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1 Department of Molecular Oncology Division of Molecular and Cellular Biology Institute on Ageing and Adaptation, Shinshu University Graduate School of Medicine, 3-1-1 Asahi, Matsumoto 390-8621, Japan
2 Department of Cell Biology, Cancer Research Institute of the Japanese Foundation of Cancer Research, 1-37-1 Kamiikebukuro, Toshima-ku, Tokyo 170-8455, Japan
3 Department of Immunology and Medical Zoology, Hyogo College of Medicine, 1-1 Mukogawa, Nishinomiya 663-8501, Japan
4 Core Research of Evolutional Science and Technology, Japan Science and Technology agency, 4-1-8 Motomati, Kawaguchi 332-0012, Japan
5 Department of Host Defense, Research Institute for Microbial Diseases, Osaka University, 3-1, Yamada-oka, Suita, Osaka 565-0871, Japan
6 Department of Molecular Genetics, Tohoku University School of Medicine, 2-1 Seiryo-cho, Aoba-Ku, Sendai 980-8575, Japan
7 Mouse Functional Genomics Research Group, Institute of Physical and Chemical Research (Japan) (RIKEN) Genomic Sciences Center, 214 Maeda-cho, Totsuka-ku, Yokohama, Kanagawa 244-0804, Japan
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Abstract |
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and IL-6. MyD88–/– and Trif–/– macrophages showed normal activation of caspase-1, demonstrating a dispensable role for MyD88 and Trif. After, LPS-challenged Asc–/– mice lacked serum elevation of IL-1ß and IL-18. Moreover, the Asc–/– mice exhibited neither acute liver injury nor lethal shock. These results demonstrate critical roles for ASC in the release of IL-1ß/IL-18 via activation of caspase-1 and provide new insights into the inflammatory responses for host defence and diseases. |
Introduction |
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B (NF-B), leading to microbial eradication. Lipopolysaccharide (LPS), a major component of Gram-negative bacteria, is recognized by TLR4 (Hoshino et al. 1999). Many investigators have shown myeloid differentiation factor 88 (MyD88) and/or Toll/IL-1 receptor domain-containing adaptor-inducing IFN-ß (Trif) as an essential adaptor molecule for intracellular signallings, differentially utilized by the TLRs (Kaisho et al. 2001; Kawai et al. 2001; Takeda et al. 2003; Yamamoto et al. 2003). In response to LPS, MyD88-deficient (MyD88–/–) macrophages fail to induce productions of IL-1ß, IL-6, IL-12 and tumour necrosis factor- (TNF-). However, the MyD88-deficient cells can secrete IL-18. IL-18 is constitutively produced as biologically inactive precursors and needs additional processing by caspase-1 to secrete and become an active form. In contrast, IL-1ß is newly produced as a biologically inactive precursor in a MyD88-dependent manner upon LPS stimulation, although, like IL-18, becoming an active form in a caspase-1-dependent manner thereafter. Therefore, caspase-1 seems to be activated through a MyD88-independent pathway after stimulation of TLR4 by LPS (Kawai et al. 2001; Seki et al. 2001). Therefore, it is important to know whether Trif, a second adaptor for the TLR4-mediated signalling pathway, is essential for the activation of caspase-1. Caspase-1 is present in the cytoplasm as an inactive precursor and must itself be activated by LPS (Ayala et al. 1994; Schumann et al. 1998). Therefore, IL-1ß and IL-18 releases are regulated post-translationally, particularly at the levels of intracellular activation of caspase-1. Caspase members, the cystein protease carrying a caspase recruitment domain (CARD) at their N-terminals, are classified into two populations in terms of their biological functions; apoptosis-associated caspases and cytokine-processing ones (Alnemri et al. 1996). Although the activation cascade of the apoptosis-associated caspases, including caspase-3, -8 and -9, toward cell-death signalling is well established, the exact mechanism(s) by which caspase-1 is activated remain unresolved (Bouchier-Hayes & Martin 2002).
We originally identified ASC (apoptosis-associated speck-like protein containing a CARD) as an intracellular protein partitioned into an insoluble cytoskeltal fraction termed ‘specks’ in human pro-myelocytic leukaemia HL-60 cells undergoing apoptosis (Masumoto et al. 1999). ASC carries a CARD at the C-terminal and a domain (PYD) homologous to pyrin, a gene product responsible for the inherited systemic disease characterized by the recurrent episodes of fever and inflammation, namely Familial Mediterranean Fever (The International FMF Consortium 1997) at the N-terminal. We and others have clarified the homophilic interaction of PYD of ASC with various adaptor molecules (Masumoto et al. 2001b; Liu et al. 2003), the preferential expression of ASC in apoptosis-prone sites in human tissues (Masumoto et al. 2001a) and the up-regulation of ASC by both inflammatory and apoptotic inducers (Martinon et al. 2002; Shiohara et al. 2002). These findings led us to assume that ASC is involved in both inflammatory reactions and apoptosis as an adaptor protein. Homophilic CARD/CARD interactions among pro-caspases and adapter proteins, presumably including ASC, are accepted to be necessary for activity and/or activation of caspases. Indeed, several lines of evidence strongly indicate involvement of ASC in inflammatory responses as well as the apoptosis process (Masumoto et al. 1999, 2001a; Shiohara et al. 2002; Liu et al. 2003). In particular, ASC has been reported to have the capacity to activate procaspase-1, but only in in vitro experiments (Martinon et al. 2002; Srinivasula et al. 2002; Wang et al. 2002; Kahlenberg & Dubyak 2004). To assess the physiological roles of ASC, we generated ASC-deficient mice (Asc–/–) by gene targeting and examined whether endogenous ASC is essential for processing procaspase-1, pro-IL-1ß and pro-IL-18 in vivo and in vitro.
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Result |
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We generated mice lacking the Asc gene (Fig. 1A). The mouse Asc gene consists of three exons. We constructed a targeting vector so that the entire exons were replaced with the nLacZ and floxed pMC1 neo poly(A) genes (Fig. 1A). Correctly targeted embryonic stem (ES) cell clones were used to generate mice with the mutated allele (Fig. 1B). To exclude the possibility that the expression of neor may result in any particular phenotype, we crossed male chimeric mice with CAG-Cre females expressing Cre recombinase to generate a neo(–)-targeted allele (Yao et al. 2002). The floxed neor was deleted by this cross (Fig. 1B), and offspring that did not contain the Cre gene were used for further studies. In the present study, the same results were obtained in both ASC-deficient mice carrying floxed pMC1 and ASC-deficient mice without neo by cre-enzyme treatment. The deletion of the Asc gene in mice was confirmed by Southern (Fig. 1B) and Western (Fig. 1C) blot analyses of several tissues. Therefore, the disruption of the Asc gene abolishes expression of the ASC protein. Mutant mice homozygous for the disrupted Asc allele were born at the expected Mendelian ratio and grew healthily under specific-pathogen-free conditions.
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We first investigated whether ASC is essential for LPS-induced IL-1ß secretion. To test this, we incubated peritoneal exudate macrophages (PECs) prepared from various genotypes of mice with LPS and measured IL-1ß in each supernatant by ELISA. Asc+/+ and Asc+/– PECs, but not Asc–/– PECs, secreted IL-1ß in response to LPS (Fig. 2A), indicating the requirement of ASC for LPS-induced IL-1ß release. When being incubated with a broad caspase inhibitor, z-VAD-fmk (zVAD), or a caspase-1-inhibitor, Ac-YVAD-CHO (YVAD), both Asc+/+ and Asc+/– PECs secreted significantly diminished levels of IL-1ß (Fig. 2A). Further, we investigated whether ASC is required for the release of other pro-inflammatory cytokines. To address this, we incubated Kupffer cells, tissue macrophages in the liver, with LPS and measured both caspase-1-dependent and -independent cytokines in each supernatant. Asc+/+ Kupffer cells dose-dependently secreted IL-1ß and IL-18 after stimulation with LPS, whereas Asc–/– Kupffer cells did not secrete either of them, indicating the requirement of ASC for the release of IL-1ß and IL-18 after LPS stimulation. In contrast, there were no significant differences of TNF- and IL-6 production between Asc+/+ and Asc–/– Kupffer cells after stimulation with LPS (Fig. 2B). These observations are consistent with results seen in previous studies with caspase-1 knockout mice (Kuida et al. 1995; Li et al. 1995; Gu et al. 1997), suggesting that ASC is essential for the production of caspase-1 dependent cytokines such as IL-1ß and IL-18 on stimulation with LPS.
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Next, we investigated the requirement of ASC for the activation of procaspase-1. Despite our intensive efforts, we did not detect activated caspase-1 in cell lysate prepared from LPS-activated Asc+/+ PECs. To identify small amounts of active caspase-1, we performed experiments in a cell-free system as previously described (Martinon et al. 2002; Kahlenberg & Dubyak 2004). In LPS-primed Asc+/+ PECs, procaspase-1 was clearly processed; that is, the density of the original 45-kDa band of procaspase-1 (p45 pro) was gradually decreased, while the density of 10 kDa (p10), a component of active caspase-1, intensified depending on the incubation time. Noticeably, no change in the density of p45 procaspase-1 and no appearance of p10 in Asc–/– PECs indicate that the processing of procaspase-1 did not occur without endogenous ASC. The processing of caspase-1 is completely inhibited by 50 µM zVAD or YVAD and anti-ASC antibody (1 µg), indicating caspase- and ASC-dependent procaspase-1 activation after stimulation with LPS (Fig. 3). In particular, prototype IL-1ß of 34 kDa (p34 pro) was detected in both the LPS-stimulated Asc+/+ and Asc–/– extracts at time 0, indicating a dispensable role for ASC in the production of pro-IL-1ß. Depending on the duration of incubation, active IL-1ß of 17 kDa (p17) cumulated in Asc+/+ PECs but not in Asc–/– PECs (Fig. 3). As expected, additional caspase inhibitors or anti-ASC antibody completely and partly, respectively, inhibited IL-1ß processing.
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In the present study, the cells were activated subsequently with LPS and ATP, raising the question of whether IL-1ß processing might occur independently of caspase-1. Therefore, we tested this using caspase-1–/– PECs. The cell extract prepared from caspase-1–/– PECs receiving the same procedures lacked IL-1ß processing but exhibited normal expression of pro-IL-1ß (Fig. 4), indicating caspase-1-dependent IL-1ß processing after stimulation with LPS in this cell-free system as well. Therefore, maturation of IL-1ß by LPS/ATP is dependent on activated caspase-1 that is promoted by endogenous ASC.
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By incubating the nuclear-free lysate from LPS-stimulated PECs, both the cellular procaspase-1 and the substrate pro-IL-1ß were converted into their active forms, indicating that de novo protein synthesis was not necessary for the processing of procaspase-1 and pro-IL-1ß in this nuclear-free lysate. As previously reported, Kupffer cells constitutively producing both procaspase-1 and pro-IL-18, but little pro-IL-1ß exhibit activation of procaspase-1 to secrete IL-18, but not IL-1ß, in response to LPS, even in the presence of transcriptional or translational inhibitors (Seki et al. 2001). These findings suggest that preformed protein/protein interactions might occur to induce the activation of procaspase-1 without de novo protein synthesis. To directly demonstrate the MyD88-independent activation of caspase-1, we performed the same experiment using PECs derived from MyD88-deficient mice. As expected, MyD88–/– PECs, like wild-type cells, expressed procaspase-1 but little pro-IL-1ß under normal conditions (Fig. 4, left panels). After stimulation with LPS, wild-type PECs exhibited induction of pro-IL-1ß, while MyD88–/– cells were impaired in it, demonstrating MyD88-dependent pro-IL-1ß production (Fig. 4, right panels). After LPS stimulation, MyD88–/– PECs showed comparable activation of procaspase-1 as in wild-type cells (Fig. 4, right panels), clearly demonstrating MyD88-independent activation of procaspase-1. It is convincing that MyD88–/– cells normally process IL-1ß because the active IL-1ß/pro-IL-1ß ratio in the mutant cells seems to be comparable in the wild-type cells, although the real amounts of IL-1ß in the former is much smaller than that in the latter (Fig. 4, right panels). Intriguingly, both ASC and procaspase-1 are constitutively produced in a MyD88-independent manner, as expected from our previous work. In fact, similar levels of ASC protein was observed in the cell extract prepared from both types of the cells (Fig. 4). Collectively, these results demonstrate the above possibility raised from the previous our studies (Seki et al. 2001) and that MyD88 is dispensable for expression of procaspase-1 and ASC.
Trif-independent activation of caspase-1
Next, we investigated whether Trif, a second adaptor molecule for the TLR4 signalling (Yamamoto et al. 2003), is required for the activation of procaspase-1. Trif–/– PECs, like wild-type and MyD88–/– cells, constitutively expressed both procaspase-1 and ASC (Fig. 5, left panels), indicating again MyD88/Trif-independent production of ASC and procaspase-1. LPS-stimulated Trif–/– PECs showed a similar activation of procaspase-1 to MyD88–/– PECs as well as wild-type cells. These results clearly indicate MyD88/Trif-independent activation of procaspase-1 upon LPS stimulation. Wild-type PECs produced large amounts of pro-IL-1ß, whereas MyD88–/– cells produced little of it again (Fig. 5, right panels). Trif–/– PECs produced intermediate amounts of pro-IL-1ß after stimulation with LPS (Fig. 5, right panels), indicating that Trif, in contrast to MyD88, is only partly involved in the induction of production of pro-IL-1ß. To substantiate further this possibility in vivo, we compared serum levels of IL-1ß and IL-18 between wild-type and Trif–/– mice after LPS challenge. The Trif–/– mice showed lower and almost equivalent levels of serum IL-1ß and IL-18, respectively, when compared with the wild-type mice (see supplementary Figs S1C and D online), indicating again minor roles for Trif in the activation of procaspase-1 in vivo. Collectively, these results demonstrate that Trif and MyD88 are dispensable for the TLR4-mediated activation of procaspase-1.
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We next analysed in vivo
roles of ASC in the induction of various pro-inflammatory cytokines. As previously reported, heat-killed Propionibacterium acnes
renders mice susceptible to a low dose of LPS (Tsutsui et al. 1997, 2000). We injected lethal doses of LPS (500 ng) into various genotypes of mice primed with P. acnes
and measured serum levels of pro-inflammatory cyotokines at the indicated time points. No significant differences of IL-12, IL-6 and TNF- elevation were found among Asc
+/+
, Asc
+/– and Asc
–/–
mice. By contrast, serum IL-1ß and IL-18 levels were not increased in P. acnes
-primed Asc
–/–
mice after treatment with LPS (Fig. 6). The increase of IFN-
was also profoundly suppressed in Asc
–/–
mice (Fig. 6). This is because IL-18 is a potent inducer of IFN- and plays an essential role for induction of IFN- after in vivo
treatment with LPS (Okamura et al. 1995; Tsutsui et al. 2000). Indeed, IL-18–/–
or caspase-1–/–
mice exhibit impaired IFN- increase after sequential treatment with P. acnes
and LPS (Okamura et al. 1995; Sakao et al. 1999). Collectively, ASC might play an essential role in the secretion of caspase-1-dependent IL-1ß and IL-18 release, but not in other independent production of cytokines in vivo
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Discussion |
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TopAbstractIntroductionResultDiscussionExperimental proceduresSupplementary materialReferences |
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Our present study has shown that Asc–/– mice have serious impairment in both the activation of procaspase-1 and the release of IL-1ß/IL-18 in vivo and in vitro after stimulation with LPS. These results clearly demonstrate that ASC is responsible for the activation of procaspase-1 and the consequent secretion of IL-1ß and IL-18, at least after stimulation with LPS. Intriguingly, Asc+/– mice produced about half of the mature IL-1ß and IL-18 in Asc+/+ mice (Figs 2A and 5), implying that the rate of IL-1ß and IL-18 release is proportional to Asc gene dosage. This suggests that ASC-promoted caspase-1 activity could be a rate-limiting step that determines the amount of mature IL-1ß and IL-18 released in vivo.
Apparently, all of these adptor proteins, ASC, MyD88 and Trif are essential for release of IL-1ß after LPS stimulation. After being stimulated with LPS, MyD88–/– Kupffer cells that constitutively store pro-IL-18, but not pro-IL-1ß, can secrete IL-18 but not IL-1ß equally, irrespective of the presence or absence of transcriptional or translational inhibitors, suggesting that MyD88-mediated pathways might be essential for induction of pro-IL-1ß. When we compared serum levels of IL-1ß and IL-18 between wild-type and MyD88–/– mice after LPS challenge, consistent with previous reports (Kawai et al. 2001; Seki et al. 2001), MyD88–/– mice showed normal capacity to produce IL-18 but not IL-1ß (see supplementary Fig. S1A,B online). Collectively, these observations led us to propose the possibility that MyD88-independent signal cascade via domain/domain interactions among the preformed proteins might be necessary and sufficient for the activation of procaspase-1. Preformation of pro-IL-18 but little pro-IL-1ß might determine the preferential secretion of IL-18 from the MyD88–/– Kupffer cells. Therefore, we wanted to know whether this was the case and whether Trif and/or ASC was involved in the LPS-induced production of pro-IL-1ß. Little pro-IL-1ß was detected in the cell extract of LPS-stimulated MyD88–/– PECs (Fig. 4). The amounts of pro-IL-1ß in LPS-treated Trif–/– PEC extract seem lower than those in wild-type preparation (Fig. 5). LPS-stimulated Asc–/– cell extract showed the similar levels of pro-IL-1ß as in the wild-type cells (Fig. 3). Collectively, these results indicate that the de novo synthesis of pro-IL-1ß profoundly and partially requires MyD88 and Trif, respectively, but not entirely ASC. In contrast, the activation of procaspase-1 requires ASC but not MyD88 or Trif (Figs 3, 4 and 5), because both MyD88–/– and Trif–/– cells, but not Asc–/– cells, produced comparable levels of active caspase-1 as in wild-type cells (Figs 3, 4 and 5). Therefore, it is convincing that the lack in production of mature IL-1ß in Asc–/– cells is as a result of failure in the activation of caspase-1 but not the induction of pro-IL-1ß (Fig. 3). In contrast, the lack in mature IL-1ß in MyD88–/– cells is as a result of failure in the induction of pro-IL-1ß but not activation of caspase-1 (Fig. 4). Therefore, ASC, MyD88 and Trif act on the definitely different processes for IL-1ß release. Intriguingly, gene expression of both Asc and procaspase-1 is regulated without being affected by the MyD88- and Trif-mediated pathways (Figs 3, 4 and 5). As these results indicate dispensable roles for MyD88 and Trif for activation of procaspase-1 after stimulation with LPS, further study is needed to identify adaptor molecules that transduce the signal from TLRs to the intracellular machinery in which procaspase-1 is activated, namely ‘inflammasome’ (Martinon et al. 2001, 2002).
ASC is also reported to have the potential to modulate NF-B activation via affecting degradation of the substrate of IB kinase after activation with various stimuli, such as TNF-, IL-1ß or LPS (Stehlik et al. 2002; Masumoto et al. 2003a). However, no significant differences in inflammatory cytokine production, such as of IL-6 and TNF-, were found between LPS-stimulated Asc+/+ and Asc–/– Kupffer cells (Fig. 2B). Furthermore, when we performed an experiment in the cell-free system, the amount of intracellular pro-IL-1ß which is produced in response to LPS, was much the same in both of PECs from Asc+/+ and Asc–/– mice (Fig. 3). These results suggest that ASC might not be profoundly involved in the de novo production of secreting cytokines, presumably via activation of NF-B.
It is obvious that much research in this area remains to be done; as the upstream stimuli need to be identified and the specific reactions between upstream proteins and ASC in various cells still need clarification. Several PYD- or CARD-containing proteins, such as pyrin, cryoprin/PYPAF1/NALP3 (gene product responsible for familial cold urticaria, etc. Agostini et al. 2004). According to several two-hybrid screening and over expression studies, Ipaf has been suggested as being associated with either caspase-1 and/or ASC and to be indispensable for caspase-1 activation (Poyet et al. 2001; Richards et al. 2001; Manji et al. 2002; Inohara & Nuñez 2003; Masumoto et al. 2003b). Very recently, it was reported that Ipaf-deficient macrophages activate caspase-1 in response to TLR stimulation but not intracellular pathogen (Salmonella typhimurium), while ASC is essential for caspase-1 activation in response to both extra- and intracellular pathogens (Mariathasan et al. 2004). In contrast, previous in vitro studies have suggested that a CARD protein, Rip2/Rick/CARDIAK binds and activates procaspase-1 via CARD/CARD interaction. However, it was reported that LPS-stimulated macrophages of Rip2-deficient mice exhibited no reduction in IL-1ß production, suggesting that Rip2 is not involved in caspase-1 activation downstream of TLRs (Lin et al. 2000). The interaction of ASC with those inflammatory proteins is thought to be an upstream reaction to regulate caspase-1 activity, though this remains to be further characterized.
In addition to caspase-1 activation, a wide array of functions has been proposed for ASC, such as triggering apoptosis. In zebrafish, a morphological abnormality was detected when ASC was knocked down with a morpholino anti-sense oligonucleotide (Masumoto et al. 2003b). In the present study, however, we have not yet detected any apparent abnormality in the morphology of Asc–/– mice. However, non-physiological circumstances, an exposure to toxic reagents for instance, could make ASC play a pro-apoptotic role in vertebrates, at least in murine ones. There are several lines of evidence that ASC can function as an adaptor protein in a p53-Bax mitochondrial pathway of apoptosis and, in a caspase-dependent manner, as a pro-apoptotic protein (McConnell & Vertino 2000; Ohtsuka et al. 2004). Suppression of ASC because of methylation in CpG islands is widely observed in human malignant tumours (Guan et al. 2003), suggesting a tumour suppressor role for ASC (McConnell & Vertino 2004).
Finally, many cell types, such as macrophages and dendritic cells, co-express ASC, procaspase-1 and pro-IL-18/pro-IL-1ß under normal conditions and increase production of these molecules upon stimulation with microbes and their products. We need further study to examine co-localization of ASC and pro-IL-1ß/pro-IL-18 in various cell types, including both immune and non-immune cells. This positive circuit for release of IL-18/IL-1ß in response to the microbe products via activation of TLRs seems to contribute to the development of host defence. Therefore, ASC plays a critical role in joining TLR signals and IL-18/IL-1ß activation. Conversely, persistent and/or excessive activation of IL-18/IL-1ß is profoundly involved in the development of various inflammatory diseases. Therefore, ASC could be a key molecule to determine health or disease. Our present study promises that ASC may become a novel target for both the efficient development of host defence against intractable microbial infection and the treatment of inflammatory and/or immune diseases.
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Experimental procedures |
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LPS from Escherichia coli Serotype O55: B5 was purchased from Sigma. z-VAD-fmk (zVAD) and Ac-YVAD-CHO (YVAD) were purchased from Peptido Institute Inc. and Calbiochem, respectively. Anti-caspase-1, anti-IL-1ß and anti-p44/42 MAP kinase (ERK1/2) antibodies were purchased from Santa Cruz, R & D systems and Cell Signalling, respectively. Polyclonal anti-ASC antibody was raised against the polypeptide corresponding to amino acids 77–193 of mouse ASC for immunoblotting. As a second antibody, anti-Rat IgG antibody and anti-Goat IgG antibody were purchased from Bio-Rad.
Generation of ASC-deficient mice
A genomic clone encompassing entire exons of the Asc gene was isolated from a genomic library of 129SV/J mouse. The targeting vector was constructed by replacing a 1.9-kb fragment encoding entire Asc ORFs with nLacZ and floxed pMC1 neo poly(A) for positive selection, and diphtheria toxin A-chain gene was inserted into the genomic fragment for negative selection. An nLacZ fragment was inserted in-frame in the NcoI site of exon 1. After the targeting vector was transfected in J1 ES cells, G418 resistant colonies were selected and screened by Southern blot analysis. Two of homologous recombinants were injected into C57BL/6 blastocysts, resulting in the birth of male chimera mice. Germ-line transmission of the disrupted Asc allele was achieved by mating with C57BL/6 females. ASC-deficient mice and their wild-type littermates from these intercrosses were used for experiments. MyD88–/– (Adachi et al. 1998) and Trif–/– mice (Yamamoto et al. 2003) were kindly provided by Dr Shizuo Akira at Osaka University (Osaka, Japan). Caspase–/– mice were a kind gift from Dr Keisuke Kuida at Vertex Pharmaceuticals (Cambridge, MA). All experiments were initiated with 8–10-week-old mice and all animals received humane care as outlined in the Guide for the Care and Use of Experimental Animals (Shinshu University Care Committee, Cancer Research Institute Care Committee, Hyogo College of Medicine Care Committee).
Cell preparation
Mice were injected i.p. with 3 mL Brewer's thioglycollate medium. After 3 days, mice were killed and peritoneal exudates were obtained by washing the peritoneum with 9 mL of pre-chilled phosphate-buffered saline (PBS). Kupffer cells were isolated from mice with various genetic backgrounds according to a method previously described (Seki et al. 2001). These cells (1 x 106/mL) were cultured in RPMI 1640 supplemented with 10% FCS and with the indicated concentrations of LPS for 24 h. The culture supernatants were collected for measuring cytokine levels.
Construction and transfection of ASC-expression plasmid
The entire open reading frame of mouse Asc was inserted into pcDNA3 (Invitrogen) at HindIII and XbaI sites. The prepared expression vector of ASC was called pcDNA-mASC. The DNA construct was confirmed by sequencing. Transfection of pcDNA-mASC into an HEK293T cell was done using 12 µg of DNA per plate and Lipofectin (Life Technologies, Inc.) according to the manufacturer's protocol.
Western blot analysis on LPS-induced caspase-1 activation and IL-1ß maturation in ASC-deficient PECs
In vitro processing assay was performed in a cell-free system as previously described (Martinon et al. 2002). Briefly, 5 x 107 PECs were primed with or without 500 ng/mL LPS for 4 h. Cells were scrapped and washed in pre-chilled PBS and re-suspended in basic salt solution (BSS) of 130 mM NaCl, 5 mM KCl, 20 mM HEPES, 5 mM glucose, 0.01% BSA, 1.5 mM CaCl2, 1 mM MgCl2 and 1 mM ATP and then incubated for 5 min at 37 °C. The cells were washed in pre-chilled PBS and re-suspended in pre-chilled 1 mL buffer W (20 mM HEPES pH 7.5, 10 mM KCl, 1.5 mM MgCl2, 1 mM EGTA, and 1 mM EDTA) supplemented with 2 mM DTT, 2 µg/mL leupeptin, 100 µg/mL phenylmethylsulfonyl fluoride (PMSF) and 2.5 µg/mL aprotinin. The cells were pelleted at 300 g for 30 s and all but approximately 50 µL of the buffer was removed. The cells were then allowed to swell for 15 min on ice and were disrupted by 20 passages through a 23 G needle. Lysate was then spun at 15 000 g for 15 min and the supernatant collected into a new tube and kept on ice. Ten microlitres of lysate (up to 
120 µg protein) were placed at 30 °C for the indicated times. Processing reaction was stopped by adding a one-fifth volume of 5 x sample buffer. Lysate was run on 15% SDS–PAGE and transferred to Hybond ECL (Amersham Biosciences). Western blot analyses were carried out with the following antibody concentrations: anti-Caspase-1 2 µg/mL, anti-IL-1ß 0.1 µg/mL, anti-mASC 12.5 µg/mL.
Assay of cytokines by ELISA
IL-1ß and IL-18 levels were measured by a commercially available ELISA kit purchased from R & D Systems and MBL, respectively. The concentrations of IFN-, IL-6, IL-12p40 and TNF- levels were determined by ELISA kits (Genzyme). Concentrations of several cytokines in the culture supernatant and the serum were measured by ELISA according to manufacturer's instructions.
Induction of endotoxin-induced liver injury and septic shock
To study liver injury, we followed a method previously described (Tsutsui et al. 1997). Briefly, the mice (9–10 weeks old) were i.p. administered 0.67 mg of heat killed Propionibacterium acnes. Seven days later, they were injected with 50 ng of LPS via a tail vein. Liver specimens were sampled 24 h after the LPS challenge and fixed in 3.5% formaldehyde in PBS. The samples were stained with haematoxylin and eosin (HE). Simultaneously, sera sampled for the measurement of liver enzymes, GPT, were performed by commercial service. For investigation of LPS-induced septic shock, the mice were monitored for signs of endotoxaemia and death at least 24 h after LPS challenge. To investigate the systemic release of cytokines after LPS challenge, P. acnes-primed mice were injected with 500 ng of LPS that was found to cause 100% death within 12 h after LPS challenge, as previously described (Gu et al. 1997). Serum was collected at indicated times after LPS challenge and cytokine levels measured by ELISA. Three to six mice were used for each experimental group.
Statistics
All data are shown as the mean ± SD of triplicate samples. Significance between the control group and a treated group was examined with the unpaired Student's t-test. P-values less than 0.05 were considered significant.
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Acknowledgements |
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Footnotes |
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a Requests for mice: E-mail: tnoda{at}ims.u-tokyo.ac.jp
* Correspondence: E-mail: stangch{at}sch.md.shinshu-u.ac.jp |
References |
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Received: 30 June 2004
Accepted: 4 August 2004
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