New insights into the mechanism of IL-1β maturation

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New insights into the mechanism of IL-1b maturation Kimberly Burns, Fabio Martinon and JuÈrg Tschopp The pro-in¯ammatory cytokine IL-1b is initially synthesised in an inactive precursor form and therefore requires processing by caspase-1 for activation. Recently, several investigators have identi®ed a number of proteins that are implicated in caspase-1 activation, and other proteins that negatively regulate both caspase-1 activation and IL-1b processing have also been identi®ed. Drugs that target these components may have therapeutic bene®ts for in¯ammatory diseases.

the formation of a membrane proximal signalling complex that activates IkB kinase (IKK) and mitogen-activated protein kinase (MAPK) pathways [4]. This leads to the activation of the transcription factors nuclear factor (NF)-kB and AP-1, resulting in the induction of genes encoding chemokines, cytokines, acute-phase proteins, cell adhesion molecules and enzymes involved in the production of small pro-in¯ammatory substances [5].

Addresses Institute of Biochemistry, University of Lausanne, Chemin des Boveresses 155, CH-1066 Epalinges, Switzerland  e-mail: [email protected]

IL-1b faces a challenge not encountered by most other secreted polypeptides because of the absence of a signal peptide. Its passage through the classical secretory pathway (i.e. endoplasmic reticulum to Golgi) is therefore precluded. IL-1b is instead translated in the cytosol, where it gains access to the extracellular environment [6±9]. Further complicating its production, IL-1b is initially synthesised as an inactive 31 kDa precursor molecule (proIL-1b) that must be proteolytically processed to generate the active mature 17 kDa form, which is the major form delivered to the extracellular environment [10]. The mechanism by which IL-1b is processed and secreted is not well understood, but recent developments in this ®eld provide new insights into the processing of proIL-1b and will be the focus of this review.

Current Opinion in Immunology 2003, 15:26±30 This review comes from a themed issue on Innate immunity Edited by Ruslan Medzhitov and Christine A Biron 0952-7915/03/$ ± see front matter ß 2003 Elsevier Science Ltd. All rights reserved. DOI 10.1016/S0952-7915(02)00017-1 Abbreviations ASC apoptosis-associated speck-like protein containing a CARD CARD caspase recruitment domain ICE IL-1b converting enzyme IL interleukin Ipaf ICE-protease activating factor LPS lipopolysaccharide LRR leucine-rich repeat NALP1 Nacht, LRR and PYD containing protein 1 PI proteinase inhibitor PYD Pyrin domain RIP2 receptor interacting protein 2 TLR Toll-like receptor TNF tumour necrosis factor

Introduction

IL-1b is a pro-in¯ammatory cytokine produced by activated macrophages and monocytes. It functions in the generation of systemic and local responses to infection, injury and immunological challenges by generating fever, activating lymphocytes and promoting infusion of leukocytes into the sites of injury or infection (reviewed by [1]). It is the primary cause of chronic and acute in¯ammation and, as an endogenous pyrogen, it is a key player in the febrile response [2,3]. IL-1b affects almost every cell type. It exerts its activities extracellularly by binding to a high af®nity receptor, IL-1RI. Upon IL-1b binding, the IL-1RI aggregates with a related polypeptide chain (IL-1RAcP), thereby triggering Current Opinion in Immunology 2003, 15:26±30

Production of IL-1b requires two distinct stimuli: priming and processing/secretion

The synthesis, processing and release of mature IL-1b are tightly regulated events. proIL-1b is generally not detected in unstimulated monocytes and macrophages, but must be induced at the transcriptional level [11]. This synthesis is under the control of transcription factors activated in many cell types when challenged with microbes, microbial products and other environmental stimuli that necessitate the engagement or ampli®cation of in¯ammatory responses. Multiple signalling pathways are involved in the transcriptional upregulation of proIL-1b, including pathways triggered by IL-1b itself, by other in¯ammatory cytokines, such as TNF, or by various Tolllike receptor (TLR) ligands, such as lipopolysaccharide (LPS), which signals via TLR4 [1]. These signals can also be mimicked by phorbol 12-myristate 13-acetate (PMA) treatment [12]. However, in the absence of a secondary or `secretion' stimulus, processing of proIL-1b and release of mature IL-1b is generally inef®cient, resulting either in the retention or degradation of proIL-1b. There are multiple agents that induce the post-translational processing of proIL-1b, such as nigericin, hypotonic stress, bacterial toxins (e.g. Shigella ¯exneri protein IpaB), antimicrobial www.current-opinion.com

Mechanisms of IL-1b maturation Burns, Martinon and Tschopp 27

peptides, mechanical disruption of the integrity of the cell, ATP and LPS [13±19]; many of these secretion stimuli may not be of highest physiological relevance. In addition, the majority of these stimuli induce apoptosis or necrosis of the cytokine-producing cells, processes that have a controversial requirement for IL-1b release [20]. LPS is somewhat special in that it has been reported to act both as a priming stimulus and as a processing/secretion stimulus, yet it does not appear to be an ef®cient trigger of both [19,21]. Although the nature, and in some cases the necessity, for a second stimulus remains unclear, it may provide a mechanism whereby a potent mediator of in¯ammation, such as IL-1b, can be externalised on a need-only, tightly regulated basis.

In¯ammatory caspases involved in IL-1 processing

Conversion of proIL-1b to the active mature form of IL-1b requires the action of the IL-1b converting enzyme (ICE; also called caspase-1), which cleaves proIL-1b after the aspartic acid residue at position 116 [22,23]. Caspase-1 is a prototypic member of a family of in¯ammatory caspases (including the human caspases 4 and 5, and the mouse caspases 11 and 12) that contain amino-terminal prodomains with a caspase recruitment domain (CARD). In mice, caspase-11 is required in addition to caspase-1 for IL-1b processing, as con®rmed by the inability of mice de®cient in caspase-11 or caspase-1 to process IL-1b [24,25]. The human homologue of murine caspase-11 is unknown, but it closely resembles human caspase-5, sharing 54% identical amino acid residues [26]. proIL-1b processing was recently found to occur more ef®ciently in a cell-free system when both caspase-1 and caspase-5 were co-activated [21], suggesting that in humans, similar to in mice, more than one pro-in¯ammatory caspase is implicated in the generation of active IL-1b.

Activation of caspase-1 and caspase-5

Similar to other caspases, the 45 kDa precursor of caspase-1 is initially synthesised as an inactive zymogen that becomes activated by cleavage at aspartic residues to generate the enzymatically active hetrodimer composed of a 10 kDa and a 20 kDa chain. The exact mechanism by which this occurs remains unclear, but recent observations concur with the mechanism proposed for the apoptototic caspases, such as that initiated by caspase-9 (Figure 1). Caspase-9 autoactivates as a result of induced proximity, triggered through oligomerisation (via CARD±CARD interactions) with the multidomain adaptor protein Apaf-1 (Figure 1; [27]). In an analogous manner, caspase-1 and caspase-5 may become activated through oligomerisation induced via association with one of several recently identi®ed candidate adaptor proteins: RIP2 (receptor interacting protein 2), Ipaf (ICE-protease activating factor, also called CARD12) or NALP1±ASC complexes. www.current-opinion.com

RIP2 is a serine-threonine kinase with an amino-terminal CARD that has the capacity to induce caspase-1 processing following overexpression [28±30]. However, mice de®cient in RIP2 are not defective in caspase-1 processing, indicating that RIP-2 may not be essential for this function in macrophages [31]. Ipaf/CARD12 [32,33] is a multidomain protein resembling NALP1 and Apaf1 (Figure 1) that also contains a CARD. Through CARD±CARD interactions, Ipaf was found to trigger caspase-1 cleavage in overexpression [33], but the capacity for Ipaf-activated caspase-1 to process proIL-1b has not yet been demonstrated. The term in¯ammasome was coined to describe the complex assembled between NALP1 (Nacht, LRR and PYD [Pyrin domain]-containing protein 1), ASC (apoptosis-associated speck-like protein containing a CARD; also called Pycard, for PYD and CARD-containing protein), caspase-1 and interestingly also caspase-5, supporting the idea discussed previously that caspase-5 may be involved in proIL-1b processing [21]. NALP1 is the member of a family of proteins called the NALPs (also called PANs [34] or PYPAFs [35]). It has modular structure comprising ®ve distinct domains, including an amino-terminal PYD and a carboxy-terminal CARD (Figure 1). Via these domains, NALP1 forms two interactions: ®rst, with the PYD of ASC, a bipartite adaptor protein containing an amino-terminal PYD and a carboxy-terminal CARD, and second with the CARD of caspase-5. The CARD module of ASC in turn recruits capase-1 (Figure 1; [21,36,37]). NALP1 self-associates [38] and probably forms a multisubunit complex [21], bringing several caspase-1 and caspase-5 monomers into close proximity. In co-immunoprecipitation experiments, caspase-1 associated with the full-length and mature p20 fragment of caspase-5, suggesting the potential for cross-activation. Of the various adaptor molecules identi®ed so far, the most compelling evidence exists for the involvement of ASC in IL-1b processing; that is, a dominant negative version of ASC (containing only the PYD domain) blocked LPS-induced IL-1b maturation in THP-1 monocytic cells and the removal of ASC in a cell-free system abolished caspase-1 and caspase-5 activation [21,36].

Negative regulation of caspase activation and IL-1b processing

Although the production of IL-1b is critical for the control of pathogenic infections, excessive cytokine production is harmful to the host and can even be fatal [39]. In recognition of this it is not surprising that several `safe guards' have been placed at distinct steps (e.g. at the level of gene expression, synthesis, secretion and receptor association) to control the activity of IL-1b (reviewed in [40]). To this list must be added several proteins (discussed below) that interfere with the processing of proIL-1b. Two of these proteins, ICEBERG and proteinase inhibitor 9 (PI-9) are Current Opinion in Immunology 2003, 15:26±30

28 Innate immunity

Figure 1

Ligand

Ligand binding site

Oligomerizing platform NALP1

LRR

Caspase

Pycard/ASC

NACHT

PYD

FIIND

CARD

PYD

CARD

CARD

CASPASE-1

CARD

CASPASE-5

?

IL-1β processing Inflammation Ipaf LPS?

LRR

NACHT

CARD

CARD

CASPASE-1

CARD

CARD

CASPASE-9

Apaf Cyt c

WD

NB-ARC

Apoptosis

Current Opinion in Immunology

Adaptor molecules involved in caspase activation. NALP1 and Ipaf have modular structures composed of several distinct domains linked in tandem. Their modular organisation closely resembles that of Apaf, the adaptor which induces oligomerisation and auto-activation of caspase-9. For NALP1 and Ipaf, the ligand that triggers adaptor±caspase association is unknown; however, this unknown ligand (possibly LPS) is proposed to bind to the LRRs of NALP1 and Ipaf, similar to the way in which Cytochrome c (Cyt) binds to the WD-40 repeats (WB), which are localised to the amino terminus of Apaf. The domains of each protein have been schematically represented. The CARD domain is a protein±protein interaction domain that binds to the same domain found in the amino-terminal prodomains of caspases-1, -5 and -9. The PYD is also a protein±protein interaction domain. It links NALP1 with caspase-1 via the bipartite adaptor ASC. The NACHT is an oligomerisation domain and may promote subunit aggregation in the proteins, similar to the nucleotide-binding ARC (apoptosis repressor with CARD) domain (NB±ARC) of Apaf. The function of the FIIND domain of NALP1 is unknown, but it probably binds to the NACHT domain. Via their respective domains, NALP1/ASC or Ipaf associate with caspase-1/5 and Apaf associates with capase-9 to induce the activation of the caspases. This in turn results either in the processing of proIL-1b and inflammation, or a cascade of caspase activation and apoptosis as a final outcome.

known to be inducibly expressed by pro-in¯ammatory agents (i.e. LPS and TNF) and they may be part of negative feedback loops. Pseudo-ICE (also called COP, for CARD-only protein) and ICEBERG contain CARDs closely resembling the prodomain of caspase-1. Via CARD±CARD interactions they presumably negatively regulate proIL-1b processing by preventing direct interactions between the caspase and its adaptor(s), thus inhibiting caspase-1/5 oligomerisation [41,42,43]. In a similar manner, a protein we call ASCI (also called ASC2), may block caspase-1 activation. ASCI looks like ASC but lacks ASC's carboxy-terminal CARD. By binding NALP1, ASCI prevents caspase-1 recruitment to the in¯ammasome (F Martinon and J Tschopp, unpublished data). In contrast to the negative regulators previously described, PI-9 inhibits caspase-1 activation by blocking its active site [44,45,46] rather than by preventing caspase±adaptor association. The secretion of activated caspase-1 and caspase-5 may also be a means of negatively regulating IL-1b processing. Current Opinion in Immunology 2003, 15:26±30

Surprisingly, although activation of IL-1b leads to the concomitant processing of caspases-1 and -5, the processed caspase fragments are not observed in the corresponding cellular extracts. This appears to be because the activated caspases-1 and -5 are rapidly and speci®cally released into the supernatant, at least in THP-1 cells following LPS stimulation [21,47]. Secretion has not been observed for other activated caspases, but this may explain why pro-in¯ammatory caspases are generally not pro-apoptotic.

Conclusions

On the basis of recent investigations into IL-1b processing, it seems safe to conclude that the in¯ammatory caspases are activated by oligomerisation induced by speci®c adaptor molecules, and that caspase activation is regulated through the use of molecules that block these interactions. The relative importance of the adapters and inhibitors identi®ed to date awaits the assessment of mice de®cient in the respective genes. Importantly, how the various processing/secretion stimuli trigger association of caspase-1/5 with its adaptor(s) remains enigmatic, and will www.current-opinion.com

Mechanisms of IL-1b maturation Burns, Martinon and Tschopp 29

undoubtedly be the topic of intense research. In the cases of NALP1 and Ipaf, it is easy to speculate how this may occur. Both molecules contain LRRs, such as those found in the TLRs. The LRRs of TLRs are involved in the recognition of pathogen-associated molecular patterns (PAMPs; [48]) and/or endogenous non-foreign `alarm signals' (e.g. mammalian DNA and heat shock proteins [49]). In support of the idea that the LRRs of NALP1 and Ipaf detect pathogen-derived molecules is the observation that removal of the LRRs renders both proteins constitutively active. Binding of a LRR ligand could release this inhibition and thereby trigger caspase/adaptor assembly. NALP1 is a member of an expanding family of proteins, containing 14 members, two of which (NALP3 and NALP12) have already been shown to bind ASC [35,37]. Different members of this family may therefore serve to activate caspase-1/5 in response to different LRR ligands.

References and recommended reading

Papers of particular interest, published within the annual period of review, have been highlighted as:  of special interest  of outstanding interest

14. Hogquist KA, Nett MA, Unanue ER, Chaplin DD: Interleukin 1 is processed and released during apoptosis. Proc Natl Acad Sci USA 1991, 88:8485-8489. 15. Kostura MJ, Tocci MJ, Limjuco G, Chin J, Cameron P, Hillman AG, Chartrain NA, Schmidt JA: Identi®cation of a monocyte speci®c pre-interleukin 1 beta convertase activity. Proc Natl Acad Sci USA 1989, 86:5227-5231. 16. Perregaux D, Gabel CA: Interleukin-1 beta maturation and release in response to ATP and nigericin. Evidence that potassium depletion mediated by these agents is a necessary and common feature of their activity. J Biol Chem 1994, 269:15195-15203. 17. Chen Y, Smith MR, Thirumalai K, Zychlinsky A: A bacterial invasin induces macrophage apoptosis by binding directly to ICE. EMBO J 1996, 15:3853-3860. 18. Perregaux DG, Bhavsar K, Contillo L, Shi J, Gabel CA: Antimicrobial peptides initiate IL-1 beta posttranslational processing: a novel role beyond innate immunity. J Immunol 2002, 168:3024-3032. 19. Schumann RR, Belka C, Reuter D, Lamping N, Kirschning CJ, Weber JR, Pfeil D: Lipopolysaccharide activates caspase-1 (interleukin-1-converting enzyme) in cultured monocytic and endothelial cells. Blood 1998, 91:577-584. 20. MacKenzie A, Wilson HL, Kiss-Toth E, Dower SK, North RA,  Surprenant A: Rapid secretion of interleukin-1beta by microvesicle shedding. Immunity 2001, 15:825-835. The authors demonstrate that IL-1b is secreted in vesicles that are shed before cells undergo apoptosis.

1.

Dinarello CA: Interleukin-1 beta, interleukin-18, and the interleukin-1 beta converting enzyme. Ann New York Acad Sci 1998, 856:1-11.

2.

Dinarello CA: Proin¯ammatory cytokines. Chest 2000, 118:503-508.

21. Martinon F, Burns K, Tschopp J: The in¯ammasome: a molecular  platform triggering activation of in¯ammatory caspases and processing of proIL-beta. Mol Cell 2002, 10:417-426. This study describes an in vitro system for the formation of an in¯ammasome involved in in¯ammatory caspase activation, and identi®es ASC as a critical component of the in¯ammosome.

3.

Dreskin SC, Thomas GW, Dale SN, Heasley LE: Isoforms of Jun kinase are differentially expressed and activated in human monocyte/macrophage (THP-1) cells. J Immunol 2001, 166:5646-5653.

22. Cerretti DP, Kozlosky CJ, Mosley B, Nelson N, Van Ness K, Greenstreet TA, March CJ, Kronheim SR, Druck T, Cannizzaro LA et al.: Molecular cloning of the interleukin-1 beta converting enzyme. Science 1992, 256:97-100.

4.

Sims JE: IL-1 and IL-18 receptors, and their extended family. Curr Opin Immunol 2002, 14:117-122.

5.

Luheshi GN: Cytokines and fever. Mechanisms and sites of action. Ann N Y Acad Sci 1998, 856:83-89.

6.

Rubartelli A, Cozzolino F, Talio M, Sitia R: A novel secretory pathway for interleukin-1 beta, a protein lacking a signal sequence. EMBO J 1990, 9:1503-1510.

23. Thornberry NA, Bull HG, Calaycay JR, Chapman KT, Howard AD, Kostura MJ, Miller DK, Molineaux SM, Weidner JR, Aunins J et al.: A novel heterodimeric cysteine protease is required for interleukin-1 beta processing in monocytes. Nature 1992, 356:768-774.

7.

Gardella S, Andrei C, Costigliolo S, Olcese L, Zocchi MR, Rubartelli A: Secretion of bioactive interleukin-1beta by dendritic cells is modulated by interaction with antigen speci®c T cells. Blood 2000, 95:3809-3815.

8.

Andrei C, Dazzi C, Lotti L, Torrisi MR, Chimini G, Rubartelli A: The secretory route of the leaderless protein interleukin 1beta involves exocytosis of endolysosome-related vesicles. Mol Biol Cell 1999, 10:1463-1475.

9.

Hamon Y, Luciani MF, Becq F, Verrier B, Rubartelli A, Chimini G: Interleukin-1beta secretion is impaired by inhibitors of the Atp binding cassette transporter, ABC1. Blood 1997, 90:2911-2915.

10. Hazuda DJ, Lee JC, Young PR: The kinetics of interleukin 1 secretion from activated monocytes. Differences between interleukin 1 alpha and interleukin 1 beta. J Biol Chem 1988, 263:8473-8479. 11. Fenton MJ: Review: transcriptional and post-transcriptional regulation of interleukin 1 gene expression. Int J Immunopharmacol 1992, 14:401-411.

24. Kuida K, Lippke JA, Ku G, Harding MW, Livingston DJ, Su MS, Flavell RA: Altered cytokine export and apoptosis in mice de®cient in interleukin-1 beta converting enzyme. Science 1995, 267:2000-2003. 25. Wang S, Miura M, Jung YK, Zhu H, Li E, Yuan J: Murine caspase-11, an ICE-interacting protease, is essential for the activation of ICE. Cell 1998, 92:501-509. 26. Lin XY, Choi MS, Porter AG: Expression analysis of the human caspase-1 subfamily reveals speci®c regulation of the CASP5 gene by lipopolysaccharide and interferon-gamma. J Biol Chem 2000, 275:39920-39926. 27. Acehan D, Jiang X, Morgan DG, Heuser JE, Wang X, Akey CW:  Three-dimensional structure of the apoptosome: implications for assembly, procaspase-9 binding, and activation. Mol Cell 2002, 9:423-432. This elegant study proposed the ®rst structure of a caspase-activating complex. 28. Humke EW, Shriver SK, Starovasnik MA, Fairbrother WJ, Dixit VM: ICEBERG: a novel inhibitor of interleukin-1beta generation. Cell 2000, 103:99-111.

12. Fenton MJ, Vermeulen MW, Clark BD, Webb AC, Auron PE: Human pro-IL-1 beta gene expression in monocytic cells is regulated by two distinct pathways. J Immunol 1988, 140:2267-2273.

29. Inohara N, del Peso L, Koseki T, Chen S, Nunez G: RICK, a novel protein kinase containing a caspase recruitment domain, interacts with CLARP and regulates CD95-mediated apoptosis. J Biol Chem 1998, 273:12296-12300.

13. Chin J, Kostura MJ: Dissociation of IL-1 beta synthesis and secretion in human blood monocytes stimulated with bacterial cell wall products. J Immunol 1993, 151:5574-5585.

30. Thome M, Hofmann K, Burns K, Martinon F, Bodmer JL, Mattmann C, Tschopp J: Identi®cation of CARDIAK, a RIP-like kinase that associates with caspase-1. Curr Biol 1998, 8:885-888.

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30 Innate immunity

31. Kobayashi K, Inohara N, Hernandez LD, Galan JE, Nunez G,  Janeway CA, Medzhitov R, Flavell RA: RICK/Rip2/CARDIAK mediates signalling for receptors of the innate and adaptive immune systems. Nature 2002, 416:194-199. This paper uses genetic methods to demonstrate the important role of RIP2 in controlling adaptive and innate immune responses. 32. Geddes BJ, Wang L, Huang WJ, Lavellee M, Manji GA, Brown M, Jurman M, Cao J, Morgenstern J, Merriam S et al.: Human CARD12 is a novel CED4/Apaf-1 family member that induces apoptosis. Biochem Biophys Res Commun 2001, 284:77-82. 33. Poyet JL, Srinivasula SM, Tnani M, Razmara M, Fernandes-Alnemri  T, Alnemri ES: Identi®cation of Ipaf, a human caspase-1-activating protein related to Apaf-1. J Biol Chem 2001, 276:28309-28313. This study identi®ed Ipaf as a potential player in the activation of caspase-1. 34. Pawlowski K, Pio F, Chu Z, Reed JC, Godzik A: PAAD ± a new protein domain associated with apoptosis, cancer and autoimmune diseases. Trends Biochem Sci 2001, 26:85-87. 35. Manji GA, Wang L, Geddes BJ, Brown M, Merriam S, Al-Garawi A,  Mak S, Lora JM, Briskin M, Jurman M et al.: PYPAF1, a PYRIN-containing Apaf1-like protein that assembles with ASC and regulates activation of NF-kappa B. J Biol Chem 2002, 277:11570-11575. See annotation to [37]. 36. Srinivasula SM, Poyet JL, Razmara M, Datta P, Zhang Z, Alnemri ES: The PYRIN-CARD protein ASC is an activating adaptor for caspase-1. J Biol Chem 2002, 277:21119-21122. 37. Wang L, Manji GA, Grenier JM, Al-Garawi A, Merriam S, Lora JM,  Geddes BJ, Briskin M, DiStefano PS, Bertin J: PYPAF7, a novel PYRIN-containing Apaf1-like protein that regulates activation of NF-kappa B and caspase-1-dependent cytokine processing. J Biol Chem 2002, 277:29874-29880. This paper, together with [35], shows the interaction of NALP proteins with ASC, suggesting that different NALPs maybe involved in the activation of in¯ammatory caspases. 38. Chu ZL, Pio F, Xie Z, Welsh K, Krajewska M, Krajewski S, Godzik A, Reed JC: A novel enhancer of the Apaf1 apoptosome involved in cytochrome c-dependent caspase activation and apoptosis. J Biol Chem 2001, 276:9239-9245. 39. Beutler B, Cerami A: The endogenous mediator of endotoxic shock. Clin Res 1987, 35:192-197.

Current Opinion in Immunology 2003, 15:26±30

40. Dinarello CA: Biologic basis for interleukin-1 in disease. Blood 1996, 87:2095-2147. 41. Druilhe A, Srinivasula SM, Razmara M, Ahmad M, Alnemri ES:  Regulation of IL-1beta generation by Pseudo-ICE and ICEBERG, two dominant negative caspase recruitment domain proteins. Cell Death Differ 2001, 8:649-657. See annotation to [43]. 42. Green DR, Melino G: ICE heats up. Cell Death Differ 2001, 8:549-550. 43. Lee SH, Stehlik C, Reed JC: Cop, a caspase recruitment  domain-containing protein and inhibitor of caspase-1 activation processing. J Biol Chem 2001, 276:34495-34500. These authors identi®ed small CARD-containing proteins that inhibit caspase-1 activation (see also [28] and [43]). 44. Annand RR, Dahlen JR, Sprecher CA, De Dreu P, Foster DC, Mankovich JA, Talanian RV, Kisiel W, Giegel DA: Caspase-1 (interleukin-1beta-converting enzyme) is inhibited by the human serpin analogue proteinase inhibitor 9. Biochem J 1999, 342:655-665. 45. Kannan-Thulasiraman P, Shapiro DJ: Modulators of in¯ammation  use NF-kappa B and AP-1 sites to induce the caspase-1 and granzyme B inhibitor, proteinase inhibitor 9. J Biol Chem 2002, 277:41230-41239. This paper identi®ed PI-9 as an NF-kB responsive gene involved in an in¯ammatory negative feedback loop. 46. Young JL, Sukhova GK, Foster D, Kisiel W, Libby P, Schonbeck U: The serpin proteinase inhibitor 9 is an endogenous inhibitor of interleukin 1beta-converting enzyme (caspase-1) activity in human vascular smooth muscle cells. J Exp Med 2000, 191:1535-1544. 47. Singer II, Scott S, Chin J, Bayne EK, Limjuco G, Weidner J, Miller DK, Chapman K, Kostura MJ: The interleukin-1 beta-converting enzyme (ICE) is localized on the external cell surface membranes and in the cytoplasmic ground substance of human monocytes by immuno-electron microscopy. J Exp Med 1995, 182:1447-1459. 48. Girardin SE, Sansonetti PJ, Philpott DJ: Intracellular vs extracellular recognition of pathogens - common concepts in mammals and ¯ies. Trends Microbiol 2002, 10:193-199. 49. GallucciS,LolkemaM,MatzingerP:Naturaladjuvants:endogenous activators of dendritic cells. Nat Med 1999, 5:1249-1255.

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