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Possible involvement of protein kinase C in apoptotic cell death of macrophages infected with Actinobacillus actinomycetemcomitans
FEMS Microbiology Letters 159 (1998) 247^254
Possible involvement of protein kinase C in apoptotic cell death of macrophages infected with Actinobacillus actinomycetemcomitans Koji Nonaka a;b , Akira Ishisaki a , Miyuki Muro b , Satsuki Kato b , Mari Oido b , Keisuke Nakashima b , Yusuke Kowashi b , Tatsuji Nishihara a; * a b
Department of Oral Science, National Institute of Infectious Diseases, 1-23-1 Toyama, Shinjuku-ku, Tokyo 162, Japan Department of Periodontology, School of Dentistry, Health Sciences University of Hokkaido, Hokkaido 061-02, Japan Received 12 November 1997; revised 19 December 1997; accepted 19 December 1997
Abstract We have previously reported the evidence for apoptosis in the mouse macrophage cell line J774.1 by the periodontopathic bacterium Actinobacillus actinomycetemcomitans. In this study, we examined the role of protein kinases in the induction of apoptosis in A. actinomycetemcomitans-infected J774.1 cells by the MTT assay, fluorescence microscopy and flow cytometric analysis. After J774.1 cells were precultured with protein kinase C (PKC) activator, phorbol 12-myristate 13-acetate (PMA), J774.1 cells infected with A. actinomycetemcomitans showed the increased percentage of apoptotic cells. On the contrary, protein kinase A (PKA) activators, such as forskolin and dibutyryl cAMP, do not mimic the effect of PMA. PKC inhibitors, such as staurosporine, calphostin C, chelerythrine chloride, and H7 were found to suppress apoptotic cell death in J774.1 cells infected with A. actinomycetemcomitans. However, HA1004, known as PKA inhibitor, had no effect on apoptosis in infected macrophages. The results presented here suggest that the signals through PKC may play crucial roles in the modulation of apoptosis in macrophages infected with A. actinomycetemcomitans. z 1998 Federation of European Microbiological Societies. Published by Elsevier Science B.V. Keywords : Apoptosis ; Actinobacillus actinomycetemcomitans; Macrophage; Protein kinase C
1. Introduction The initial event in the pathogenesis of most bacterial diseases is microbial adherence to host cells and tissues. Actinobacillus actinomycetemcomitans has been implicated in the pathogenesis of several forms of periodontitis. Extracellular components from Actinobacillus actinomycetemcomitans are potent mediators of the adherence of this bacterial * Corresponding author. Tel.: (81) (3) 5285-1111; Fax: (81) (3) 5285-1172; E-mail: [email protected]
strain to human oral epithelial cells [1]. After adhesion to host tissue, some bacteria can invade host cells and cause several disease symptoms in humans [2]. A. actinomycetemcomitans was found to invade oral epithelial cells in vitro [3]. We developed an in vitro cell culture infection model for A. actinomycetemcomitans and provided evidence for apoptotic cell death of murine macrophages by A. actinomycetemcomitans infection [4]. Recently, we reported the involvement of CD14 molecules in the phagocytosis of A. actinomycetemcomitans by macrophages and in the subsequent induction of apoptosis [5].
0378-1097 / 98 / $19.00 ß 1998 Federation of European Microbiological Societies. Published by Elsevier Science B.V. PII S 0 3 7 8 - 1 0 9 7 ( 9 7 ) 0 0 5 7 8 - 8
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Apoptosis is a cellular event which underlies a wide variety of normal physiological phenomena including the clonal selection of lymphocytes [6], and the removal of tissue in£ammatory cells [7]. Recently, it has been shown that Shigella £exneri, the etiologic agent of dysentery, and Bordetella pertussis, the causative agent of whooping cough in humans, and periodontopathic bacterium A. actinomycetemcomitans, induce apoptosis in macrophages [4,8,9]. Despite the potential importance of apoptosis in infected macrophages as a pathological mechanism, our understanding of its molecular basis and regulation is still minimal. There is evidence that apoptosis is regulated at the level of signal transduction pathways. Among these pathways, protein kinases are well known to be attractive targets for modulation of apoptosis [10]. In this study, we investigated the meditative role of protein kinases in apoptotic cell death of macrophages infected with A. actinomycetemcomitans. For this purpose, we examined the e¡ects of activators and inhibitors of protein kinase C (PKC) or protein kinase A (PKA) on the induction of apoptosis in macrophages infected with A. actinomycetemcomitans. In addition, we have explored a correlation between Bcl-2 family products and the regulation of apoptosis in A. actinomycetemcomitans-infected macrophages by PKC.
2. Materials and methods 2.1. Reagents Forskolin, dibutyryl cAMP, and phorbol 12-myristate 13-acetate (PMA) were purchased from WAKO Pure Chemical Co. (Osaka, Japan). H7 and HA1004 were purchased from Seikagaku Kogyo Co. (Tokyo, Japan). Staurosporine, calphostin C, and chelerythrine chloride were obtained from Sigma Chemical Co. (St. Louis, MO). Rabbit anti-mouse Bcl-2, anti-mouse Bcl-XL and anti-mouse Bax sera were obtained from Santa Cruz Biotechnology Inc. (Santa Cruz, CA). 2.2. Cells and bacterial strains The murine macrophage cell line J774.1 was ob-
tained from the Japanese Cancer Research Resources Bank. J774.1 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 ml31 ) and streptomycin (100 Wg ml31 ) at 37³C in an atmosphere of 5% CO2 in air. A. actinomycetemcomitans Y4 was grown in Todd-Hewitt broth (Difco Laboratories, Detroit, MI) supplemented with 1% (wt/vol) yeast extract at 37³C for 2 days in an atmosphere of 5% CO2 in air [4]. 2.3. Routine procedure for in vitro infection of macrophages J774.1 cells were plated in a 96-well plate (Corning Glass Works, Corning, NY) at a concentration of 2U104 cells well31 (50 Wl well31 in RPMI 1640 medium containing 10% FCS and antibiotics) 1 day before the experiment. In some experiments, J774.1 cells were preincubated with PMA, forskolin, or dibutyryl cAMP for 18 h. A. actinomycetemcomitans Y4 was harvested by centrifugation and suspended in RPMI 1640 medium without antibiotics to an optical density of 0.55 at 550 nm, corresponding to approximately 5U109 bacteria ml31 . The bacterial suspension was added to the wells, and the plates were centrifuged at 1000Ug for 10 min at 4³C prior to incubation at 37³C for 1 h. J774.1 cells infected with A. actinomycetemcomitans Y4 at ¢nal bacterium/cell ratios of 5:1, 50:1, 500:1 and 5000:1 were washed three times with RPMI 1640 medium containing penicillin G, streptomycin and gentamicin (200 Wg ml31 ) to remove extracellular bacteria. The infected J774.1 cells were cultured with RPMI 1640 medium countering 5% FCS and antibiotics for 24 or 48 h [4]. 2.4. Cytotoxicity assay After J774.1 cells were infected with A. actinomycetemcomitans Y4, the cells were cultured with various concentrations of staurosporine, calphostin C, chelerythrine chloride, H7, or HA1004 for 48 h, and stock MTT (3-[4,5-dimethylthizol-2-yl]-2,5-diphenyltetrazolium bromide, 2.5 mg ml31 ; Sigma) solution (20 Wl well31 ) was added to the wells. The plates were read on a Multiskan bichromatic micro-
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plate reader (Labsystems, Helsinki, Finland) by using a test wavelength of 570 nm and reference wavelength 620 nm (MTT assay) [11]. The percent cytotoxicity was calculated by the following formula: 100U(13optical density at 570 to 620 nm with infection/optical density at 570 to 620 nm without infection). The percent inhibition was calculated by the following formula: percent inhibition = 100U(13percent cytotoxicity with stimulants/percent cytotoxicity without stimulants). 2.5. Detection of apoptotic cells To detect apoptotic nuclei, J774.1 cells (106 ) were suspended in hypotonic solution (3.4 mM sodium citrate, 0.1% Triton X-100, 0.1 mM EDTA, 1 mM Tris-HCl; pH 8.0), stained with 5 Wg of propidium iodide per ml, and analyzed by a FACScan (Becton Dickinson Immunocytometry Systems, San Jose, CA). For the Hoechst staining, the infected J774.1 cells were ¢xed with 1% glutaraldehyde for 1 h, and washed with phosphate-bu¡ered saline (pH 7.2). Samples were stained with 56 Wg ml31 of Hoechst dye 33342, and mounted on a slide glass. Nuclei were visualized by £uorescence microscopy, and the number of J774.1 cells with apoptotic nuclei was counted.
Fig. 1. Cell death of J774.1 cells infected with A. actinomycetemcomitans. J774.1 cells (2U104 ) were preincubated in the absence (white columns) or presence of forskolin (10 WM, hatched columns), dibutyryl cAMP (10 WM, shaded columns) or PMA (50 ng ml31 , black columns), and then infected with A. actinomycetemcomitans Y4 at bacterium/cell ratios of 5:1, 50:1 and 500:1. The percent cytotoxicity was determined at 48 h postinfection by the MTT assay and calculated as described in the text. *P 6 0.001 compared with the corresponding controls by Student's t-test.
conditions, PMA increased the cytotoxicity of A. actinomycetemcomitans Y4 on J774.1 cells at bacterium/cell ratios of 5:1, 50:1 and 500:1, but forskolin and dibutyryl cAMP showed no e¡ect on cell viability of A. actinomycetemcomitans-infected J774.1 cells
2.6. Immunoblot analysis J774.1 cells infected with A. actinomycetemcomitans were lysed in ice-cold lysis bu¡er (40 mM Tris-HCl, pH 7.4, 140 mM NaCl, 1% NP-40) and 15 Wg of protein extracts were electrophoresed in 12.5% SDS-polyacrylamide gels, electroblotted on PVDF membranes and reacted with primary antibodies. Signals were detected by an ECL detection kit (Amersham, Buckinghamshire, UK).
3. Results 3.1. E¡ect of PKC on cytotoxicity of J774.1 cells infected with A. actinomycetemcomitans We explored the cytotoxic e¡ects of A. actinomycetemcomitans Y4 on J774.1 cells preincubated with PMA, forskolin, or dibutyryl cAMP. Under these
Fig. 2. Inhibitory e¡ects of PKC inhibitors on cytotoxicity of J774.1 cells infected with A. actinomycetemcomitans. After J774.1 cells (2U104 ) were infected with A. actinomycetemcomitans Y4 at bacterium/cell ratios 50:1 (F) and 500 :1 (E), the cells were incubated with H7 (20 WM), staurosporine (40 nM), calphostin C (350 nM), chelerythrine chloride (1 WM), or HA1004 (20 WM) for 48 h. The percent cytotoxicity was determined by the MTT assay. The percent inhibition was calculated as described in the text.
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(Fig. 1). In addition, we examined the e¡ects of ¢ve protein kinase inhibitors such as staurosporine, calphostin C, chelerythrine chloride, H7 and HA1004 on cytotoxicity of J774.1 cells infected with A. actinomycetemcomitans by the MTT assay. PKC inhibitors such as staurosporine, calphostin C, chelerythrine chloride and H7 suppressed the cytotoxicity of J774.1 cells infected with A. actinomycetemcomitans at bacterium/cell ratios of 50:1 and 500:1. On the contrary, PKA inhibitor, HA1004, showed no e¡ect on cytotoxicity of J774.1 cells induced by A. actinomycetemcomitans Y4 infection (Fig. 2). 3.2. Detection of apoptotic cells We examined the percentage of apoptotic J774.1 cells with hypodiploid DNA by a £ow cytometer. The propidium iodide-stained histogram clearly distinguished nuclei with normal diploid DNA from
Fig. 3. Analysis of apoptosis in J774.1 cells infected with A. actinomycetemcomitans by a £ow cytometer. J774.1 cells (106 ) were preincubated in the absence (E) or presence (F) of PMA (50 ng ml31 ), and then infected with A. actinomycetemcomitans Y4 at bacterium/cell ratios of 5:1 and 50:1. The cells were cultured for 48 h, stained with propidium iodide and analyzed by a £ow cytometer. The percents of hypodiploid nuclei in J774.1 cells were indicated as the percent apoptosis. *P 6 0.05 compared with the corresponding control by Student's t-test. **P 6 0.001 compared with the corresponding control by Student's t-test.
Fig. 4. Analysis of DNA content in J774.1 cells infected with A. actinomycetemcomitans. After J774.1 cells (106 ) were infected with A. actinomycetemcomitans Y4 at a bacterium/cell ratio of 500:1, the cells were incubated with or without H7 (20 WM) for 48 h. The cells were stained with propidium iodide and analyzed by a £ow cytometer. The percents of hypodiploid nuclei in J774.1 cells are indicated.
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Y4, a large number of apoptotic J774.1 cells were detected (Fig. 7). Hoechst staining revealed that H7 and staurosporine decreased the numbers of apoptotic J774.1 cells infected with A. actinomycetemcomitans Y4 (Fig. 7). 3.4. Expression of Bcl-2, Bcl-XL and BAX in J774.1 cells infected with A. actinomycetemcomitans
Fig. 5. Inhibitory e¡ects of PKC inhibitors on apoptosis in J774.1 cells infected with A. actinomycetemcomitans. After J774.1 cells (106 ) were infected with A. actinomycetemcomitans Y4 at bacterium/cell ratios of 50:1 (F) and 500:1 (E), the cells were incubated with PKC inhibitors for 48 h. The percents of hypodiploid nuclei in J774.1 cells were expressed as the percent apoptosis. The percent inhibition was calculated by the following formula : the percent inhibition = 100U(13the percent apoptosis with PKC inhibitors/the percent apoptosis without PKC inhibitors).
We examined whether A. actinomycetemcomitans infection modulates the expression levels of the Bcl2 family proteins by immunoblot analysis. The expression levels of Bcl-2, Bcl-XL, and Bax in J774.1 cells were unchanged after being infected with A. actinomycetemcomitans. Cultivation with staurospor-
apoptotic nuclei with hypodiploid DNA. J774.1 cells were preincubated with PMA, and infected with A. actinomycetemcomitans Y4 at bacterium/cell ratios of 5:1 and 50:1. Preincubation of PMA signi¢cantly increased the population of the cells with hypodiploid DNA (Fig. 3). When J774.1 cells were infected with A. actinomycetemcomitans Y4 at a bacterium/ cell ratio of 500:1, the percentage of the cells with hypodiploid DNA was 31.0%. H7 (20 WM) remarkably decreased the percentage of apoptotic J774.1 cells infected with A. actinomycetemcomitans Y4 (Fig. 4). In addition, we found that not only H7 but also staurosporine, calphostin C and chelerythrine chloride inhibited the induction of apoptosis in J774.1 cells infected with A. actinomycetemcomitans Y4 as determined by a £ow cytometer (Fig. 5). 3.3. Analysis of nuclear morphology of apoptotic cells After J774.1 cells were infected with A. actinomycetemcomitans Y4, the cells were stained with Hoechst dye, and visualized by £uorescence microscopy. As shown in Fig. 6, the apoptotic cells were identi¢ed according to the characteristic cell morphology such as condensation and degradation of the nuclei. After being incubated with PMA for 18 h, and then infected with A. actinomycetemcomitans
Fig. 6. Representative morphology of J774.1 cells infected with A. actinomycetemcomitans. J774.1 cells were infected with A. actinomycetemcomitans Y4 at a bacterium/cell ratio of 500:1. After being cultured for 36 or 48 h, the cells were stained with the DNA-speci¢c £uorochrome Hoechst dye 33342. Apoptotic cells exhibiting the characteristic chromatin condensation were observed by £uorescence microscopy (U250). A: Non-infected J774.1 cells were cultured for 48 h. B: J774.1 cells infected with A. actinomycetemcomitans Y4 at a bacterium/cell ratio of 500:1 were cultured for 48 h.
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Fig. 7. E¡ects of PKC inhibitors and PKC activator on apoptosis in J774.1 cells infected with A. actinomycetemcomitans. J774.1 cells were treated with PKC activator and PKC inhibitors as described in the legends of Figs. 2 and 3, respectively. The cells were infected with A. actinomycetemcomitans Y4 at a bacterium/ cell ratio of 500 :1, cultured for 48 h and stained with Hoechst dye 33342. A hundred cells were observed by £uorescence microscopy (U250), and apoptotic cells exhibiting the characteristic chromatin condensation were detected. Data were expressed as percent apoptosis. *P 6 0.001 compared with the corresponding control by Student's t-test.
ine or H7 did not a¡ect the levels of these proteins. In addition, the expression levels of these proteins in A. actinomycetemcomitans-infected J774.1 cells were not altered, even when being preincubated with PMA (Fig. 8).
4. Discussion There is abundant evidence that the tumor promoter PMA causes di¡erentiation of human leukemia cells to macrophage-like cells [12]. We have previously reported that PMA or lipopolysaccharide stimulates murine macrophage cell lines P388D1 and J774.1, to produce cytokines including interleukin-1 (IL-1), IL-1 receptor antagonist, tumor necrosis factor, and activin A [13^15]. It is also well known that activated macrophages exhibit strong bactericidal activity [16]. First, we sought to examine whether PMA pretreatment a¡ects cell death in macrophages infected with periodontopathic bacterium A. actinomycetemcomitans by the MTT assay. As shown in Fig. 1, PMA showed strong e¡ect on cell viability of J774.1 cells infected with A. actinomyce-
Fig. 8. Expression of Bcl-2, Bcl-XL and Bax in J774.1 cells infected with A. actinomycetemcomitans. J774.1 cells were treated with PKC activator and PKC inhibitors as described in the legends of Figs. 2 and 3, respectively. J774.1 cells infected with A. actinomycetemcomitans Y4 at a bacterium/cell ratio of 5000:1 were cultured for 24 h and analyzed for the expression of Bcl-2, Bcl-XL and Bax by immunoblotting.
temcomitans. In addition, PMA-preincubated J774.1 cells showed a signi¢cant increase of apoptotic cells after being infected with A. actinomycetemcomitans (Figs. 3 and 7). These ¢ndings suggest that the pretreatment of PMA increases apoptotic cell death in J774.1 cells infected with A. actinomycetemcomitans. The use of PMA in several studies of apoptosis has led to contradictory conclusions. Although tumorpromoting phorbol diesters such as PMA are well known to activate PKC [17], PMA has been shown to prevent apoptosis in many cell types [18], but also to induce apoptosis in other cells [19]. In this study, we detected the increase of apoptotic cell death of J774.1 cells infected with A. actinomycetemcomitans, after being precultured with PKC activator, PMA, but not PKA activators, such as forskolin and dibutyryl cAMP (Fig. 1). These ¢ndings led us to investigate the role of PKC in the regulation of apoptosis in macrophages infected with A. actinomycetemcomitans by using several protein kinase inhibitors. In this study, we examined the e¡ects of PKC inhibitors, such as H7, staurosporine, calphostin C and chelerythrine chloride on apoptosis in J774.1 cells infected with A. actinomycetemcomitans at bacte-
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rium/cell ratios of 50:1 and 500:1. We ¢rst performed the MTT assay to explore whether PKC inhibitors a¡ect cytotoxicity of J774.1 cells infected with A. actinomycetemcomitans Y4. All PKC inhibitors used in this study remarkably suppressed the cytotoxicity of infected J774.1 cells, but the PKA inhibitor HA1004 did not (Fig. 2). Flow cytometric analysis revealed that PKC inhibitors strongly decreased the percentage of apoptotic J774.1 cells infected with A. actinomycetemcomitans (Fig. 4). Furthermore, we con¢rmed the nuclear morphology of apoptotic J774.1 cells by Hoechst dye staining. Incubation of infected J774.1 cells with H7 or staurosporine decreased the number of apoptotic cells (Fig. 7). H7 and staurosporine are known to be pharmaceutical PKC inhibitors. H7 has inhibition constants of 6 WM for PKC and 3 WM for cAMP-dependent kinase [20]. Tamaoki et al. [21] reported that staurosporine was a more potent inhibitor of PKC than tri£uoperazine, chlorpromazine and polymyxin B. Chelerythrine chloride and calphostin C, which are strong PKC inhibitors, compete for the conserved catalytic sites and regulatory domains of PKC, respectively [22,23]. Taken together, these ¢ndings suggest the regulation of apoptosis in macrophages infected with A. actinomycetemcomitans by PKC. The role of Bcl-2 related molecules, such as Bcl-2, Bcl-XL and Bax, in regulating apoptosis has been characterized for several years [24]. The ¢rst member of the family, Bcl-2, is a 26-kDa protein localized to outer mitochodria, nuclear envelope and endoplasmic reticula [25]. Bcl-X has long (Bcl-XL) and short (Bcl-Xs) forms that di¡er by 63 amino acids due to alternative splicing. Both Bcl-2 and Bcl-XL have been shown to protect cells from apoptosis in a variety of cellular systems [26]. Another homologue, Bax, has been known to inhibit the function of Bcl-2 and to form heterodimers with Bcl-2 [27]. Recently, Itano et al. [28] reported that the level of Bcl2 protein was increased by the treatment of PKC activator, PMA, but decreased by the treatment of PKA activator, dibutyryl cAMP, suggesting that the expression of Bcl-2 protein is regulated by PKC and PKA in a positive and negative manner, respectively. In this study, we examined the expression levels of Bcl-2, Bcl-XL and Bax by immunoblot analysis. The relative protein expression of Bcl-2 family was unaltered by the induction of apoptosis in J774.1 cells
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infected with A. actinomycetemcomitans. In addition, we found no appreciable change in Bcl-2 family protein levels in J774.1 cells infected with A. actinomycetemcomitans, even when being treated with staurosporine, H7 or PMA (Fig. 8). These ¢ndings suggest that the induction of apoptosis in J774.1 cells infected with A. actinomycetemcomitans and the inhibition of apoptosis by PKC inhibitors may be regulated by a Bcl-2-independent mechanism. In conclusion, the results presented by £ow cytometric analysis and Hoechst staining revealed that PKC inhibitors suppressed apoptosis in J774.1 cells infected with A. actinomycetemcomitans, but that PKA inhibitor did not. Furthermore, PKC activator was found to enhance apoptosis in J774.1 cells infected with A. actinomycetemcomitans. PKA activator showed no e¡ect on the induction of apoptosis in infected J774.1 cells. Taken together, our study indicates the involvement of PKC in the apoptosis of macrophages infected with periodontopathic bacterium A. actinomycetemcomitans.
Acknowledgments This work was supported partially by Grants-inAid from the Ministry of Education, Science and Culture of Japan, and the Ministry of Health and Welfare of Japan.
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Possible involvement of protein kinase C in apoptotic cell death of macrophages infected with Actinobacillus actinomycetemcomitans Koji Nonaka a;b , Akira Ishisaki a , Miyuki Muro b , Satsuki Kato b , Mari Oido b , Keisuke Nakashima b , Yusuke Kowashi b , Tatsuji Nishihara a; * a b
Department of Oral Science, National Institute of Infectious Diseases, 1-23-1 Toyama, Shinjuku-ku, Tokyo 162, Japan Department of Periodontology, School of Dentistry, Health Sciences University of Hokkaido, Hokkaido 061-02, Japan Received 12 November 1997; revised 19 December 1997; accepted 19 December 1997
Abstract We have previously reported the evidence for apoptosis in the mouse macrophage cell line J774.1 by the periodontopathic bacterium Actinobacillus actinomycetemcomitans. In this study, we examined the role of protein kinases in the induction of apoptosis in A. actinomycetemcomitans-infected J774.1 cells by the MTT assay, fluorescence microscopy and flow cytometric analysis. After J774.1 cells were precultured with protein kinase C (PKC) activator, phorbol 12-myristate 13-acetate (PMA), J774.1 cells infected with A. actinomycetemcomitans showed the increased percentage of apoptotic cells. On the contrary, protein kinase A (PKA) activators, such as forskolin and dibutyryl cAMP, do not mimic the effect of PMA. PKC inhibitors, such as staurosporine, calphostin C, chelerythrine chloride, and H7 were found to suppress apoptotic cell death in J774.1 cells infected with A. actinomycetemcomitans. However, HA1004, known as PKA inhibitor, had no effect on apoptosis in infected macrophages. The results presented here suggest that the signals through PKC may play crucial roles in the modulation of apoptosis in macrophages infected with A. actinomycetemcomitans. z 1998 Federation of European Microbiological Societies. Published by Elsevier Science B.V. Keywords : Apoptosis ; Actinobacillus actinomycetemcomitans; Macrophage; Protein kinase C
1. Introduction The initial event in the pathogenesis of most bacterial diseases is microbial adherence to host cells and tissues. Actinobacillus actinomycetemcomitans has been implicated in the pathogenesis of several forms of periodontitis. Extracellular components from Actinobacillus actinomycetemcomitans are potent mediators of the adherence of this bacterial * Corresponding author. Tel.: (81) (3) 5285-1111; Fax: (81) (3) 5285-1172; E-mail: [email protected]
strain to human oral epithelial cells [1]. After adhesion to host tissue, some bacteria can invade host cells and cause several disease symptoms in humans [2]. A. actinomycetemcomitans was found to invade oral epithelial cells in vitro [3]. We developed an in vitro cell culture infection model for A. actinomycetemcomitans and provided evidence for apoptotic cell death of murine macrophages by A. actinomycetemcomitans infection [4]. Recently, we reported the involvement of CD14 molecules in the phagocytosis of A. actinomycetemcomitans by macrophages and in the subsequent induction of apoptosis [5].
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Apoptosis is a cellular event which underlies a wide variety of normal physiological phenomena including the clonal selection of lymphocytes [6], and the removal of tissue in£ammatory cells [7]. Recently, it has been shown that Shigella £exneri, the etiologic agent of dysentery, and Bordetella pertussis, the causative agent of whooping cough in humans, and periodontopathic bacterium A. actinomycetemcomitans, induce apoptosis in macrophages [4,8,9]. Despite the potential importance of apoptosis in infected macrophages as a pathological mechanism, our understanding of its molecular basis and regulation is still minimal. There is evidence that apoptosis is regulated at the level of signal transduction pathways. Among these pathways, protein kinases are well known to be attractive targets for modulation of apoptosis [10]. In this study, we investigated the meditative role of protein kinases in apoptotic cell death of macrophages infected with A. actinomycetemcomitans. For this purpose, we examined the e¡ects of activators and inhibitors of protein kinase C (PKC) or protein kinase A (PKA) on the induction of apoptosis in macrophages infected with A. actinomycetemcomitans. In addition, we have explored a correlation between Bcl-2 family products and the regulation of apoptosis in A. actinomycetemcomitans-infected macrophages by PKC.
2. Materials and methods 2.1. Reagents Forskolin, dibutyryl cAMP, and phorbol 12-myristate 13-acetate (PMA) were purchased from WAKO Pure Chemical Co. (Osaka, Japan). H7 and HA1004 were purchased from Seikagaku Kogyo Co. (Tokyo, Japan). Staurosporine, calphostin C, and chelerythrine chloride were obtained from Sigma Chemical Co. (St. Louis, MO). Rabbit anti-mouse Bcl-2, anti-mouse Bcl-XL and anti-mouse Bax sera were obtained from Santa Cruz Biotechnology Inc. (Santa Cruz, CA). 2.2. Cells and bacterial strains The murine macrophage cell line J774.1 was ob-
tained from the Japanese Cancer Research Resources Bank. J774.1 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 ml31 ) and streptomycin (100 Wg ml31 ) at 37³C in an atmosphere of 5% CO2 in air. A. actinomycetemcomitans Y4 was grown in Todd-Hewitt broth (Difco Laboratories, Detroit, MI) supplemented with 1% (wt/vol) yeast extract at 37³C for 2 days in an atmosphere of 5% CO2 in air [4]. 2.3. Routine procedure for in vitro infection of macrophages J774.1 cells were plated in a 96-well plate (Corning Glass Works, Corning, NY) at a concentration of 2U104 cells well31 (50 Wl well31 in RPMI 1640 medium containing 10% FCS and antibiotics) 1 day before the experiment. In some experiments, J774.1 cells were preincubated with PMA, forskolin, or dibutyryl cAMP for 18 h. A. actinomycetemcomitans Y4 was harvested by centrifugation and suspended in RPMI 1640 medium without antibiotics to an optical density of 0.55 at 550 nm, corresponding to approximately 5U109 bacteria ml31 . The bacterial suspension was added to the wells, and the plates were centrifuged at 1000Ug for 10 min at 4³C prior to incubation at 37³C for 1 h. J774.1 cells infected with A. actinomycetemcomitans Y4 at ¢nal bacterium/cell ratios of 5:1, 50:1, 500:1 and 5000:1 were washed three times with RPMI 1640 medium containing penicillin G, streptomycin and gentamicin (200 Wg ml31 ) to remove extracellular bacteria. The infected J774.1 cells were cultured with RPMI 1640 medium countering 5% FCS and antibiotics for 24 or 48 h [4]. 2.4. Cytotoxicity assay After J774.1 cells were infected with A. actinomycetemcomitans Y4, the cells were cultured with various concentrations of staurosporine, calphostin C, chelerythrine chloride, H7, or HA1004 for 48 h, and stock MTT (3-[4,5-dimethylthizol-2-yl]-2,5-diphenyltetrazolium bromide, 2.5 mg ml31 ; Sigma) solution (20 Wl well31 ) was added to the wells. The plates were read on a Multiskan bichromatic micro-
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plate reader (Labsystems, Helsinki, Finland) by using a test wavelength of 570 nm and reference wavelength 620 nm (MTT assay) [11]. The percent cytotoxicity was calculated by the following formula: 100U(13optical density at 570 to 620 nm with infection/optical density at 570 to 620 nm without infection). The percent inhibition was calculated by the following formula: percent inhibition = 100U(13percent cytotoxicity with stimulants/percent cytotoxicity without stimulants). 2.5. Detection of apoptotic cells To detect apoptotic nuclei, J774.1 cells (106 ) were suspended in hypotonic solution (3.4 mM sodium citrate, 0.1% Triton X-100, 0.1 mM EDTA, 1 mM Tris-HCl; pH 8.0), stained with 5 Wg of propidium iodide per ml, and analyzed by a FACScan (Becton Dickinson Immunocytometry Systems, San Jose, CA). For the Hoechst staining, the infected J774.1 cells were ¢xed with 1% glutaraldehyde for 1 h, and washed with phosphate-bu¡ered saline (pH 7.2). Samples were stained with 56 Wg ml31 of Hoechst dye 33342, and mounted on a slide glass. Nuclei were visualized by £uorescence microscopy, and the number of J774.1 cells with apoptotic nuclei was counted.
Fig. 1. Cell death of J774.1 cells infected with A. actinomycetemcomitans. J774.1 cells (2U104 ) were preincubated in the absence (white columns) or presence of forskolin (10 WM, hatched columns), dibutyryl cAMP (10 WM, shaded columns) or PMA (50 ng ml31 , black columns), and then infected with A. actinomycetemcomitans Y4 at bacterium/cell ratios of 5:1, 50:1 and 500:1. The percent cytotoxicity was determined at 48 h postinfection by the MTT assay and calculated as described in the text. *P 6 0.001 compared with the corresponding controls by Student's t-test.
conditions, PMA increased the cytotoxicity of A. actinomycetemcomitans Y4 on J774.1 cells at bacterium/cell ratios of 5:1, 50:1 and 500:1, but forskolin and dibutyryl cAMP showed no e¡ect on cell viability of A. actinomycetemcomitans-infected J774.1 cells
2.6. Immunoblot analysis J774.1 cells infected with A. actinomycetemcomitans were lysed in ice-cold lysis bu¡er (40 mM Tris-HCl, pH 7.4, 140 mM NaCl, 1% NP-40) and 15 Wg of protein extracts were electrophoresed in 12.5% SDS-polyacrylamide gels, electroblotted on PVDF membranes and reacted with primary antibodies. Signals were detected by an ECL detection kit (Amersham, Buckinghamshire, UK).
3. Results 3.1. E¡ect of PKC on cytotoxicity of J774.1 cells infected with A. actinomycetemcomitans We explored the cytotoxic e¡ects of A. actinomycetemcomitans Y4 on J774.1 cells preincubated with PMA, forskolin, or dibutyryl cAMP. Under these
Fig. 2. Inhibitory e¡ects of PKC inhibitors on cytotoxicity of J774.1 cells infected with A. actinomycetemcomitans. After J774.1 cells (2U104 ) were infected with A. actinomycetemcomitans Y4 at bacterium/cell ratios 50:1 (F) and 500 :1 (E), the cells were incubated with H7 (20 WM), staurosporine (40 nM), calphostin C (350 nM), chelerythrine chloride (1 WM), or HA1004 (20 WM) for 48 h. The percent cytotoxicity was determined by the MTT assay. The percent inhibition was calculated as described in the text.
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(Fig. 1). In addition, we examined the e¡ects of ¢ve protein kinase inhibitors such as staurosporine, calphostin C, chelerythrine chloride, H7 and HA1004 on cytotoxicity of J774.1 cells infected with A. actinomycetemcomitans by the MTT assay. PKC inhibitors such as staurosporine, calphostin C, chelerythrine chloride and H7 suppressed the cytotoxicity of J774.1 cells infected with A. actinomycetemcomitans at bacterium/cell ratios of 50:1 and 500:1. On the contrary, PKA inhibitor, HA1004, showed no e¡ect on cytotoxicity of J774.1 cells induced by A. actinomycetemcomitans Y4 infection (Fig. 2). 3.2. Detection of apoptotic cells We examined the percentage of apoptotic J774.1 cells with hypodiploid DNA by a £ow cytometer. The propidium iodide-stained histogram clearly distinguished nuclei with normal diploid DNA from
Fig. 3. Analysis of apoptosis in J774.1 cells infected with A. actinomycetemcomitans by a £ow cytometer. J774.1 cells (106 ) were preincubated in the absence (E) or presence (F) of PMA (50 ng ml31 ), and then infected with A. actinomycetemcomitans Y4 at bacterium/cell ratios of 5:1 and 50:1. The cells were cultured for 48 h, stained with propidium iodide and analyzed by a £ow cytometer. The percents of hypodiploid nuclei in J774.1 cells were indicated as the percent apoptosis. *P 6 0.05 compared with the corresponding control by Student's t-test. **P 6 0.001 compared with the corresponding control by Student's t-test.
Fig. 4. Analysis of DNA content in J774.1 cells infected with A. actinomycetemcomitans. After J774.1 cells (106 ) were infected with A. actinomycetemcomitans Y4 at a bacterium/cell ratio of 500:1, the cells were incubated with or without H7 (20 WM) for 48 h. The cells were stained with propidium iodide and analyzed by a £ow cytometer. The percents of hypodiploid nuclei in J774.1 cells are indicated.
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Y4, a large number of apoptotic J774.1 cells were detected (Fig. 7). Hoechst staining revealed that H7 and staurosporine decreased the numbers of apoptotic J774.1 cells infected with A. actinomycetemcomitans Y4 (Fig. 7). 3.4. Expression of Bcl-2, Bcl-XL and BAX in J774.1 cells infected with A. actinomycetemcomitans
Fig. 5. Inhibitory e¡ects of PKC inhibitors on apoptosis in J774.1 cells infected with A. actinomycetemcomitans. After J774.1 cells (106 ) were infected with A. actinomycetemcomitans Y4 at bacterium/cell ratios of 50:1 (F) and 500:1 (E), the cells were incubated with PKC inhibitors for 48 h. The percents of hypodiploid nuclei in J774.1 cells were expressed as the percent apoptosis. The percent inhibition was calculated by the following formula : the percent inhibition = 100U(13the percent apoptosis with PKC inhibitors/the percent apoptosis without PKC inhibitors).
We examined whether A. actinomycetemcomitans infection modulates the expression levels of the Bcl2 family proteins by immunoblot analysis. The expression levels of Bcl-2, Bcl-XL, and Bax in J774.1 cells were unchanged after being infected with A. actinomycetemcomitans. Cultivation with staurospor-
apoptotic nuclei with hypodiploid DNA. J774.1 cells were preincubated with PMA, and infected with A. actinomycetemcomitans Y4 at bacterium/cell ratios of 5:1 and 50:1. Preincubation of PMA signi¢cantly increased the population of the cells with hypodiploid DNA (Fig. 3). When J774.1 cells were infected with A. actinomycetemcomitans Y4 at a bacterium/ cell ratio of 500:1, the percentage of the cells with hypodiploid DNA was 31.0%. H7 (20 WM) remarkably decreased the percentage of apoptotic J774.1 cells infected with A. actinomycetemcomitans Y4 (Fig. 4). In addition, we found that not only H7 but also staurosporine, calphostin C and chelerythrine chloride inhibited the induction of apoptosis in J774.1 cells infected with A. actinomycetemcomitans Y4 as determined by a £ow cytometer (Fig. 5). 3.3. Analysis of nuclear morphology of apoptotic cells After J774.1 cells were infected with A. actinomycetemcomitans Y4, the cells were stained with Hoechst dye, and visualized by £uorescence microscopy. As shown in Fig. 6, the apoptotic cells were identi¢ed according to the characteristic cell morphology such as condensation and degradation of the nuclei. After being incubated with PMA for 18 h, and then infected with A. actinomycetemcomitans
Fig. 6. Representative morphology of J774.1 cells infected with A. actinomycetemcomitans. J774.1 cells were infected with A. actinomycetemcomitans Y4 at a bacterium/cell ratio of 500:1. After being cultured for 36 or 48 h, the cells were stained with the DNA-speci¢c £uorochrome Hoechst dye 33342. Apoptotic cells exhibiting the characteristic chromatin condensation were observed by £uorescence microscopy (U250). A: Non-infected J774.1 cells were cultured for 48 h. B: J774.1 cells infected with A. actinomycetemcomitans Y4 at a bacterium/cell ratio of 500:1 were cultured for 48 h.
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Fig. 7. E¡ects of PKC inhibitors and PKC activator on apoptosis in J774.1 cells infected with A. actinomycetemcomitans. J774.1 cells were treated with PKC activator and PKC inhibitors as described in the legends of Figs. 2 and 3, respectively. The cells were infected with A. actinomycetemcomitans Y4 at a bacterium/ cell ratio of 500 :1, cultured for 48 h and stained with Hoechst dye 33342. A hundred cells were observed by £uorescence microscopy (U250), and apoptotic cells exhibiting the characteristic chromatin condensation were detected. Data were expressed as percent apoptosis. *P 6 0.001 compared with the corresponding control by Student's t-test.
ine or H7 did not a¡ect the levels of these proteins. In addition, the expression levels of these proteins in A. actinomycetemcomitans-infected J774.1 cells were not altered, even when being preincubated with PMA (Fig. 8).
4. Discussion There is abundant evidence that the tumor promoter PMA causes di¡erentiation of human leukemia cells to macrophage-like cells [12]. We have previously reported that PMA or lipopolysaccharide stimulates murine macrophage cell lines P388D1 and J774.1, to produce cytokines including interleukin-1 (IL-1), IL-1 receptor antagonist, tumor necrosis factor, and activin A [13^15]. It is also well known that activated macrophages exhibit strong bactericidal activity [16]. First, we sought to examine whether PMA pretreatment a¡ects cell death in macrophages infected with periodontopathic bacterium A. actinomycetemcomitans by the MTT assay. As shown in Fig. 1, PMA showed strong e¡ect on cell viability of J774.1 cells infected with A. actinomyce-
Fig. 8. Expression of Bcl-2, Bcl-XL and Bax in J774.1 cells infected with A. actinomycetemcomitans. J774.1 cells were treated with PKC activator and PKC inhibitors as described in the legends of Figs. 2 and 3, respectively. J774.1 cells infected with A. actinomycetemcomitans Y4 at a bacterium/cell ratio of 5000:1 were cultured for 24 h and analyzed for the expression of Bcl-2, Bcl-XL and Bax by immunoblotting.
temcomitans. In addition, PMA-preincubated J774.1 cells showed a signi¢cant increase of apoptotic cells after being infected with A. actinomycetemcomitans (Figs. 3 and 7). These ¢ndings suggest that the pretreatment of PMA increases apoptotic cell death in J774.1 cells infected with A. actinomycetemcomitans. The use of PMA in several studies of apoptosis has led to contradictory conclusions. Although tumorpromoting phorbol diesters such as PMA are well known to activate PKC [17], PMA has been shown to prevent apoptosis in many cell types [18], but also to induce apoptosis in other cells [19]. In this study, we detected the increase of apoptotic cell death of J774.1 cells infected with A. actinomycetemcomitans, after being precultured with PKC activator, PMA, but not PKA activators, such as forskolin and dibutyryl cAMP (Fig. 1). These ¢ndings led us to investigate the role of PKC in the regulation of apoptosis in macrophages infected with A. actinomycetemcomitans by using several protein kinase inhibitors. In this study, we examined the e¡ects of PKC inhibitors, such as H7, staurosporine, calphostin C and chelerythrine chloride on apoptosis in J774.1 cells infected with A. actinomycetemcomitans at bacte-
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rium/cell ratios of 50:1 and 500:1. We ¢rst performed the MTT assay to explore whether PKC inhibitors a¡ect cytotoxicity of J774.1 cells infected with A. actinomycetemcomitans Y4. All PKC inhibitors used in this study remarkably suppressed the cytotoxicity of infected J774.1 cells, but the PKA inhibitor HA1004 did not (Fig. 2). Flow cytometric analysis revealed that PKC inhibitors strongly decreased the percentage of apoptotic J774.1 cells infected with A. actinomycetemcomitans (Fig. 4). Furthermore, we con¢rmed the nuclear morphology of apoptotic J774.1 cells by Hoechst dye staining. Incubation of infected J774.1 cells with H7 or staurosporine decreased the number of apoptotic cells (Fig. 7). H7 and staurosporine are known to be pharmaceutical PKC inhibitors. H7 has inhibition constants of 6 WM for PKC and 3 WM for cAMP-dependent kinase [20]. Tamaoki et al. [21] reported that staurosporine was a more potent inhibitor of PKC than tri£uoperazine, chlorpromazine and polymyxin B. Chelerythrine chloride and calphostin C, which are strong PKC inhibitors, compete for the conserved catalytic sites and regulatory domains of PKC, respectively [22,23]. Taken together, these ¢ndings suggest the regulation of apoptosis in macrophages infected with A. actinomycetemcomitans by PKC. The role of Bcl-2 related molecules, such as Bcl-2, Bcl-XL and Bax, in regulating apoptosis has been characterized for several years [24]. The ¢rst member of the family, Bcl-2, is a 26-kDa protein localized to outer mitochodria, nuclear envelope and endoplasmic reticula [25]. Bcl-X has long (Bcl-XL) and short (Bcl-Xs) forms that di¡er by 63 amino acids due to alternative splicing. Both Bcl-2 and Bcl-XL have been shown to protect cells from apoptosis in a variety of cellular systems [26]. Another homologue, Bax, has been known to inhibit the function of Bcl-2 and to form heterodimers with Bcl-2 [27]. Recently, Itano et al. [28] reported that the level of Bcl2 protein was increased by the treatment of PKC activator, PMA, but decreased by the treatment of PKA activator, dibutyryl cAMP, suggesting that the expression of Bcl-2 protein is regulated by PKC and PKA in a positive and negative manner, respectively. In this study, we examined the expression levels of Bcl-2, Bcl-XL and Bax by immunoblot analysis. The relative protein expression of Bcl-2 family was unaltered by the induction of apoptosis in J774.1 cells
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infected with A. actinomycetemcomitans. In addition, we found no appreciable change in Bcl-2 family protein levels in J774.1 cells infected with A. actinomycetemcomitans, even when being treated with staurosporine, H7 or PMA (Fig. 8). These ¢ndings suggest that the induction of apoptosis in J774.1 cells infected with A. actinomycetemcomitans and the inhibition of apoptosis by PKC inhibitors may be regulated by a Bcl-2-independent mechanism. In conclusion, the results presented by £ow cytometric analysis and Hoechst staining revealed that PKC inhibitors suppressed apoptosis in J774.1 cells infected with A. actinomycetemcomitans, but that PKA inhibitor did not. Furthermore, PKC activator was found to enhance apoptosis in J774.1 cells infected with A. actinomycetemcomitans. PKA activator showed no e¡ect on the induction of apoptosis in infected J774.1 cells. Taken together, our study indicates the involvement of PKC in the apoptosis of macrophages infected with periodontopathic bacterium A. actinomycetemcomitans.
Acknowledgments This work was supported partially by Grants-inAid from the Ministry of Education, Science and Culture of Japan, and the Ministry of Health and Welfare of Japan.
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