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Involvement of caspase activation through release of cytochrome c from mitochondria in apoptotic cell death of macrophages infected with Actinobacillus actinomycetemcomitans
FEMS Microbiology Letters 233 (2004) 29–35 www.fems-microbiology.org
Involvement of caspase activation through release of cytochrome c from mitochondria in apoptotic cell death of macrophages infected with Actinobacillus actinomycetemcomitans Hironori Kasai
a,b
, Kozo Yamamoto a, Takeyoshi Koseki c, Makoto Yokota b, Tatsuji Nishihara a,*
a
c
Department of Oral Microbiology, Kyushu Dental College, 2-6-1 Manazuru, Kokurakita-ku, Kitakyushu 803-8580, Japan b Department of Periodontology and Endodontology, Kyushu Dental College, Kitakyushu 803-8580, Japan Department of Oral Health and Development Sciences, Graduate School of Dentistry, Tohoku University, Sendai 980-8575, Japan Received 6 August 2003; received in revised form 19 November 2003; accepted 20 January 2004 First published online 6 February 2004
Abstract We previously reported that infection with the periodontopathic bacterium Actinobacillus actinomycetemcomitans induced apoptosis in a mouse macrophage cell line J774.1. In the present study, we examined the involvement of cytochrome c and caspases in the induction of apoptosis in A. actinomycetemcomitans-infected J774.1 cells. Following infection, the expression levels of cytochrome c, and cleaved forms of caspase-3 and caspase-9 in the cells were examined using immunoblot analysis. Cytochrome c was released from mitochondria into the cytoplasm after A. actinomycetemcomitans-infected J774.1 cells were cultured for 6 h, and caspase-3 and caspase-9 were found to be cleaved forms in the cells. Further, caspase-9 activity was markedly increased, and phosphorylated p53 was detected in the cells 30 h following infection. These results suggest that apoptosis in A. actinomycetemcomitans-infected J774.1 cells is regulated by the release of cytochrome c from mitochondria into cytoplasm and the subsequent activation of caspases through phosphorylation of p53. Ó 2004 Federation of European Microbiological Societies. Published by Elsevier B.V. All rights reserved. Keywords: Apoptosis; Actinobacillus actinomycetemcomitans; Caspases; Cytochrome c; p53; Macrophage
1. Introduction The initial event in the pathogenesis of most bacterial infectious diseases is microbial invasion of host cells after the microorganisms adhere to some host tissues, either directly or via some intermediary microorganisms [1]. Actinobacillus actinomycetemcomitans, a periodontopathic bacterium, has been implicated in the pathogenesis of several forms of periodontitis, and extracellular components from the bacterium are known to be potent mediators of its adherence to human oral epithelial cells [2]. It has also been reported that A. ac*
Corresponding author. Tel.: +81-93-582-1131x5485; fax: +81-93581-4984. E-mail address: [email protected] (T. Nishihara).
tinomycetemcomitans can invade periodontal tissues. Immunofluorescence microscopic analysis revealed the presence of A. actinomycetemcomitans in biopsy samples from periodontitis patients [3]. It is very important to elucidate whether periodontopathic bacteria can invade host cells and contribute to the initiation and progression of periodontitis, and many studies have shown evidence that A. actinomycetemcomitans can invade mammalian cells. Sreenivasan et al. [4] also provided direct evidence of in vitro invasion of human epithelial cells by A. actinomycetemcomitans. It is not difficult for periodontopathic bacteria to invade scavenger cells, also known as professional phagocytic cells, by phagocytosis. Shigella flexneri, the etiologic agent of dysentery, Bordetella pertussis, the causative agent of whooping cough in humans, and the
0378-1097/$22.00 Ó 2004 Federation of European Microbiological Societies. Published by Elsevier B.V. All rights reserved. doi:10.1016/j.femsle.2004.01.034
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H. Kasai et al. / FEMS Microbiology Letters 233 (2004) 29–35
periodontopathic bacterium A. actinomycetemcomitans, have each been shown to induce apoptosis in macrophages [5–7]. There is also evidence that apoptosis in S. flexneri-infected macrophages is dependent on caspase-1 [8,9]. Recently, we reported that A. actinomycetemcomitans (serotype a, b, and c) induced apoptosis in mouse macrophages. However, other periodontopathic bacteria, such as Poryphyromonas gingivalis 381 and W83, did not induce apoptosis [5]. These findings suggested that the ability of A. actinomycetemcomitans to promote apoptosis might be important in the initiation and development of periodontitis [5]. Further, in a series associated with that study, we found that adherence via CD14 molecules might play an essential role in the phagocytosis of A. actinomycetemcomitans by macrophages as well as in the subsequent induction of apoptosis [10]. Apoptosis plays important roles in the control of various biological systems such as immune responses, hematopoiesis, and embryonic development [11,12]. According to the current model of apoptosis, two classes of caspases, initiators and effectors, are involved in its machinery [13]. The signals for apoptosis activate initiator caspases (caspase-2, -8, and -9), and activated initiator caspases trigger the activation of effector caspases (caspase-3, -6, and -7), which in turn activate endonucleases and cause cell collapse by cleaving the specific substrates. In addition, several investigators have reported that mitochondria trigger apoptosis by releasing caspase activators, such as cytochrome c [14–16]. Another report speculated that the increase in cytosolic holocytochrome c upon apoptosis suggested that mitochondria may participate in apoptosis by releasing cytochrome c [17]. Despite the potential importance of apoptosis in A. actinomycetemcomitans-infected macrophages in severe periodontal diseases, current understanding of its molecular basis and regulation is minimal. In the present study, we investigated the involvement of cytochrome c, activation of caspases and phosphorylation of p53 in A. actinomycetemcomitansinfected macrophages.
vol) yeast extract at 37 °C for 4 days in an atmosphere of 5% CO2 in air [5]. 2.2. Routine procedure for in vitro A. actinomycetemcomitans infection of macrophages J774.1 cells were plated in a culture plate (Corning Glass Works, Corning, NY) at a concentration of 1 106 cells/ml 1 day prior to beginning of the experiment. A. actinomycetemcomitans Y4 bacterial cells were harvested by centrifugation and suspended in RPMI 1640 medium without antibiotics to an optical density of 0.55 at 550 nm, corresponding to approximately 5 109 bacteria/ml. The bacterial suspension was added to the plates, which were centrifuged at 1000g for 10 min at 4 °C prior to incubation at 37 °C for 1 h. J774.1 cells infected with A. actinomycetemcomitans Y4 at a final bacterium/cell ratio of 5000:1 were washed three times with RPMI 1640 medium containing penicillin G, streptomycin, and gentamicin (200 lg/ml) to remove extracellular bacteria. The infected J774.1 cells were then cultured in RPMI 1640 medium containing 5% FCS and antibiotics [5]. 2.3. Detection of apoptotic cells To detect apoptotic nuclei, infected J774.1 cells (106 ) were suspended in hypotonic solution (0.1% sodium citrate, 0.2% NP-40, 0.25 mg/ml RNase; pH 8.0), stained with of propidium iodide (50 lg/ml), and analyzed using an EPICS XL (Beckman Coulter, Fullerton, CA). The cell cycle phases were analyzed using Multicycle for Windows (Phoenix Flow Systems, San Diego, CA). For Annexin V/PI staining, infected cells were washed with cold phosphate-buffered saline (PBS; pH 7.2), and then resuspended in binding buffer (10 mM HEPES, 140 mM NaCl, 2.5 mM CaCl2 , pH 7.4). Fluorescein-conjugated Annexin V solution and PI solution were added and mixed gently according to the manufacturerÕs instructions (Molecular Probe, Inv. Eugene, OR). Cells were incubated at room temperature, and then analyzed using an EPICS XL.
2. Materials and methods
2.4. Cell fractionation
2.1. Cell and bacterial strains
Cytosolic extracts free of nuclei and mitochondriaenriched fractions were prepared according to the manufacturerÕs instructions (Mitochondria/Cytosol Fractionation Kit; Biovision Inc, Palo Alto, CA). In brief, A. actinomycetemcomitans-infected J774.1 cells were washed in PBS, and then suspended in digitonin lysis buffer supplemented with protease inhibitor cocktail (Sigma). After 5 min on ice, the cells were centrifuged at 14000g for 5 min at 4 °C. The clarified supernatant was transferred to a fresh tube, and the pellet was resuspended in lysis buffer. Both cytosolic and
A murine macrophage cell line J774.1 was obtained from the Japanese Cancer Research Resources Bank. Cells were cultured in RPMI 1640 medium (GIBCO Laboratories, Grand Island, NY) supplemented with 10% heat-inactivated fetal calf serum (FCS), penicillin G (100 U/ml), and streptomycin (100 lg/ml) at 37 °C in an atmosphere of 5% CO2 in air. A. actinomycetemcomitans Y4 was grown in Brain Heart Infusion broth (Difco Laboratories, Detroit, MI) supplemented with 1% (wt/
H. Kasai et al. / FEMS Microbiology Letters 233 (2004) 29–35
pellet fractions, enriched with mitochondria, were stored at )80 °C until use.
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10 min. Suspended cells were then centrifuged for 5 min, and caspase-9 activity in the cytosolic extracts was determined according to the manufacturerÕs instructions.
2.5. Immunoblot analysis J774.1 cells infected with A. actinomycetemcomitans were lysed in SDS lysis buffer (50 mM Tris–HCl, 2% SDS; pH 6.8) and 20 lg of protein extracts were electrophoresed on 10%, 12.5%, or 15% SDS-polyacrylamide gels, electroblotted on PVDF membranes. After incubation with 5% non-fat skim milk in PBS for 1 h, the membranes were reacted with primary antibodies for 1 h at room temperature. Immunodetection was performed using an ECL Western blotting detection system (Amersham Pharmacia Biotech, UK). The protein contents of samples were determined with a protein assay reagent (Bio-Rad Laboratories, Hercules, CA). Blots were stained with Coomassie brilliant blue and confirmed to contain a similar amount of protein extract in each lane. A polyclonal antibody against human cytochrome c was purchased from Santa Cruz Biotechnology Inc. (Santa Cruz, CA). A monoclonal antibody against caspase-3 was purchased from Cell Signaling Technology (Beverly, MA). Polyclonal antibodies against caspase-8 and caspase-9 were purchased from Chemicon International (Temecula, CA) and Medical and Biological Laboratories (Nagoya, Japan), respectively. A monoclonal antibody against p53 and a polyclonal antibody against phosphorylated p53 (Ser 15) were purchased from Oncogene Research Products (Cambridge, MA) and Cell Signaling Technology, respectively. 2.6. Determination of caspase-9 activity To determine caspase-9 activity in the cytosol of J774.1 cells infected with A. actinomycetemcomitans, ChemiconÕs Caspase-9 Colorimetric Activity Assay Kits (Chemicon International; Temecula, CA) were used. In brief, infected cells were lysed with lysis buffer on ice for
3. Results 3.1. Detection of apoptotic cells We determined the percentage of apoptotic J774.1 cells by examining hypodiploid DNA using a flow cytometer. When J774.1 cells were infected with A. actinomycetemcomitans Y4 at a bacterium/cell ratio of 5000:1, the percentage of cells with hypodiploid DNA was 37.7% (Fig. 1). To further investigate the nature of J774.1 cell death following infection of A. actinomycetemcomitans, we analyzed infected cells with a combination of Annexin V and PI. As shown in Fig. 2, flow cytometric analysis revealed that A. actinomycetemcomitans infection enhanced the surface expression of phosphatidylserine, as evidenced by increases Annexin V binding, compared to non-infected cells after 18 and 30 h of culture. Taken together, these results clearly indicate that J774.1 cells undergo apoptosis following A. actinomycetecomitans infection. 3.2. Cytochrome c release from mitochondria during apoptosis in J774.1 cells infected with A. actinomycetemcomitans To examine whether cytochrome c was released from mitochondria into the cytosol of A. actinomycetemcomitans-infected J774.1 cells, we investigated the expression of cytochrome c using an immunoblot analysis. Without infection, most of the cytochrome c in J774.1 cells was detected in the mitochondria. Cytochrome c in the cytosol of J774.1 cells was increased 6 h following A. actinomycetemcomitans infection, and continued to increase up to 18 h. In contrast, cytochrome c in
Fig. 1. Apoptotic cell death of J774.1 cells infected with A. actinomycetemcomitans. (a) Non-infected J774.1 cells. (b) J774.1 cells were infected with A. actinomycetemcomitans Y4 at a bacterium/cell ratio of 5000:1, and then cultured for 30 h. Shown are cells stained with propidium iodide, which were analyzed using a flow cytometer.
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Fig. 2. Annexin V/PI staining of J774.1 cells infected with A. actinomycetemcomitans. (a) Non-infected J774.1 cells. (b) J774.1 cells were infected with A. actinomycetemcomitans Y4 at a bacterium/cell ratio of 5000:1, and then cultured for 1 h. (c) Infected cells were cultured for 18 h. (d) Infected cells were cultured for 30 h. Stained cells were analyzed using a flow cytometer.
mitochondria showed a corresponding decrease at 18 h after infection (Fig. 3). 3.3. Detection of cleaved forms of caspase-3 and caspase9, and phosphorylation of p53 in A. actinomycetemcomitans-infected J774.1 cells After J774.1 cells were infected with A. actinomycetemcomitans, we examined the cleavage by caspase-3
and caspase-9 using an immunoblot analysis. Procaspase-3 and procaspase-9 were detected in the cells as protein bands with a molecular mass of 32 and 45 kDa, respectively, whereas non-infected J774.1 cells contained undetectable levels of those cleaved forms (data not shown). Cleaved forms of caspase-3 (17 kDa) and caspase-9 (35 kDa) were detected at 18 h after infection (Fig. 4(a) and (b)). Since flow cytometric analysis revealed that many apoptotic cells were detected in J774.1 cells at 30 h after A. actinomycetemcomitans infection, we also examined the expression levels of p53 and phosphorylated p53 in those cells by immunoblot analysis. p53 was detected in both infected and non-infected cells, while phosphorylated p53 was only detected in J774.1 cells at 30 h after infection (Fig. 4(c)). 3.4. Caspase-9 activity in cytosol of A. actinomycetemcomitans-infected J774.1 cells
Fig. 3. Release of cytochrome c from mitochondria in A. actinomycetemcomitans-infected macrophages. A. actinomycetemcomitans-infected J774.1 cells were cultured for the times indicated. Each sample was prepared as described in Section 2, and separated on a 15% polyacrylamide gel containing 0.1% SDS. Proteins were electroblotted on PVDF membranes and analyzed for the expression of cytochrome c by immunoblotting.
Caspase-9 activity in the cytosol of A. actinomycetemcomitans-infected J774.1 cells was examined at 12, 18 and 24 h of culture. An extract of non-infected J774.1 cells showed a very low level of caspase-9 activity (data not shown). At the beginning of the culture, an extract
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4. Discussion
Fig. 4. Expressions of caspase-3, caspase-9 and p53 in J774.1 cells infected with A. actinomycetemcomitans. A. actinomycetemcomitansinfected J774.1 cells were cultured for the times indicated. Each sample (20 lg of protein) was separated on a 15%, 12.5% and 10% polyacrylamide gel containing 0.1% SDS to detect caspase-3 (a), caspase-9 (b) and p53 and phosphorylated p53 (c), respectively. Proteins were electroblotted on PVDF membranes and then analyzed by immunoblotting.
Caspase-9 activity (OD405 )
of infected cells showed a low level of caspase-9 activity, which gradually increased after infection to about 2.7fold at 24 h, as compared with that in non-infected cells. Further, caspase-9 activity in the cytosol was inhibited by the treatment with a caspase-9 inhibitor (Fig. 5). 0.4
0.3
0.2
0.1
0.0 0
12
18
24
Culture time (h) Fig. 5. Caspase-9 activity in cytosol of A. actinomycetemcomitans-infected J774.1 cells. The cytosolic extracts of J774.1 cells infected with A. actinomycetemcomitans were incubated with (black bar) or without (white bar) caspase-9 inhibitor (Ac-LEHD-CHO; 2.5 lM) for 10 min at room temperature before adding caspase-9 substrate solution. Caspase-9 activity in cytosol was determined as described in Section 2.
Several previous studies have shown that the products of A. actinomycetemcomitans kill mammalian cells. Among these bacterial products, the leukotoxin is well known to be a potent virulence factor of A. actinomycetemcomitans. It has been reported that A. actinomycetemcomitans clinical isolates produce the leukotoxin able to lyse human neutrophils and monocytes [18]. The leukotoxin is a 110 kDa pore-forming, basic exotoxin which destroys human phagocytic cells by osmotic lysis by the pathway resembling necrosis [19]. Interestingly, Taichman et al. [20] have reported that the leukotoxin destroys human leukocytes and monocytes, but not the cells derived from rabbits, rats, mice and guinea pigs. In the previous studies, we have demonstrated that A. actinomycetemcomitans-infected mouse macrophages induce apoptosis, but not necrosis [5,10,21]. Several regulatory components of the apoptotic pathway have been identified in various types of cells. In mammalian cells, cysteine proteases related to the Caenorhabditis elegans CED-3 protein are designated as caspases and appear to be major effectors of the apoptotic process. To date, more than 10 caspases have been identified and partially characterized, several of which have been implicated in the induction of apoptosis [22]. To understand apoptosis signal pathways, we investigated the involvement of caspase-1 and -3 in apoptosis in macrophages infected with the periodontopathic bacterium A. actinomycetemcomitans [21]. However, the current study of caspases stimulated interest in determining other cascades, initiator caspases (caspase-2, -8 and -9) and effector caspases (caspase-3, -6 and -7) that induce apoptotic cell death of macrophages infected with that periodontopathic bacterium. The complex cascade of caspase activation is well known to be specific to apoptosis, and caspase-3, -8 and -9 have been found to play pivotal roles in the execution of apoptosis. Caspase-8 or caspase-9 activates caspase-3 which in turn cleaves several intracellular proteins including other caspases, and leads to the execution of apoptosis [23]. Further, several investigators have provided novel evidence for the involvement of cytochrome c which is released from mitochondria during the execution of apoptosis [24]. In addition, it was recently reported that cytochrome c promotes the activation of caspase-9 by forming a complex with apoptosis protease activating factor-1 (Apaf-1) in the presence of ATP [25], and that the interaction provokes a conformational change in Apaf-1 that recruits and activates procaspase-9 [26]. It is well known that caspases exist in cells as inactive proenzymes, and activated and cleaved in response to apoptotic stimulation. In the present study, cleaved forms of caspase-3 (17 kDa) and caspase-9 (35 kDa) were detected in A. actinomycetemcomitans-infected
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J774.1 cells by immunoblot analysis (Fig. 4), and activated caspase-9 proteases were seen after 18 and 24 h of culture (Fig. 5). On the other hand, activated caspase-8 proteases were not detected (data not shown). We previously reported the involvement of caspase-3 in the induction of apoptosis in A. actinomycetemcomitans-infected J774.1 cells [21]. In addition, flow cytometric analysis revealed that A. actinomycetemcomitans infection remarkably increased the population of apoptotic J774.1 cells. These findings suggest that caspase activation through release of cytochrome c from mitochondria participates in the induction of apoptosis in macrophages infected with the periodontopathic bacterium A. actinomycetemcomitans. p53 is a tumor suppressor protein, that is a major gatekeeper for growth and division of cells, and plays an important role in triggering apoptosis under several physiological conditions [27]. For example, thymocytes from wild-type mice undergo apoptosis in response to DNA damage, whereas those from p53 knock-out mice do not undergo apoptosis in response to the same stimulus [28]. Recently, it has been reported that p53 can directly engage the apoptotic pathways in cells and stimulate both death receptor signaling and mitochondrial perturbations, including cytochrome c release [29]. Soengas et al. [30] reported that loss of caspase-9 or Apaf-1 rendered mouse fibroblasts resistant to p53-dependent apoptosis. It has also been shown that DNA damage induces phosphorylation of p53 at Ser 15 and leads to reduced interaction between p53 and its negative regulatory protein MDM2 [31]. In the present study, A. actinomycetemcomitans infection induced phosphorylation of p53 at the Ser 15 residue at 30 h after infection. At this time, we have no explanation for the discrepancy of detection time between caspase-9 (24 h) and phosphorylated p53 (30 h), but think it is caused by the involvement of other signal pathways. These findings suggest that phosphorylation of p53 might play a role in apoptotic cell death of A. actinomycetemcomitans-infected macrophages. Recently, it was proposed that p53 is phosphorylated at multiple sites in response to DNA damage, and that its phosphorylation is regulated by some kinases, such as ERK and p38 MAPK [32]. Additional studies are under way to determine the role of p53 and other signalling molecules in A. actinomycetemcomitans-infected macrophages during the process of apoptosis. In conclusion, flow cytometric analysis revealed that A. actinomycetemcomitans infection induced apoptosis in J774.1 cells. During the process of apoptosis, cytochrome c, caspase-9 and caspase-3 were found to transduce signals in the infected cells. Further, p53 was found to be phosphorylated in A. actinomycetemcomitans-infected macrophages, suggesting the involvement of activated p53 in apoptosis induction. Taken together, these findings provide important new insights to
better understand the mechanism by which A. actinomycetemcomitans infection induces apoptosis in macrophages.
Acknowledgements This work was supported partially by Grants-in Aid from the ministry of Education, Science and Culture of Japan, and the Ministry of Health and Welfare of Japan.
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Involvement of caspase activation through release of cytochrome c from mitochondria in apoptotic cell death of macrophages infected with Actinobacillus actinomycetemcomitans Hironori Kasai
a,b
, Kozo Yamamoto a, Takeyoshi Koseki c, Makoto Yokota b, Tatsuji Nishihara a,*
a
c
Department of Oral Microbiology, Kyushu Dental College, 2-6-1 Manazuru, Kokurakita-ku, Kitakyushu 803-8580, Japan b Department of Periodontology and Endodontology, Kyushu Dental College, Kitakyushu 803-8580, Japan Department of Oral Health and Development Sciences, Graduate School of Dentistry, Tohoku University, Sendai 980-8575, Japan Received 6 August 2003; received in revised form 19 November 2003; accepted 20 January 2004 First published online 6 February 2004
Abstract We previously reported that infection with the periodontopathic bacterium Actinobacillus actinomycetemcomitans induced apoptosis in a mouse macrophage cell line J774.1. In the present study, we examined the involvement of cytochrome c and caspases in the induction of apoptosis in A. actinomycetemcomitans-infected J774.1 cells. Following infection, the expression levels of cytochrome c, and cleaved forms of caspase-3 and caspase-9 in the cells were examined using immunoblot analysis. Cytochrome c was released from mitochondria into the cytoplasm after A. actinomycetemcomitans-infected J774.1 cells were cultured for 6 h, and caspase-3 and caspase-9 were found to be cleaved forms in the cells. Further, caspase-9 activity was markedly increased, and phosphorylated p53 was detected in the cells 30 h following infection. These results suggest that apoptosis in A. actinomycetemcomitans-infected J774.1 cells is regulated by the release of cytochrome c from mitochondria into cytoplasm and the subsequent activation of caspases through phosphorylation of p53. Ó 2004 Federation of European Microbiological Societies. Published by Elsevier B.V. All rights reserved. Keywords: Apoptosis; Actinobacillus actinomycetemcomitans; Caspases; Cytochrome c; p53; Macrophage
1. Introduction The initial event in the pathogenesis of most bacterial infectious diseases is microbial invasion of host cells after the microorganisms adhere to some host tissues, either directly or via some intermediary microorganisms [1]. Actinobacillus actinomycetemcomitans, a periodontopathic bacterium, has been implicated in the pathogenesis of several forms of periodontitis, and extracellular components from the bacterium are known to be potent mediators of its adherence to human oral epithelial cells [2]. It has also been reported that A. ac*
Corresponding author. Tel.: +81-93-582-1131x5485; fax: +81-93581-4984. E-mail address: [email protected] (T. Nishihara).
tinomycetemcomitans can invade periodontal tissues. Immunofluorescence microscopic analysis revealed the presence of A. actinomycetemcomitans in biopsy samples from periodontitis patients [3]. It is very important to elucidate whether periodontopathic bacteria can invade host cells and contribute to the initiation and progression of periodontitis, and many studies have shown evidence that A. actinomycetemcomitans can invade mammalian cells. Sreenivasan et al. [4] also provided direct evidence of in vitro invasion of human epithelial cells by A. actinomycetemcomitans. It is not difficult for periodontopathic bacteria to invade scavenger cells, also known as professional phagocytic cells, by phagocytosis. Shigella flexneri, the etiologic agent of dysentery, Bordetella pertussis, the causative agent of whooping cough in humans, and the
0378-1097/$22.00 Ó 2004 Federation of European Microbiological Societies. Published by Elsevier B.V. All rights reserved. doi:10.1016/j.femsle.2004.01.034
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periodontopathic bacterium A. actinomycetemcomitans, have each been shown to induce apoptosis in macrophages [5–7]. There is also evidence that apoptosis in S. flexneri-infected macrophages is dependent on caspase-1 [8,9]. Recently, we reported that A. actinomycetemcomitans (serotype a, b, and c) induced apoptosis in mouse macrophages. However, other periodontopathic bacteria, such as Poryphyromonas gingivalis 381 and W83, did not induce apoptosis [5]. These findings suggested that the ability of A. actinomycetemcomitans to promote apoptosis might be important in the initiation and development of periodontitis [5]. Further, in a series associated with that study, we found that adherence via CD14 molecules might play an essential role in the phagocytosis of A. actinomycetemcomitans by macrophages as well as in the subsequent induction of apoptosis [10]. Apoptosis plays important roles in the control of various biological systems such as immune responses, hematopoiesis, and embryonic development [11,12]. According to the current model of apoptosis, two classes of caspases, initiators and effectors, are involved in its machinery [13]. The signals for apoptosis activate initiator caspases (caspase-2, -8, and -9), and activated initiator caspases trigger the activation of effector caspases (caspase-3, -6, and -7), which in turn activate endonucleases and cause cell collapse by cleaving the specific substrates. In addition, several investigators have reported that mitochondria trigger apoptosis by releasing caspase activators, such as cytochrome c [14–16]. Another report speculated that the increase in cytosolic holocytochrome c upon apoptosis suggested that mitochondria may participate in apoptosis by releasing cytochrome c [17]. Despite the potential importance of apoptosis in A. actinomycetemcomitans-infected macrophages in severe periodontal diseases, current understanding of its molecular basis and regulation is minimal. In the present study, we investigated the involvement of cytochrome c, activation of caspases and phosphorylation of p53 in A. actinomycetemcomitansinfected macrophages.
vol) yeast extract at 37 °C for 4 days in an atmosphere of 5% CO2 in air [5]. 2.2. Routine procedure for in vitro A. actinomycetemcomitans infection of macrophages J774.1 cells were plated in a culture plate (Corning Glass Works, Corning, NY) at a concentration of 1 106 cells/ml 1 day prior to beginning of the experiment. A. actinomycetemcomitans Y4 bacterial cells were harvested by centrifugation and suspended in RPMI 1640 medium without antibiotics to an optical density of 0.55 at 550 nm, corresponding to approximately 5 109 bacteria/ml. The bacterial suspension was added to the plates, which were centrifuged at 1000g for 10 min at 4 °C prior to incubation at 37 °C for 1 h. J774.1 cells infected with A. actinomycetemcomitans Y4 at a final bacterium/cell ratio of 5000:1 were washed three times with RPMI 1640 medium containing penicillin G, streptomycin, and gentamicin (200 lg/ml) to remove extracellular bacteria. The infected J774.1 cells were then cultured in RPMI 1640 medium containing 5% FCS and antibiotics [5]. 2.3. Detection of apoptotic cells To detect apoptotic nuclei, infected J774.1 cells (106 ) were suspended in hypotonic solution (0.1% sodium citrate, 0.2% NP-40, 0.25 mg/ml RNase; pH 8.0), stained with of propidium iodide (50 lg/ml), and analyzed using an EPICS XL (Beckman Coulter, Fullerton, CA). The cell cycle phases were analyzed using Multicycle for Windows (Phoenix Flow Systems, San Diego, CA). For Annexin V/PI staining, infected cells were washed with cold phosphate-buffered saline (PBS; pH 7.2), and then resuspended in binding buffer (10 mM HEPES, 140 mM NaCl, 2.5 mM CaCl2 , pH 7.4). Fluorescein-conjugated Annexin V solution and PI solution were added and mixed gently according to the manufacturerÕs instructions (Molecular Probe, Inv. Eugene, OR). Cells were incubated at room temperature, and then analyzed using an EPICS XL.
2. Materials and methods
2.4. Cell fractionation
2.1. Cell and bacterial strains
Cytosolic extracts free of nuclei and mitochondriaenriched fractions were prepared according to the manufacturerÕs instructions (Mitochondria/Cytosol Fractionation Kit; Biovision Inc, Palo Alto, CA). In brief, A. actinomycetemcomitans-infected J774.1 cells were washed in PBS, and then suspended in digitonin lysis buffer supplemented with protease inhibitor cocktail (Sigma). After 5 min on ice, the cells were centrifuged at 14000g for 5 min at 4 °C. The clarified supernatant was transferred to a fresh tube, and the pellet was resuspended in lysis buffer. Both cytosolic and
A murine macrophage cell line J774.1 was obtained from the Japanese Cancer Research Resources Bank. Cells were cultured in RPMI 1640 medium (GIBCO Laboratories, Grand Island, NY) supplemented with 10% heat-inactivated fetal calf serum (FCS), penicillin G (100 U/ml), and streptomycin (100 lg/ml) at 37 °C in an atmosphere of 5% CO2 in air. A. actinomycetemcomitans Y4 was grown in Brain Heart Infusion broth (Difco Laboratories, Detroit, MI) supplemented with 1% (wt/
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pellet fractions, enriched with mitochondria, were stored at )80 °C until use.
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10 min. Suspended cells were then centrifuged for 5 min, and caspase-9 activity in the cytosolic extracts was determined according to the manufacturerÕs instructions.
2.5. Immunoblot analysis J774.1 cells infected with A. actinomycetemcomitans were lysed in SDS lysis buffer (50 mM Tris–HCl, 2% SDS; pH 6.8) and 20 lg of protein extracts were electrophoresed on 10%, 12.5%, or 15% SDS-polyacrylamide gels, electroblotted on PVDF membranes. After incubation with 5% non-fat skim milk in PBS for 1 h, the membranes were reacted with primary antibodies for 1 h at room temperature. Immunodetection was performed using an ECL Western blotting detection system (Amersham Pharmacia Biotech, UK). The protein contents of samples were determined with a protein assay reagent (Bio-Rad Laboratories, Hercules, CA). Blots were stained with Coomassie brilliant blue and confirmed to contain a similar amount of protein extract in each lane. A polyclonal antibody against human cytochrome c was purchased from Santa Cruz Biotechnology Inc. (Santa Cruz, CA). A monoclonal antibody against caspase-3 was purchased from Cell Signaling Technology (Beverly, MA). Polyclonal antibodies against caspase-8 and caspase-9 were purchased from Chemicon International (Temecula, CA) and Medical and Biological Laboratories (Nagoya, Japan), respectively. A monoclonal antibody against p53 and a polyclonal antibody against phosphorylated p53 (Ser 15) were purchased from Oncogene Research Products (Cambridge, MA) and Cell Signaling Technology, respectively. 2.6. Determination of caspase-9 activity To determine caspase-9 activity in the cytosol of J774.1 cells infected with A. actinomycetemcomitans, ChemiconÕs Caspase-9 Colorimetric Activity Assay Kits (Chemicon International; Temecula, CA) were used. In brief, infected cells were lysed with lysis buffer on ice for
3. Results 3.1. Detection of apoptotic cells We determined the percentage of apoptotic J774.1 cells by examining hypodiploid DNA using a flow cytometer. When J774.1 cells were infected with A. actinomycetemcomitans Y4 at a bacterium/cell ratio of 5000:1, the percentage of cells with hypodiploid DNA was 37.7% (Fig. 1). To further investigate the nature of J774.1 cell death following infection of A. actinomycetemcomitans, we analyzed infected cells with a combination of Annexin V and PI. As shown in Fig. 2, flow cytometric analysis revealed that A. actinomycetemcomitans infection enhanced the surface expression of phosphatidylserine, as evidenced by increases Annexin V binding, compared to non-infected cells after 18 and 30 h of culture. Taken together, these results clearly indicate that J774.1 cells undergo apoptosis following A. actinomycetecomitans infection. 3.2. Cytochrome c release from mitochondria during apoptosis in J774.1 cells infected with A. actinomycetemcomitans To examine whether cytochrome c was released from mitochondria into the cytosol of A. actinomycetemcomitans-infected J774.1 cells, we investigated the expression of cytochrome c using an immunoblot analysis. Without infection, most of the cytochrome c in J774.1 cells was detected in the mitochondria. Cytochrome c in the cytosol of J774.1 cells was increased 6 h following A. actinomycetemcomitans infection, and continued to increase up to 18 h. In contrast, cytochrome c in
Fig. 1. Apoptotic cell death of J774.1 cells infected with A. actinomycetemcomitans. (a) Non-infected J774.1 cells. (b) J774.1 cells were infected with A. actinomycetemcomitans Y4 at a bacterium/cell ratio of 5000:1, and then cultured for 30 h. Shown are cells stained with propidium iodide, which were analyzed using a flow cytometer.
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Fig. 2. Annexin V/PI staining of J774.1 cells infected with A. actinomycetemcomitans. (a) Non-infected J774.1 cells. (b) J774.1 cells were infected with A. actinomycetemcomitans Y4 at a bacterium/cell ratio of 5000:1, and then cultured for 1 h. (c) Infected cells were cultured for 18 h. (d) Infected cells were cultured for 30 h. Stained cells were analyzed using a flow cytometer.
mitochondria showed a corresponding decrease at 18 h after infection (Fig. 3). 3.3. Detection of cleaved forms of caspase-3 and caspase9, and phosphorylation of p53 in A. actinomycetemcomitans-infected J774.1 cells After J774.1 cells were infected with A. actinomycetemcomitans, we examined the cleavage by caspase-3
and caspase-9 using an immunoblot analysis. Procaspase-3 and procaspase-9 were detected in the cells as protein bands with a molecular mass of 32 and 45 kDa, respectively, whereas non-infected J774.1 cells contained undetectable levels of those cleaved forms (data not shown). Cleaved forms of caspase-3 (17 kDa) and caspase-9 (35 kDa) were detected at 18 h after infection (Fig. 4(a) and (b)). Since flow cytometric analysis revealed that many apoptotic cells were detected in J774.1 cells at 30 h after A. actinomycetemcomitans infection, we also examined the expression levels of p53 and phosphorylated p53 in those cells by immunoblot analysis. p53 was detected in both infected and non-infected cells, while phosphorylated p53 was only detected in J774.1 cells at 30 h after infection (Fig. 4(c)). 3.4. Caspase-9 activity in cytosol of A. actinomycetemcomitans-infected J774.1 cells
Fig. 3. Release of cytochrome c from mitochondria in A. actinomycetemcomitans-infected macrophages. A. actinomycetemcomitans-infected J774.1 cells were cultured for the times indicated. Each sample was prepared as described in Section 2, and separated on a 15% polyacrylamide gel containing 0.1% SDS. Proteins were electroblotted on PVDF membranes and analyzed for the expression of cytochrome c by immunoblotting.
Caspase-9 activity in the cytosol of A. actinomycetemcomitans-infected J774.1 cells was examined at 12, 18 and 24 h of culture. An extract of non-infected J774.1 cells showed a very low level of caspase-9 activity (data not shown). At the beginning of the culture, an extract
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4. Discussion
Fig. 4. Expressions of caspase-3, caspase-9 and p53 in J774.1 cells infected with A. actinomycetemcomitans. A. actinomycetemcomitansinfected J774.1 cells were cultured for the times indicated. Each sample (20 lg of protein) was separated on a 15%, 12.5% and 10% polyacrylamide gel containing 0.1% SDS to detect caspase-3 (a), caspase-9 (b) and p53 and phosphorylated p53 (c), respectively. Proteins were electroblotted on PVDF membranes and then analyzed by immunoblotting.
Caspase-9 activity (OD405 )
of infected cells showed a low level of caspase-9 activity, which gradually increased after infection to about 2.7fold at 24 h, as compared with that in non-infected cells. Further, caspase-9 activity in the cytosol was inhibited by the treatment with a caspase-9 inhibitor (Fig. 5). 0.4
0.3
0.2
0.1
0.0 0
12
18
24
Culture time (h) Fig. 5. Caspase-9 activity in cytosol of A. actinomycetemcomitans-infected J774.1 cells. The cytosolic extracts of J774.1 cells infected with A. actinomycetemcomitans were incubated with (black bar) or without (white bar) caspase-9 inhibitor (Ac-LEHD-CHO; 2.5 lM) for 10 min at room temperature before adding caspase-9 substrate solution. Caspase-9 activity in cytosol was determined as described in Section 2.
Several previous studies have shown that the products of A. actinomycetemcomitans kill mammalian cells. Among these bacterial products, the leukotoxin is well known to be a potent virulence factor of A. actinomycetemcomitans. It has been reported that A. actinomycetemcomitans clinical isolates produce the leukotoxin able to lyse human neutrophils and monocytes [18]. The leukotoxin is a 110 kDa pore-forming, basic exotoxin which destroys human phagocytic cells by osmotic lysis by the pathway resembling necrosis [19]. Interestingly, Taichman et al. [20] have reported that the leukotoxin destroys human leukocytes and monocytes, but not the cells derived from rabbits, rats, mice and guinea pigs. In the previous studies, we have demonstrated that A. actinomycetemcomitans-infected mouse macrophages induce apoptosis, but not necrosis [5,10,21]. Several regulatory components of the apoptotic pathway have been identified in various types of cells. In mammalian cells, cysteine proteases related to the Caenorhabditis elegans CED-3 protein are designated as caspases and appear to be major effectors of the apoptotic process. To date, more than 10 caspases have been identified and partially characterized, several of which have been implicated in the induction of apoptosis [22]. To understand apoptosis signal pathways, we investigated the involvement of caspase-1 and -3 in apoptosis in macrophages infected with the periodontopathic bacterium A. actinomycetemcomitans [21]. However, the current study of caspases stimulated interest in determining other cascades, initiator caspases (caspase-2, -8 and -9) and effector caspases (caspase-3, -6 and -7) that induce apoptotic cell death of macrophages infected with that periodontopathic bacterium. The complex cascade of caspase activation is well known to be specific to apoptosis, and caspase-3, -8 and -9 have been found to play pivotal roles in the execution of apoptosis. Caspase-8 or caspase-9 activates caspase-3 which in turn cleaves several intracellular proteins including other caspases, and leads to the execution of apoptosis [23]. Further, several investigators have provided novel evidence for the involvement of cytochrome c which is released from mitochondria during the execution of apoptosis [24]. In addition, it was recently reported that cytochrome c promotes the activation of caspase-9 by forming a complex with apoptosis protease activating factor-1 (Apaf-1) in the presence of ATP [25], and that the interaction provokes a conformational change in Apaf-1 that recruits and activates procaspase-9 [26]. It is well known that caspases exist in cells as inactive proenzymes, and activated and cleaved in response to apoptotic stimulation. In the present study, cleaved forms of caspase-3 (17 kDa) and caspase-9 (35 kDa) were detected in A. actinomycetemcomitans-infected
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J774.1 cells by immunoblot analysis (Fig. 4), and activated caspase-9 proteases were seen after 18 and 24 h of culture (Fig. 5). On the other hand, activated caspase-8 proteases were not detected (data not shown). We previously reported the involvement of caspase-3 in the induction of apoptosis in A. actinomycetemcomitans-infected J774.1 cells [21]. In addition, flow cytometric analysis revealed that A. actinomycetemcomitans infection remarkably increased the population of apoptotic J774.1 cells. These findings suggest that caspase activation through release of cytochrome c from mitochondria participates in the induction of apoptosis in macrophages infected with the periodontopathic bacterium A. actinomycetemcomitans. p53 is a tumor suppressor protein, that is a major gatekeeper for growth and division of cells, and plays an important role in triggering apoptosis under several physiological conditions [27]. For example, thymocytes from wild-type mice undergo apoptosis in response to DNA damage, whereas those from p53 knock-out mice do not undergo apoptosis in response to the same stimulus [28]. Recently, it has been reported that p53 can directly engage the apoptotic pathways in cells and stimulate both death receptor signaling and mitochondrial perturbations, including cytochrome c release [29]. Soengas et al. [30] reported that loss of caspase-9 or Apaf-1 rendered mouse fibroblasts resistant to p53-dependent apoptosis. It has also been shown that DNA damage induces phosphorylation of p53 at Ser 15 and leads to reduced interaction between p53 and its negative regulatory protein MDM2 [31]. In the present study, A. actinomycetemcomitans infection induced phosphorylation of p53 at the Ser 15 residue at 30 h after infection. At this time, we have no explanation for the discrepancy of detection time between caspase-9 (24 h) and phosphorylated p53 (30 h), but think it is caused by the involvement of other signal pathways. These findings suggest that phosphorylation of p53 might play a role in apoptotic cell death of A. actinomycetemcomitans-infected macrophages. Recently, it was proposed that p53 is phosphorylated at multiple sites in response to DNA damage, and that its phosphorylation is regulated by some kinases, such as ERK and p38 MAPK [32]. Additional studies are under way to determine the role of p53 and other signalling molecules in A. actinomycetemcomitans-infected macrophages during the process of apoptosis. In conclusion, flow cytometric analysis revealed that A. actinomycetemcomitans infection induced apoptosis in J774.1 cells. During the process of apoptosis, cytochrome c, caspase-9 and caspase-3 were found to transduce signals in the infected cells. Further, p53 was found to be phosphorylated in A. actinomycetemcomitans-infected macrophages, suggesting the involvement of activated p53 in apoptosis induction. Taken together, these findings provide important new insights to
better understand the mechanism by which A. actinomycetemcomitans infection induces apoptosis in macrophages.
Acknowledgements This work was supported partially by Grants-in Aid from the ministry of Education, Science and Culture of Japan, and the Ministry of Health and Welfare of Japan.
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