Journal of Leukocyte Biology eBioscience full spectrum cell analysis
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Originally published online as doi:10.1189/jlb.1006627 on April 2, 2007

Published online before print April 2, 2007
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
jlb.1006627v1
82/1/177    most recent
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Pan, Q.
Right arrow Articles by Ulevitch, R. J.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Pan, Q.
Right arrow Articles by Ulevitch, R. J.
(Journal of Leukocyte Biology. 2007;82:177-183.)
© 2007 by Society for Leukocyte Biology

MDP-induced interleukin-1ß processing requires Nod2 and CIAS1/NALP3

Qilin Pan*, John Mathison*, Colleen Fearns*, Vladimir V. Kravchenko*, Jean Da Silva Correia*, Hal M. Hoffman{dagger}, Koichi S. Kobayashi{ddagger}, John Bertin§,1, Ethan P. Grant§,1, Anthony J. Coyle§,2, Fayyaz S. Sutterwala||, Yasunori Ogura||, Richard A. Flavell|| and Richard J. Ulevitch*,3

* Department of Immunology, The Scripps Research Institute, La Jolla, California, USA;
{dagger} Ludwig Institute of Cancer Research and University of California at San Diego, La Jolla, California, USA;
{ddagger} Department of Cancer Immunology & AIDS, Dana-Farber Cancer Institute, Harvard School of Public Health, Boston, Massachusetts, USA;
§ Millennium Pharmaceuticals, Inc., Cambridge, Massachusetts, USA; and
|| Section of Immunobiology, Yale University School of Medicine, New Haven, Connecticut, USA

3 Correspondence: Department of Immunology, IMM12, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, CA 92037, USA. E-mail: ulevitch{at}scripps.edu


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Nucleotide-binding oligomerization domain (Nod)2 is a sensor of muramyl dipeptides (MDP) derived from bacterial peptidoglycan. Nod2 also plays a role in some autoinflammatory diseases. Cold-induced autoinflammatory syndrome 1 (CIAS1)/NACHT domain, leucine-rich repeat, and pyrin domain-containing protein 3 (NALP3) has been suggested to be sufficient for MDP-dependent release of mature IL-1ß, but the role of Nod2 in this process is unclear. Using mice bearing selective gene deletions, we provide in vitro and in vivo data showing that MDP-induced IL-1ß release requires Nod2 and CIAS1/NALP3 as well as receptor-interacting protein-2 (Rip2), apoptosis-associated speck-like protein containing a caspase activation and recruitment domain (ASC), and caspase-1. In contrast, MDP-dependent IL-6 production only requires Nod2 and Rip2. Together, our data provide a new understanding of this important pathway of IL-1ß production and allow for further studies of the role of these proteins within the broader context of inflammatory disease.

Key Words: cytokine • inflammation • PAMP • NLR


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Historically, the major function of the innate immune system was thought to be the removal of opsonized pathogens or foreign materials. Compared with myriad recognition specificities in adaptive immunity, innate immunity was thought to be regulated by a limited number of nonselective receptors. However, the past 5 years have witnessed remarkable advances in our understanding of innate immunity at the molecular level. Central to this has been the identification of two protein families, which serve as sensors for viral and bacterial components. The most well-studied family is that comprised of transmembrane proteins known as TLRs [1 ]. A second important family consists of intracellular proteins and is known as the nucleotide-binding oligomerization domain (Nod)/caspase activation and recruitment domain, transcription enhancer, R (purine)-binding, pyrin, lots of leucine repeats (caterpillar)/Nod-like receptor (NLR) family [2 3 4 ]. It is important that the TLRs and NLRs recognize nonoverlapping ligands and do not share their proximal signaling pathways [5 ].

Genetic studies in man have linked mutations in NLR family members with various human diseases characterized by dysregulated, inflammatory responses. For example, mutations in Nod2 have been linked to Blau syndrome [6 ] or to increased susceptibility to Crohn’s disease [7 , 8 ]. Mutations in another NLR family member, CIAS1/NACHT domain, leucine-rich repeat, and pyrin domain-containing protein 3 (NALP3), are linked to three autoinflammatory disorders, collectively known as cryopyrinopathies, which include familial cold autoinflammatory syndrome, Muckle-Wells syndrome, and neonatal onset multisystem inflammatory disease [9 , 10 ]. Hyperproduction of IL-1ß has been suggested to be causal in at least some of these diseases. Studies of CIAS1/NALP3 have provided support for a multiprotein complex, the inflammasome, and its role in processing of IL-1ß [11 , 12 ]. Most recently, in studies with CIAS1/NALP3-deficient mice, a variety of proinflammatory agonists was shown to require CIAS1/NALP3 for caspase-1-dependent processing of pro-IL-1ß [13 14 15 16 ]. It has also been suggested that muramyl dipeptide (MDP)-induced IL-1ß requires CIAS1/NALP3 [17 ]. However, MDP was first shown to be a specific activator of another NLR family member, Nod2 [18 ]. Despite this, several recent reviews suggested that MDP induces IL-1ß release solely through a CIAS1/NALP3-dependent pathway [19 , 20 ] and surprisingly, do not consider any role for Nod2. At present, the relationships among Nod2, CIAS1/NALP3, and other proteins linked to Nod2 and CIAS1/NALP3 pathways have not been studied directly in the context of processing of pro-IL-1ß to mature IL-1ß. This has been a significant gap in our knowledge needing clarification because of the potential roles for Nod2 and CIAS1/NALP3 in human disease.

IL-ß is an important mediator of chronic inflammatory diseases including rheumatoid arthritis, psoriasis, and Crohn’s disease [21 ]. In contrast to many other cytokines, which are almost exclusively regulated at the transcriptional level, mature IL-1ß release is controlled by a key post-transcriptional event involving caspase-1-dependent proteolysis of pro-IL-ß to the mature form. As caspase-1 is present in cells in a precursor form, its activation must occur to process pro-IL-1ß. Here, we have used bone marrow-derived macrophages (BMDM) from wild-type and mice bearing specific gene deletions in members of the NLR family or in other genes thought to be linked to pathways involving NLR family members to clarify the requirements for MDP-induced IL-1ß release. Data from in vivo and in vitro studies support our contention that Nod2, together with CIAS1/NALP3, in conjunction with a number of additional proteins, including caspase-1, apoptosis-associated speck-like protein containing a caspase activation and recruitment domain (Asc), and receptor-interacting protein-2 (Rip2), is required for MDP-induced, IL-1ß release. The totality of our data indicates that Nod2 and CIAS1/NALP3 have essential, nonredundant roles in processing pro-IL-1ß when there is a source of MDP present in the extracellular space.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Mice, cells, and reagents
Dr. Vishva Dixit (Genentech, Inc., South San Francisco, CA, USA) provided Asc–/– BMDM [22 ] and IL-1ß-converting enzyme protease-activating factor (IPAF)–/– BMDM [22 ]. Dr. Koichi Kobayashi and Dr. Richard Flavell (Yale University School of Medicine, New Haven, CT, USA) generously provided Nod2–/– BMDM and mice [23 ], Rip2–/– BMDM [24 ], CIAS1/NALP3–/– BMDM [16 ], and caspase-1–/– BMDM [25 ]. Finally, studies with CIAS1/NALP3–/– mice [13 , 16 ] were performed in DR. H. M. Hoffman’s lab in the Ludwig Institute of Cancer Research at the University of California San Diego (UCSD; La Jolla, CA, USA). The Scripps Research Institute (La Jolla, CA, USA) and UCSD Institutional Animal Care and Use Committees approved all animal experimental protocols. MDP was purchased from Sigma Chemical Co. (St. Louis, MO, USA); ATP was purchased from Roche (Indianapolis, IN, USA); LPS, isolated from Escherichia coli O111:B4, was purchased from List Biological Laboratories (Campbell, CA, USA).

Macrophage cell culture
BMDM were isolated and cultured as described [26 ]. Briefly, BM was flushed from mouse femurs and cultured in BM macrophage growth media (DMEM containing 10% heat-inactivated FBS, 30% L929 cell-conditioned media, 2 mM L-glutamine, 100 µg ml–1 streptomycin, and 100 units ml–1 penicillin) for 7 days at 37°C to allow differentiation and maturation. The cells were observed to be >94% CD11b positive by FACS.

EMSA
Preparation of nuclear extracts and EMSA were performed as described previously [27 ]. Briefly, cells were lysed in buffer A (10 mM HEPES, pH 7.9, 10 mM KCl, 0.1 mM EDTA, 0.1 mM EGTA, 1 mM DTT, 0.5 mM PMSF) on ice. Nuclei were separated from cytosol by centrifugation and were resuspended in Buffer B (20 mM HEPES, pH 7.9, 0.4 M NaCl, 1 mM EDTA, 1 mM EGTA, 0.1 mM PMSF). The supernatants containing nuclear proteins after a second centrifugation were transferred to new vials. Aliquots of the nuclear extracts (2.5 µg) were incubated with 12 µl binding buffer for 10 min at room temperature. Then, 32P-labeled NF-{kappa}B oligonucleotide probe was added, and the mixture was incubated at room temperature for 10 min. The samples were analyzed on nondenaturing 5% acrylamide gels. Gel contents were transferred to Whatman DE-81 paper, dried, and exposed overnight at –80°C with an intensifying screen.

In vivo IL-1ß release assay
Mice (8–12 weeks old) were injected i.p. with 10 µg LPS from E. coli (Serotype 0111:B4, List Biological Laboratories). Three hours later, these mice received an additional injection of MDP (300 µg in 50 µl saline), ATP (50 µl 100 mM in saline), or saline (50 µl). Serum samples were collected by retro-orbital bleed 1.5 h after the second injection.

Macrophage infection with Salmonella typhimurium
BMDM were plated in a 96-well plate at 5 x 104 cells per well in BM macrophage growth media and primed with 0.5 ng/ml LPS for 6 h to induce intracellular pro-IL-1ß. Cells were washed with fresh media, which lacked antibiotics, and infected with wild-type S. typhimurium CS401 (kindly provided by Dr. Samuel Miller, University of Washington, Seattle, WA, USA) at a multiplicity of infection of 50/cell for 30 min.

Cytokine measurements
Culture supernatants from infected/stimulated macrophages or serum from activated mice were assayed for IL-1ß and IL-6 by ELISA (PharMingen, San Diego, CA, USA).

Detection of caspase-1 activation and IL-1ß procession
Ten milliliters of culture supernatants from MDP-activated or S. typhimurium-infected macrophages was precipitated with 25 µg rabbit anticaspase-1 (SC514, Santa Cruz Biotechnology, Santa Cruz, CA, USA) or 15 µg goat antimouse IL-1ß (AF-401-NA, R&D Systems, Minneapolis, MN, USA) in the presence of protein-G beads. The complexes were resolved in 4–20% Tris-glycine gradient gels (Invitrogen) and transferred to Immobilon-P [polyvinylidene difluoride (PVDF)] membranes (Millipore, Bedford, MA, USA) by electroblotting. The membranes were immunoblotted with rabbit anticaspase-1 (SC514, Santa Cruz Biotechnology) and hamster antimouse IL-1ß (Clone B122, BD PharMingen, San Diego, CA, USA), respectively.

Western blotting
For Western blotting analysis, the lysates from activated macrophages were resolved in 12% SDS-PAGE gel, and the following antibodies were used for immunoblotting: rabbit anti-I{kappa}B{alpha} (Santa Cruz Biotechnology), mouse anti-phospho-p38 (Cell Signaling Technology, Beverly, MA, USA), rabbit anti-p38 (Cell Signaling Technology).

Statistical analysis
The significance of experimental observations was evaluated by a two-tailed t test. A P value of less than 0.05 was considered significant.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
MDP induces release of mature IL-1ß by a Nod2-dependent pathway
We isolated murine BMDM and used these cells to study MDP-induced release of mature IL-1ß. Two distinct signals are required to induce release of mature IL-1ß. Proinflammatory signals such as LPS prime cells to synthesize intracellular pro-IL-1ß (34 kDa), and then a variety of second signals supports caspase-1-dependent proteolysis of the IL-1 precursor, leading to release of mature IL-1ß (17 kDa). Here, we describe experimental strategies developed in our laboratory to maximize the release of the mature IL-1ß from BMDM. These include the use of subnanogram amounts of highly purified LPS for priming of BMDM, followed by the addition of MDP. In addition, we add a low concentration of cycloheximide (CHX), which as we show here, and others have reported, appears to have effects on signaling pathways that are unexpected [1 , 28 29 30 31 ]. First, we showed that CHX addition enhanced MDP-induced translocation of NF-{kappa}B to the nucleus (Fig. 1A ), increased the extent and duration of MDP-induced p38 phosphorylation, and prevented the resynthesis of I{kappa}B{alpha} (Fig. 1B) . It is most important that CHX enhanced MDP-induced release of mature IL-1ß to levels substantially above that induced by MDP alone but had no effect on release of mature IL-1ß induced by ATP, a known inflammasome activator (Fig. 1C) . Moreover, we have observed enhanced cytokine production with the addition of low-dose CHX in a variety of cell types activated with MDP or with peptidic Nod1 activators containing diamino-pimelic acid but not with activators of TLRs such as LPS (R. J. Ulevitch, C. Fearns, Q. Pan, unpublished data). Although our understanding of how CHX increases the extent of MDP-induced cell activation is not complete, we have compared the combined effects of CHX and MDP with MDP alone in all of the in vitro studies presented here to enhance the pathways involved in IL-1ß production.


Figure 1
View larger version (45K):
[in this window]
[in a new window]

  		Figure 1. CHX enhances MDP signaling in BMDM. (A) CHX enhances MDP-induced NF-{kappa}B DNA-binding activity. C57BL/6 BMDM were primed with LPS (0.5 ng/ml) for 6 h, and then media were removed, and cells were stimulated with MDP (10 µg/ml), CHX (1 µg/ml), or MDP together with CHX for the indicated time-points. DNA-binding activity was determined by EMSA. Time 0 is set at the end of LPS priming. n.s., Nonspecific band. (B) CHX enhances MDP-induced p38 phosphorylation and I{kappa}B{alpha} degradation. BMDM were stimulated as described in A, and protein phosphorylation (p38) and degradation (I{kappa}B{alpha}) were detected by specific immunoblot. (C) CHX enhances MDP-induced IL-1ß release. LPS-primed BMDM were stimulated with MDP (10 µg/ml) or ATP (5 mM), alone or in the presence of CHX (1 µg/ml) for 18 h, and IL-1ß in cell supernatants was measured by ELISA. Data are representative of three independent experiments. Results represent the mean ± SD of triplicate wells.

 
We first compared MDP and highly purified LPS for induction of pro-IL-1ß protein in BMDM. It is surprising that MDP alone failed to induce pro-IL-1ß protein, and LPS was a strong inducer (Fig. 2A , see pro-IL1ß panel). Although LPS induces pro-IL-1ß potently, we were only able to detect low amounts (<0.1 ng/ml) of mature IL-1ß in the supernatants. Thus, the LPS isolate used here alone does not induce significant caspase-1 activation, and this result is consistent with findings from Martinon et al. [17 ]. MDP is also a weak inducer of NF-{kappa}B, as measured by p38 phosphorylation and I{kappa}B{alpha} degradation, when compared with the effects of LPS. The weak induction of NF-{kappa}B may provide some insight into why MDP alone fails to up-regulate pro-IL-1ß (Fig. 2A) . Finally, our data are in line with the view that multiple steps induced by distinct signals are required to produce mature IL-1ß.


Figure 2
View larger version (63K):
[in this window]
[in a new window]

  		Figure 2. MDP activates caspase-1 and induces maturation of IL-1ß in the presence of CHX. (A) LPS, but not MDP, induces intracellular pro-IL-ß. C57BL/6 BMDM were stimulated with MDP (10 µg/ml) or LPS (1 ng/ml) for different time-points. Phosphorylation of p38, degradation of I{kappa}B{alpha}, and induction of pro-IL-1ß were monitored by immunoblotting of cell lysate. The sustained p38 activation seen at 6 h compared with that observed in Figure 1 may be a result of stimulation with a higher concentration of LPS. (B) Cells were primed with LPS (0.5 ng/ml) for 6 h and then activated by MDP (M; 10 µg/ml) and CHX (C; 1 µg/ml) with freshly added media for 18 h. As a positive control, cells were also infected with S. typhimurium for 30 min. Activated caspase-1 in the culture supernatants was immunoprecipitated and immunoblotted using a rabbit anticaspase-1 antibody. Secreted, mature IL-1ß was immunoprecipitated and immunoblotted by goat and hamster anti-IL-1ß antibodies, respectively.

 
Support for this contention is derived from experiments where we compared responses of BMDM from wild-type (Nod2+/+) or Nod2–/– mice primed with LPS and then stimulated with MDP (±CHX, Fig. 2B ). As activated macrophages process 45 kDa caspase-1 into two subunits (p20, p10) and secrete these fragments into the extracellular space, we looked for the presence of mature IL-1ß and activated caspase-1 in the cell supernatants. We also infected macrophages with S. typhimurium to examine Nod2-independent pathways of caspase-1 activation and IL-1 production. The inclusion of CHX together with MDP provided a sufficiently strong response, which we were able to detect with mature IL-1ß (17 kDa) and the p10 fragment derived from activated caspase-1 when we stimulated Nod2+/+ cells but not when we used Nod2–/– cells. As expected, Nod2 deficiency did not reduce the ability of Salmonella infection to induce caspase-1 activation or release of mature IL-1ß. The amount of mature IL-1ß present in the supernatants of MDP-treated BMDM primed with LPS is detectable by ELISA (data not shown) but below the sensitivity of the combined immunoprecipitation and biochemical analyses shown here. Nonetheless, inclusion of CHX amplified the cellular response so that the released proteins were visualized in our Western blots. Thus, in their totality, our data establish the use of the BMDM model to study MDP-induced IL-1ß, as we are able to observe the induction of pro-IL1ß, caspase-1 activation, and release of mature IL-1.

Identification of proteins required for MDP-induced IL-1ß release by BMDM
Studies from several laboratories have provided support for a model, whereby oligomerization of NLRs results in recruitment of adaptor proteins into a multiprotein complex, which catalyzes caspase-1 activation [25 , 32 ]. The composition of a caspase-1-activating complex formed in responses to MDP is unknown. To address this gap in our knowledge, we used a series of murine BMDM derived from mice bearing specific gene deletions in the NLR family members Nod2 [23 , 33 ], CIAS1/NALP3 [13 , 16 ], and IPAF [34 , 35 ]; in the kinase Rip2 [24 ]; and in the adaptor protein Asc [22 ]. First, we asked whether LPS-induced, pro-IL-1ß protein expression was changed when wild-type and BMDM bearing the previous group of gene deletions are compared. We observed that the absence of Nod2, Rip2, Asc, CIAS1/NALP3, caspase-1, or IPAF (Fig. 3A 3B 3C 3D 3E 3F ) did not prevent expression of LPS-induced, pro-IL-1ß protein. In contrast, BMDM from TLR4–/– mice failed to express pro-IL-1ß protein after addition of LPS (data not shown). It is not surprising that we did note a modest increase in pro-IL-1ß protein in wild-type cells when we added MDP to the LPS-pretreated BMDM from wild-type but not from Nod2 or Rip2–/– BMDM. Moreover, we observed that the addition of CHX did not reduce pro-IL-1ß protein expression in any of the LPS-pretreated BMDM.


Figure 3
View larger version (53K):
[in this window]
[in a new window]

  		Figure 3. Intracellular pro-IL-1ß induction was not affected in Nod2–/–, Rip2–/–, Asc–/–, CIAS1/NALP3–/–, IPAF–/–, and caspase-1–/– cells. BMDM were primed with LPS (0.5 ng/ml) for 6 h, and then MDP (10 µg/ml) and CHX (1 µg/ml) were added to cells with fresh media. Cells were lysed with SDS-running buffer 6 h later. Pro-IL-1ß induction was assayed by immunoblot.

 
We next compared IL-1ß and IL-6 release using BMDM from wild-type or from mice bearing gene deletions (Fig. 4A 4B 4C 4D 4E 4F ). These data show that MDP-induced release of IL-1ß from LPS-pretreated cells requires Nod2, Rip2, Asc, CIAS1/NALP3, and caspase-1, and in contrast, IPAF is not needed. Conversely, IL-6 release only required the presence of Nod2 and Rip2 and does not depend on the presence of Asc, CIAS1/NALP3, or caspase-1. When MDP was added together with CHX, the cellular requirements for IL-1ß release were exactly the same, except that the amount released increased approximately threefold. The CHX effect is confined to IL-1ß, as the combined effects of CHX and MDP did not change IL-6 release when compared with that released by MDP alone.


Figure 4
View larger version (24K):
[in this window]
[in a new window]

  		Figure 4. MDP-induced IL-1ß release was abolished in Nod2–/–, Rip2–/–, Asc–/–, CIAS1/NALP3–/–, caspase-1–/–, but not IPAF-1–/– cells. BMDM were primed and stimulated as in Figure 2B . Cytokine release was measured by ELISA. Data are representative of three independent experiments. Results represent the mean ± SD of triplicate wells. ND, Not detected. S, Saline. Brackets indicate P < 0.05.

 
In vivo release of IL-1ß induced by MDP
To extend our studies in BMDM and to further establish the physiological significance of the roles of Nod2 and CIAS1/NALP3, we used an in vivo model. Specifically, we studied IL-1ß release with wild-type, Nod2–/–, or CIAS1/NALP3–/– mice. We first primed mice with an i.p. LPS injection and 3 h later, injected the primed mice with ATP, MDP, or saline. Blood samples for ELISA analysis of IL-1ß or IL-6 were removed 90 min postchallenge. We observed that the presence of Nod2 was required for MDP-induced IL-1ß or IL-6 but not for the response to ATP, and in contrast, the presence of CIAS1/NALP3 is required for MDP- and ATP-induced IL-1ß production but not for MDP- or ATP-induced IL-6 release (Fig. 5 ). These data are consistent with previous findings [14 , 16 ] with respect to the requirement for CIAS1/NALP3 in LPS-induced IL-1ß release in vivo and most importantly, extend our understanding of the essential roles for Nod2 and CIAS1/NALP3 when MDP stimulates IL-1 processing.


Figure 5
View larger version (7K):
[in this window]
[in a new window]

  		Figure 5. Impaired IL-1ß release in Nod2–/– and CIAS1/NALP3–/– mice following treatment with MDP. Groups of mice (n=8 per group for Nod2+/+ and Nod2–/– in A, or n=5 per group for CIAS1/NALP3+/+ and CIAS1/NALP3–/– for B) were injected i.p. with 10 µg LPS (O111:B4); 3 h later, these mice received an additional injection of 50 µl 100 mM ATP, 300 µg MDP, or saline, 1.5 h before bleeding. Cytokine induction was monitored by ELISA. Data are representative of two independent experiments. Results represent the mean ± SD. Brackets indicate P < 0.05.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Multiple lines of evidence support the contention that caspase-1 is essential in processing of pro-IL-1ß. What has not been well understood until recently is the role of various other intracellular proteins in steps leading to caspase-1 activation and processing of pro-IL-1ß. Now, studies from several laboratories provide data to support the contention that distinct members of the NLR protein family regulate activation of caspase-1 in a ligand-specific manner. For example, IPAF has been shown to play a specific role in mediating IL-1ß release by bacterial flagellin protein [34 , 35 ]. In contrast, diverse ligands such as uric acid crystals or ATP require the presence of CIAS1/NALP3 for production of mature IL-1ß [14 , 15 ]. CIAS/NALP3 is also required for MDP-induced release of IL-1ß [17 ]. Here, we investigated further the mechanisms involved in MDP-induced IL-1ß release using BMDM and in vivo models with mice bearing selective gene deletions. We established that MDP itself is a weak inducer of pro-IL-1ß, in contrast to prototypic TLR activators such as LPS, but it facilitates the release of mature IL-1ß induced by such activators. Further, the totality of our findings suggests that MDP-induced release of mature IL-1ß requires Nod2, CIAS1/NALP3, and at least two other proteins, Rip2 and Asc. The absence of any one of these proteins is sufficient to prevent MDP-induced IL-1ß release. In contrast, MDP-induced release of other cytokines such as IL-6 only requires the presence of Nod2 and Rip2 and occurs identically in wild-type and BMDM lacking Asc, CIAS1/NALP3, and caspase-1. Finally, IPAF, a key protein for IL-1ß release induced by bacterial flagellin protein, has no demonstrable role when MDP is used to activate BMDM.

These results suggests that Nod2 and Rip2 may also be a part of the NALP3 inflammmasome, a multiprotein complex comprised of NALP3, Asc, and caspase-1, which leads to activation of caspase-1 and release of mature IL-1ß. We have some evidence from overexpression systems, which begins to address this. Our studies show that CIAS/NALP3 binds to Nod2 in a CARD-independent manner (data not shown). However, further studies are required to determine whether endogenous Nod2 and Rip2 are indeed part of a complex with CIAS/NALP3 and Asc. Alternately, Nod2 may not be part of the inflammasome but affect other aspects of IL-1ß homeostasis, such as pro-IL-1ß synthesis or stability or the secretion of mature IL-1ß. Whatever the mechanism of action is, Nod2 is absolutely required for MDP-induced release of IL-1ß.

Mutations in Nod2 and/or CIAS1/NALP3 have been linked to human diseases, which are characterized as autoinflammatory diseases and include Blau syndrome and the cryopyrinopathies. Hyperproduction of IL-1ß is thought to be a central event leading to symptoms in these diseases, as IL-1-targeted therapy has been used successfully for treatment. Moreover, mutations in Nod2 have also been linked to increased susceptibility to Crohn’s disease, and recently, it was suggested that the Crohn’s-associated Nod2 mutations result in gain-of-function and increased IL-1ß production [36 ]. Here, we show an absolute dependence on CIAS1/NALP3 and Nod2 when MDP is used to activate BMDM to release mature IL-1ß. We also showed how MDP alone does not lead to induction of pro-IL-1ß, but rather, cells require priming by TLR agonists such as the prototypic TLR4 activator, LPS, or other TLR agonists (data not shown). MDP is a weak activator of NF-{kappa}B and as a consequence, a weak inducer of cytokines when compared with other proinflammatory stimuli, such as LPS. Our studies suggest a major function of MDP is to actively trigger the maturation and release of IL-1ß, provided that the cell has been preactivated by stronger proinflammatory stimuli such as LPS. Also surprising is the importance of Rip2. Studies are currently underway to evaluate whether Rip2 requires its kinase function to participate in the events leading to mature IL-1ß release. It is possible that the primary function of Rip2 is to stabilize the caspase-1-activating complex formed in response to MDP rather than to phosphorylate as-yet-unidentified proteins.

At present, we can only speculate about the source or mechanism of cell entry of MDP or related muropeptides, which activate Nod2. One source might be from phagocytosed bacteria, which have undergone degradation within a phagocytic vacuole [37 , 38 ]. However, it is important to recall that more than two decades ago, studies of Martin et al. [39 ] provided evidence for accumulation of muropeptides, which could be derived only from endogenous peptidoglycan. Although the source(s) or mechanisms associated with this accumulation were not determined, it appears there may be unique pathways involving gut bacteria and release of peptidoglycan into the systemic circulation. Future studies of such pathways may be fruitful in the quest to identify endogenous activators of IL-1ß, which occur in the absence of infection. Finally, our data suggest that investigations into the genetics of autoinflammatory syndromes in man should include studies of Nod2, CIAS1/NALP3, Asc, and Rip2, as each of these proteins makes an essential contribution to MDP-dependent, IL-1ß release.


   ACKNOWLEDGEMENTS
 
We thank the Vishva Dixit laboratory (Genentech) for their generous gifts of various sources of murine BM used to prepare BMDM; and we thank James Mueller and Amir Misaghi for technical support.


   FOOTNOTES
 
1 Current address: Synta Pharmaceuticals Corp., Lexington, MA 02421, USA. Back

2 Current address: Medimmune, Gaithersburg, MD 20878, USA. Back

Received October 10, 2006; revised February 8, 2007; accepted February 22, 2007.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Aderem, A., Ulevitch, R. J. (2000) Toll-like receptors in the induction of the innate immune response Nature 406,782-787[CrossRef][Medline]
  2. Inohara, N., Nunez, G. (2003) NODs: intracellular proteins involved in inflammation and apoptosis Nat. Rev. Immunol. 3,371-382[CrossRef][Medline]
  3. Philpott, D. J., Girardin, S. E. (2004) The role of Toll-like receptors and Nod proteins in bacterial infection Mol. Immunol. 41,1099-1108[CrossRef][Medline]
  4. Ting, J. P., Williams, K. L. (2005) The CATERPILLER family: an ancient family of immune/apoptotic proteins Clin. Immunol. 115,33-37[CrossRef][Medline]
  5. Fritz, J. H., Girardin, S. E. (2005) How Toll-like receptors and Nod-like receptors contribute to innate immunity in mammals J. Endotoxin Res. 11,390-394[Medline]
  6. Miceli-Richard, C., Lesage, S., Rybojad, M., Prieur, A. M., Manouvrier-Hanu, S., Hafner, R., Chamaillard, M., Zouali, H., Thomas, G., Hugot, J. P. (2001) CARD15 mutations in Blau syndrome Nat. Genet. 29,19-20[CrossRef][Medline]
  7. Hugot, J. P., Chamaillard, M., Zouali, H., Lesage, S., Cezard, J. P., Belaiche, J., Almer, S., Tysk, C., O’Morain, C. A., Gassull, M., Binder, V., Finkel, Y., Cortot, A., Modigliani, R., Laurent-Puig, P., Gower-Rousseau, C., Macry, J., Colombel, J. F., Sahbatou, M., Thomas, G. (2001) Association of NOD2 leucine-rich repeat variants with susceptibility to Crohn’s disease Nature 411,599-603[CrossRef][Medline]
  8. Ogura, Y., Bonen, D. K., Inohara, N., Nicolae, D. L., Chen, F. F., Ramos, R., Britton, H., Moran, T., Karaliuskas, R., Duerr, R. H., Achkar, J. P., Brant, S. R., Bayless, T. M., Kirschner, B. S., Hanauer, S. B., Nunez, G., Cho, J. H. (2001) A frameshift mutation in NOD2 associated with susceptibility to Crohn’s disease Nature 411,603-606[CrossRef][Medline]
  9. Hoffman, H. M., Mueller, J. L., Broide, D. H., Wanderer, A. A., Kolodner, R. D. (2001) Mutation of a new gene encoding a putative pyrin-like protein causes familial cold autoinflammatory syndrome and Muckle-Wells syndrome Nat. Genet. 29,301-305[CrossRef][Medline]
  10. Feldmann, J., Prieur, A. M., Quartier, P., Berquin, P., Certain, S., Cortis, E., Teillac-Hamel, D., Fischer, A., de Saint, G. B. (2002) Chronic infantile neurological cutaneous and articular syndrome is caused by mutations in CIAS1, a gene highly expressed in polymorphonuclear cells and chondrocytes Am. J. Hum. Genet. 71,198-203[CrossRef][Medline]
  11. Martinon, F., Burns, K., Tschopp, J. (2002) The inflammasome: a molecular platform triggering activation of inflammatory caspases and processing of proIL-ß Mol. Cell 10,417-426[CrossRef][Medline]
  12. Petrilli, V., Papin, S., Tschopp, J. (2005) The inflammasome Curr. Biol. 15,R581[CrossRef][Medline]
  13. Kanneganti, T. D., Ozoren, N., Body-Malapel, M., Am, A., Park, J. H., Franchi, L., Whitfield, J., Barchet, W., Colonna, M., Vandenabeele, P., Bertin, J., Coyle, A., Grant, E. P., Akira, S., Nunez, G. (2006) Bacterial RNA and small antiviral compounds activate caspase-1 through cryopyrin/Nalp3 Nature 440,233-236[CrossRef][Medline]
  14. Mariathasan, S., Weiss, D. S., Newton, K., McBride, J., O’Rourke, K., Roose-Girma, M., Lee, W. P., Weinrauch, Y., Monack, D. M., Dixit, V. M. (2006) Cryopyrin activates the inflammasome in response to toxins and ATP Nature 440,228-232[CrossRef][Medline]
  15. Martinon, F., Petrilli, V., Mayor, A., Tardivel, A., Tschopp, J. (2006) Gout-associated uric acid crystals activate the NALP3 inflammasome Nature 440,237-241[CrossRef][Medline]
  16. Sutterwala, F. S., Ogura, Y., Szczepanik, M., Lara-Tejero, M., Lichtenberger, G. S., Grant, E. P., Bertin, J., Coyle, A. J., Galan, J. E., Askenase, P. W., Flavell, R. A. (2006) Critical role for NALP3/CIAS1/Cryopyrin in innate and adaptive immunity through its regulation of caspase-1 Immunity 24,317-327[CrossRef][Medline]
  17. Martinon, F., Agostini, L., Meylan, E., Tschopp, J. (2004) Identification of bacterial muramyl dipeptide as activator of the NALP3/cryopyrin inflammasome Curr. Biol. 14,1929-1934[CrossRef][Medline]
  18. Inohara, N., Ogura, Y., Fontalba, A., Gutierrez, O., Pons, F., Crespo, J., Fukase, K., Inamura, S., Kusumoto, S., Hashimoto, M., Foster, S. J., Moran, A. P., Fernandez-Luna, J. L., Nunez, G. (2003) Host recognition of bacterial muramyl dipeptide mediated through NOD2. Implications for Crohn’s disease J. Biol. Chem. 278,5509-5512[Abstract/Free Full Text]
  19. McDonald, C., Inohara, N., Nunez, G. (2005) Peptidoglycan signaling in innate immunity and inflammatory disease J. Biol. Chem. 280,20177-20180[Free Full Text]
  20. Akira, S., Uematsu, S., Takeuchi, O. (2006) Pathogen recognition and innate immunity Cell 124,783-801[CrossRef][Medline]
  21. Reimund, J. M., Wittersheim, C., Dumont, S., Muller, C. D., Kenney, J. S., Baumann, R., Poindron, P., Duclos, B. (1996) Increased production of tumor necrosis factor-{alpha}, interleukin-1 ß, and interleukin-6 by morphologically normal intestinal biopsies from patients with Crohn’s disease Gut 39,684-689[Abstract/Free Full Text]
  22. Mariathasan, S., Newton, K., Monack, D. M., Vucic, D., French, D. M., Lee, W. P., Roose-Girma, M., Erickson, S., Dixit, V. M. (2004) Differential activation of the inflammasome by caspase-1 adaptors ASC and IPAF Nature 430,213-218[CrossRef][Medline]
  23. Kobayashi, K. S., Chamaillard, M., Ogura, Y., Henegariu, O., Inohara, N., Nunez, G., Flavell, R. A. (2005) Nod2-dependent regulation of innate and adaptive immunity in the intestinal tract Science 307,731-734[Abstract/Free Full Text]
  24. Kobayashi, K., Inohara, N., Hernandez, L. D., Galan, J. E., Nunez, G., Janeway, C. A., Medzhitov, R., Flavell, R. A. (2002) RICK/Rip2/CARDIAK mediates signaling for receptors of the innate and adaptive immune systems Nature 416,194-199[CrossRef][Medline]
  25. Kuida, K., Lippke, J. A., Ku, G., Harding, M. W., Livingston, D. J., Su, M. S., Flavell, R. A. (1995) Altered cytokine export and apoptosis in mice deficient in interleukin-1 ß converting enzyme Science 267,2000-2003[Abstract/Free Full Text]
  26. Pan, Q., Kravchenko, V., Katz, A., Huang, S., Ii, M., Mathison, J. C., Kobayashi, K., Flavell, R. A., Schreiber, R. D., Goeddel, D., Ulevitch, R. J. (2006) NF-{kappa}B-inducing kinase regulates selected gene expression in the Nod2 signaling pathway Infect. Immun. 74,2121-2127[Abstract/Free Full Text]
  27. Kravchenko, V. V., Pan, Z., Han, J., Herbert, J. M., Ulevitch, R. J., Ye, R. D. (1995) Platelet-activating factor induces NF-{kappa} B activation through a G protein-coupled pathway J. Biol. Chem. 270,14928-14934[Abstract/Free Full Text]
  28. Friedman, R. M. (1979) Induction and production of interferon Methods Enzymol. 58,292-296[Medline]
  29. Vilcek, J., Havell, E. A. (1973) Stabilization of interferon messenger RNA activity by treatment of cells with metabolic inhibitors and lowering of the incubation temperature Proc. Natl. Acad. Sci. USA 70,3909-3913[Abstract/Free Full Text]
  30. Vilcek, J., Havell, E. A., Kohase, M. (1976) Superinduction of interferon with metabolic inhibitors: possible mechanisms and practical applications J. Infect. Dis. 133(Suppl.),A22-A29[Medline]
  31. Youngner, J. S., Stinebring, W. R., Taube, S. E. (1965) Influence of inhibitors of protein synthesis on interferon formation in mice Virology 27,541-550[CrossRef][Medline]
  32. Thornberry, N. A., Bull, H. G., Calaycay, J. R., Chapman, K. T., Howard, A. D., Kostura, M. J., Miller, D. K., Molineaux, S. M., Weidner, J. R., Aunins, J. (1992) A novel heterodimeric cysteine protease is required for interleukin-1 ß processing in monocytes Nature 356,768-774[CrossRef][Medline]
  33. Pauleau, A. L., Murray, P. J. (2003) Role of nod2 in the response of macrophages to Toll-like receptor agonists Mol. Cell. Biol. 23,7531-7539[Abstract/Free Full Text]
  34. Franchi, L., Am, A., Body-Malapel, M., Kanneganti, T. D., Ozoren, N., Jagirdar, R., Inohara, N., Vandenabeele, P., Bertin, J., Coyle, A., Grant, E. P., Nunez, G. (2006) Cytosolic flagellin requires IPAF for activation of caspase-1 and interleukin 1ß in Salmonella-infected macrophages Nat. Immunol. 7,576-582[CrossRef][Medline]
  35. Miao, E. A., Alpuche-Aranda, C. M., Dors, M., Clark, A. E., Bader, M. W., Miller, S. I., Aderem, A. (2006) Cytoplasmic flagellin activates caspase-1 and secretion of interleukin 1ß via IPAF Nat. Immunol. 7,569-575[CrossRef][Medline]
  36. Maeda, S., Hsu, L. C., Liu, H., Bankston, L. A., Iimura, M., Kagnoff, M. F., Eckmann, L., Karin, M. (2005) Nod2 mutation in Crohn’s disease potentiates NF-{kappa}B activity and IL-1ß processing Science 307,734-738[Abstract/Free Full Text]
  37. Johannsen, L., Wecke, J., Obal, F., Jr, Krueger, J. M. (1991) Macrophages produce somnogenic and pyrogenic muramyl peptides during digestion of Staphylococci Am. J. Physiol. 260,R126-R133[Medline]
  38. Vermeulen, M. W., Gray, G. R. (1984) Processing of Bacillus subtilis peptidoglycan by a mouse macrophage cell line Infect. Immun. 46,476-483[Abstract/Free Full Text]
  39. Martin, S. A., Karnovsky, M. L., Krueger, J. M., Pappenheimer, J. R., Biemann, K. (1984) Peptidoglycans as promoters of slow-wave sleep. I. Structure of the sleep-promoting factor isolated from human urine J. Biol. Chem. 259,12652-12658[Abstract/Free Full Text]



This article has been cited by other articles:


Home page
 JEMHome page
J. Maelfait, E. Vercammen, S. Janssens, P. Schotte, M. Haegman, S. Magez, and R. Beyaert
Stimulation of Toll-like receptor 3 and 4 induces interleukin-1{beta} maturation by caspase-8
J. Exp. Med., September 1, 2008; 205(9): 1967 - 1973.
[Abstract] [Full Text] [PDF]

Home page
 J. Leukoc. Biol.Home page
H. L. Rosenzweig, T. M. Martin, S. R. Planck, K. Galster, M. M. Jann, M. P. Davey, K. Kobayashi, R. A. Flavell, and J. T. Rosenbaum
Activation of NOD2 in vivo induces IL-1{beta} production in the eye via caspase-1 but results in ocular inflammation independently of IL-1 signaling
J. Leukoc. Biol., August 1, 2008; 84(2): 529 - 536.
[Abstract] [Full Text] [PDF]

Home page
 J. Immunol.Home page
H. Hara, K. Tsuchiya, T. Nomura, I. Kawamura, S. Shoma, and M. Mitsuyama
Dependency of Caspase-1 Activation Induced in Macrophages by Listeria monocytogenes on Cytolysin, Listeriolysin O, after Evasion from Phagosome into the Cytoplasm
J. Immunol., June 15, 2008; 180(12): 7859 - 7868.
[Abstract] [Full Text] [PDF]

Home page
 Proc. Natl. Acad. Sci. USAHome page
L.-C. Hsu, S. R. Ali, S. McGillivray, P.-H. Tseng, S. Mariathasan, E. W. Humke, L. Eckmann, J. J. Powell, V. Nizet, V. M. Dixit, et al.
A NOD2-NALP1 complex mediates caspase-1-dependent IL-1{beta} secretion in response to Bacillus anthracis infection and muramyl dipeptide
PNAS, June 3, 2008; 105(22): 7803 - 7808.
[Abstract] [Full Text] [PDF]

Home page
 IOVSHome page
H. L. Rosenzweig, T. M. Martin, M. M. Jann, S. R. Planck, M. P. Davey, K. Kobayashi, R. A. Flavell, and J. T. Rosenbaum
NOD2, the Gene Responsible for Familial Granulomatous Uveitis, in a Mouse Model of Uveitis
Invest. Ophthalmol. Vis. Sci., April 1, 2008; 49(4): 1518 - 1524.
[Abstract] [Full Text] [PDF]

Home page
 J. Immunol.Home page
N. Marina-Garcia, L. Franchi, Y.-G. Kim, D. Miller, C. McDonald, G.-J. Boons, and G. Nunez
Pannexin-1-Mediated Intracellular Delivery of Muramyl Dipeptide Induces Caspase-1 Activation via Cryopyrin/NLRP3 Independently of Nod2
J. Immunol., March 15, 2008; 180(6): 4050 - 4057.
[Abstract] [Full Text] [PDF]

Home page
 J. Leukoc. Biol.Home page
F. Martinon
Detection of immune danger signals by NALP3
J. Leukoc. Biol., March 1, 2008; 83(3): 507 - 511.
[Abstract] [Full Text] [PDF]

Home page
 J. Immunol.Home page
C. Stehlik and A. Dorfleutner
COPs and POPs: Modulators of Inflammasome Activity
J. Immunol., December 15, 2007; 179(12): 7993 - 7998.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
jlb.1006627v1
82/1/177    most recent
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Pan, Q.
Right arrow Articles by Ulevitch, R. J.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Pan, Q.
Right arrow Articles by Ulevitch, R. J.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS