decreased the expression of IL-1b mRNA after 4 h
stimulation of PBMC with M. tuberculosis, as assessed by quantitative
PCR (one representative experiments out of three performed).
determine the relative importance of these two mechanisms. In
contrast, no role for the JNK kinases for the induction of IL-1b by
M. tuberculosis has been observed.
The processing of IL-1b is assumed to be mediated by caspase-
1 activation by a protein complex formed mainly by receptors of
the NOD-like receptor family, called the inflammasome. Several
inflammasomes activate IL-1b during bacterial infections, such as
the NALP3 inflammasome that recognizes bacterial ligands
including muramyl dipeptide [42], the NALP1b inflammasome
that recognizes anthrax lethal toxin [43], and the Salmonella-
induced Ipaf inflammasome [43]. Although M. tuberculosis
contains peptidoglycans, the bacterial structures containing
muramyl dipeptide, it is not known whether inflammasome
components recognize and are activated by mycobacterial PAMP.
We have shown that IL-1b production by human monocytes
stimulated with M. tuberculosis is caspase-1 dependent. When we
assessed the capacity of M. tuberculosis to activate caspase-1, we
observed that caspase-1 was already present in its active form in
freshly isolated human PBMC, and only moderately upregulated
by stimulation with mycobacteria. These data were supported by
the demonstration of functional caspase-1 in human primary
monocytes using a functional colorimetric assay. This implies that
www.eji-journal.eu
Eur. J. Immunol. 2009. 39: 1914–1922
A B 10 RPMI
MTB
8
IL-1beta (ng/ml)
MTB/oxATP
RPMI LPS MTB
6 and PBMC contain constitutively active caspase-1. These data
p45
4 question the necessity for inflammasome activation during
p10 2
0
intracellular
C MTB
P2X7
TLR2/6
Mal
MyD88 K+ influx
ATP
NOD2
Constitutively
ERK, p38 active caspase-1
Rip2
mRNA ProIL-1β
Figure 5. Mechanisms involved in the processing and secretion of
IL-1b. (A) Western blots of the inactive (p45) and active (p10) caspase-1
fragments in monocytes isolated from healthy volunteers, either not
stimulated (RPMI), or stimulated with LPS or M. tuberculosis (MTB). One
representative experiment out of three experiments is presented.
(B) The role of the endogenous ATP release for the stimulation of IL-1b
was investigated by blocking P2X7 receptors with oxATP (300 mM)
during the stimulation of cells for 24 h with M. tuberculosis. Mature
IL-1b concentrations were measured by ELISA in both PBMC cell lysates
and supernatants. (n 5 6, means7SD, po0.05 by Wilcoxon rank test).
(C) Diagram depicting the pathways leading to IL-1b production and
release induced by M. tuberculosis.
in human primary monocytes the inflammasome is constitutively
active, and bacterial products are able only to moderately
increase caspase-1 activation and the processing of proIL-1b.
Therefore, although inflammasome components such as NALP3
and ASC are central for caspase-1 activation, this process does
not necessarily require activation by microbial products in
human monocytes. This is in contrast with the literature from
which the concept was proposed that inflammasome activation
by danger molecules (either bacterial or not) is an essential
step for the induction of IL-1b production [44]. However, this
concept has been built upon a body of evidence derived
from monocyte/macrophage cell-lines (e.g. THP-1 cells 0 or
murine cells, and not from primary cells [42, 45]). Important
differences between THP-1 cells (no active caspase-1) and
primary human monocytes (constitutively active caspase-1) for
the activation of caspase-1 have been recently demonstrated by
our group [46]. Thus, the present study cautions against the
extrapolation of data obtained in cell-lines with the physiology
of human primary cells.
The release of IL-1b from the cells was dependent on endo-
genous ATP production from the monocytes [47], and the
blockade of the P2X7 receptor with oxATP significantly reduced
IL-1b production.
IL-1b is one of the important proinflammatory cytokines with
anti-mycobacterial activities, and in this study we show that
& 2009 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
Innate immunity 1919
M. tuberculosis is a potent inducer of IL-1b secretion from human
primary PBMC and alveolar macrophages. Interestingly, the main
regulatory step of IL-1b production is at the level of transcription,
* recognition of mycobacteria by these cells. Upon recognition of
M. tuberculosis, TLR2/TLR6 and NOD2 receptors induce IL-1b
extracellular
transcription through pathways involving Erk, p38 and Rip2
(Fig. 5C). In conclusion, we have dissected the molecular
mechanisms responsible for IL-1b production by M. tuberculosis,
IL-1β and this contributes to a better understanding of the mechanisms
leading to host cell activation by mycobacteria.
Materials and methods
Volunteers
IL-1β
Blood was collected from ten healthy, non-smoking volunteers
who had no infectious or inflammatory disease at the time of the
study. In addition, blood was collected from 74 patients with
Crohn’s disease, and the presence of the 3020insC NOD2
polymorphism was analyzed by Genescan analysis on an ABI-
Prism 3100 Genetic Analyzer, as previously described [19]. Four
patients with Crohn’s disease were found to be homozygous for
the 3020insC mutation, and they were further investigated in the
cytokine studies. None of the patients with Crohn’s disease used
immunosuppressive medication at the time of the study. The
study was approved by the Ethics Committee of the Radboud
University Nijmegen. In addition, alveolar macrophages collected
from healthy volunteers by bronchoalveaolar lavage were
suspended to a concentration of 5  106 cells/mL and stimulated
for cytokine production.
Microorganisms and reagents
Cultures of M. tuberculosis H37Rv were grown to mid-log phase
in Middlebrook 7H9 liquid medium supplemented with oleic
acid/albumin/dextrose/catalase (Difco, Becton-Dickinson, Palo
Alto, CA), washed three times in sterile saline, and resuspended
in RPMI 1640 medium at various concentrations. Separate
culture suspensions were sonicated for 10 min on ice, in order
to obtain cell lysates. The irreversible caspase-1 inhibitor
Ac-Tyr-Val-Ala-Asp-2,6 dimethylbenzoyloxymethylketone was
purchased from AlexisBiochemicals (San Diego, CA). The
inhibitor was reconstituted in 10 mM DMSO and subsequently
diluted to the desired concentration in RPMI. SB202190, p38/
Rip2 MAPK inhibitor (p38-i); SP600125, JNK1/2/3 inhibitor
(JNK-i) and U0126 MEK1/2 inhibitor (Erk-i) were purchased
from Superarray Bioscience Corporation (Bethesda, MD). In
experiments using pharmacological inhibitors, control cells were
treated with an equivalent concentration of vehicle (0.01–0.1%
DMSO).
www.eji-journal.eu
1920 Johanneke Kleinnijenhuis et al.
Isolation of PBMC and stimulation of cytokine
production
After informed consent, venous blood was drawn from the cubital
vein of patients and healthy volunteers into three 10-mL EDTA
tubes (Monoject, s-Hertogenbosch, Netherlands). Isolation of
PBMC was performed as described elsewhere [48], with minor
modifications. The PBMC fraction was obtained by density
centrifugation of blood diluted 1:1 in pyrogen-free saline over
Ficoll–Paque (Pharmacia Biotech, Uppsala, Sweden). Cells were
washed twice in saline and suspended in culture medium (RPMI
1640 DM) supplemented with 10 mg/mL gentamicin, 10 mM
L-glutamine and 10 mM pyruvate. The cells were counted in a
Coulter counter (Coulter Electronics, Mijdrecht, Netherlands),
and the number was adjusted to 5  106 cells/mL.
PBMC (5  105) were added in a 100 mL volume to round-
bottom 96-well plates (Greiner, Alphen, Netherlands), incubated
with 100 mL of culture medium (negative control) or M. tubercu-
losis bacteria (1  105 mo/mL), with or without caspase-1 inhibitor
at different concentrations. In some blocking experiments, PBMC
were preincubated for 1 h with the inhibitors p38-i 20 nM, Erk-i
10 nM, or JNK-i 25 nM, before the stimulation with M. tuberculosis.
The role of the endogenous ATP release for the stimulation of IL-1b
was investigated by blocking P2X7 receptors with oxATP (300 mM)
during the stimulation of cells for 24 h with LPS [20]. The stimuli
were checked for the contamination with LPS in the Limulus
amoebocyte lysate assay and was found to be negative. In addition,
alveolar macrophages collected from healthy volunteers by
bronchoalveaolar lavage were suspended to a concentration of
5  106 cells/mL and stimulated for cytokine production.
Cytokine production by murine peritoneal
macrophages
Resident peritoneal macrophages were harvested from either
control mice or mice deficient for the various TLR or adaptor
molecules. TLR1/, TLR2/, TLR4/, TLR6/, TLR9/ and
MyD88/ mice back-crossed to the seventh generation into the
C57Bl/6J background were kindly provided by S. Akira (Osaka
University, Japan). TRIF-defective mice (Lps2 strain) were a kind
donation of B. Beutler (Scripps Institute, San Diego). After
centrifugation and washing, the cells were resuspended in RPMI
1640 containing 1 mM pyruvate, 2 mM L-glutamine, 100 mg/mL
gentamicin and 2% fresh mouse plasma. Cells were cultured in 96-
well microtiter plates (Greiner) at 1  105 cells/well, in a volume of
100 mL. The cells were stimulated with M. tuberculosis (1  105 mo/
mL). After 24 h incubation at 371C, the supernatants were collected
and stored at 701C until cytokine assays were performed.
Cytokine and ATP measurements
Human and murine cytokine concentrations were measured by
commercial ELISA kits (Pelikine Compact, CLB, Amsterdam,
& 2009 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
Eur. J. Immunol. 2009. 39: 1914–1922
Netherlands), according to the instructions of the manufacturer.
ATP concentrations in the supernatants were assessed using a
firefly luciferase assay (ATP determination kit, Invitrogen,
Carlsbad, CA).
Caspase-1 colorimetric assay
In order to assess the functional activity of caspase-1, PBMC were
incubated for 2 h with either RPMI or various stimuli. Cells were
lysed and caspase-1 activity was assessed using a commercial
colorimetric assay (R&D Systems, Minneapolis, MN).
Western blot assays
After stimulation with either RPMI or M. tuberculosis for 2 h,
PBMC (107 cells/well in a total volume of 1 mL) were lysed in
100 mL 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
glycerophosphate, 50 mM sodium fluoride, 200 mM sodium
vanadate, 10 mg/mL leupeptin, 10 mg/mL aprotinin, 1 mM pepsta-
tin A and 1 mM phenylmethylsulfonyl fluoride). Following
centrifugation (10 rpm, 5 min), the protein content was deter-
mined by BCA protein assay (Pierce) and equal amounts of
protein were loaded on 12% SDS-PAGE and transferred onto
nitrocellulose membranes. For caspase-1 quantitations,
membranes were incubated with specific caspase-1 p10 poly-
clonal antiserum (1/500) and procaspase-1 polyclonal antiserum
(1/500) (Santa Cruz). b-actin was quantified as an internal
control using specific anti-b-actin polyclonal antiserum (1/1000)
(Santa Cruz). Blots were washed and incubated with peroxidase-
conjugated goat anti-rabbit IgG (1/1000 dilution). After washing
the blots three times with TBS-T the blots where developed with
hyperECL according to manufacturer’s instructions (GE Health-
care). In addition, supernatants of PBMC stimulated for 24 h with
either RPMI or M. tuberculosis and were assessed for the presence
of pro-IL-1b and IL-1b. Membranes were incubated with anti-
(pro)IL-1b polyclonal antibody (1/1000 dilution) (Cell Signal-
ing), while the rest of the procedure was similar with that
employed for caspase-1 Western blots.
Quantitative PCR
PBMC were stimulated as described under the section Isolation of
PBMC. After 4 h, the supernatant was removed and the cells were
resuspended in 200 mL RNAzolB RNA isolation solvent (Campro
Scientific) and stored at 801C. mRNA was isolated following the
manufacturer’s protocol. The amount and quality of mRNA were
determined using a NanoDrops ND-1000 UV–Vis Spectrophot-
ometer (Thermo Fisher Scientific, Wilmington, DE). cDNA was
synthesized from using Superscript Reverse Transcriptase (Invitro-
gen). Relative mRNA levels were determined using the Biorad I-
Cycler and SYBR Green method (Invitrogen) The following
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Eur. J. Immunol. 2009. 39: 1914–1922
primers were used: IL-1b forward (50 -TGGCCCAGGCAGTCAGA-
30 ), and reverse (50 -GGTTTGCTACAACATGGGCTACA-30 ); b2 M
forward (50 -ATGAGTATGCCTGCCGTGTG-30 ) and reverse (50 -
CCAAATGCGGCATCTTCAAAC-30 ) (Biolegio, Malden, Nether-
lands).
Statistical analysis
The differences between groups were analyzed by the
Mann–Whitney U test, and where appropriate by the Wilcoxon
rank test to determine differences between groups where
pr0.05. All experiments were performed at least twice, and the
data presented as the cumulative result of all experiments
performed. Data are given as means7SD.
Acknowledgements: This study was performed within the Top
Institute Pharma consortium (D1-101), The Netherlands. The
authors are grateful to Trees Jansen and Liesbeth Jacobs for
technical assistance. M. G. N. was supported by a Vidi grant of the
Netherlands Organization of Scientific Research.
Conflict of interest: The authors declare no financial or
commercial conflict of interest.
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Abbreviation: TB: tuberculosis
Full correspondence: Dr. Mihai G. Netea, Department of Medicine (463),
Radboud University Nijmegen Medical Center, Geert Grooteplein 8,
6525 GA Nijmegen, The Netherlands
Fax: 131-24-3541734
e-mail: m.netea@aig.umcn.nl
Received: 26/11/2008
Revised: 3/3/2009
Accepted: 20/4/2009
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