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Shigella flexneri YSH6000 induces two types of cell death, apoptosis and oncosis, in the differentiated human monoblastic cell line U937
FEMS Microbiology Letters 174 (1999) 89^95
Shigella £exneri YSH6000 induces two types of cell death, apoptosis and oncosis, in the di¡erentiated human monoblastic cell line U937 Takashi Nonaka
1;a
, Asaomi Kuwae b , Chihiro Sasakawa b , Shinobu Imajoh-Ohmi a *
a
b
Department of Bacterial Infection, The Institute of Medical Science, University of Tokyo, 4-6-1, Shirokanedai, Minato-ku, Tokyo 108-8639, Japan Department of Bacteriology, The Institute of Medical Science, University of Tokyo, 4-6-1, Shirokanedai, Minato-ku, Tokyo 108-8639, Japan Received 21 December 1998 ; received in revised form 22 February 1999; accepted 5 March 1999
Abstract Shigella flexneri, but not a non-invasive mutant derivative, rapidly induced cell death in human monoblastic U937 cells as well as in differentiated cells pretreated with interferon-Q (IFNQ) or retinoic acid (RA). We investigated the morphological and biochemical characteristics of bacterial invasion-induced cell death in these differentiated U937 cells. IFNQ-differentiated cells showed morphological changes typical of apoptosis and their DNA was cleaved giving a ladder-like electrophoretic pattern after infection by Shigellae. In contrast, swelling of the cytoplasm and blebbing of the plasma membrane were observed in RAdifferentiated and undifferentiated cells invaded by the bacteria. No condensation of nuclei was observed in these cells by light microscopy, and no internucleosomal fragmentation of DNA was detected on agarose gels, which resembled the features of oncosis. Furthermore, cleavage of poly(ADP-ribose) polymerase, a substrate for apoptotic caspases, was seen only in IFNQpretreated cells but not in RA-pretreated or undifferentiated cells. These findings suggested that virulent Shigella flexneri induces distinct types of cell death in U937 cells depending on their differentiation state. z 1999 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. Keywords : Apoptosis ; Oncosis ; Shigella £exneri; U937; Di¡erentiation
1. Introduction Shigella £exneri possesses a 230 kb plasmid which encodes virulent proteins [1] including a series of in* Corresponding author. Tel. and Fax: +81 (3) 5449-5311; E-mail: [email protected] 1
Research Fellow of the Japan Society for the Promotion of Science.
vasion plasmid antigens, IpaB, IpaC and IpaD, essential for bacterial invasion into host cells [2^5]. Among these, IpaB is thought to play a key role in induction of apoptosis following infection in the murine macrophage cell line J774 [6]. Microinjection of IpaB induces apoptosis in J774 cells presumably by binding to caspase-1 [7] (ICE; interleukin-1L converting enzyme), a member of the caspase family [8] involved in signal transduction for apoptotic cell
0378-1097 / 99 / $20.00 ß 1999 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. PII: S 0 3 7 8 - 1 0 9 7 ( 9 9 ) 0 0 1 2 5 - 1
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death [9,10]. Sequential activation of procaspases is necessary for signal transduction of apoptosis. For example, Fas, a cell surface death receptor for Fas ligand, interacts with procaspase-8 via an adaptor protein, FADD, when bound with the ligand [11]. The receptor-coupled procaspase-8 is autocatalytically activated, and caspase-8 catalyzes activation of precursors for downstream caspases such as caspase-3 and -7 [12]. Peripheral caspases cleave various proteins: DNase inhibitor inactivated by proteolysis leading to DNA breakdown [13], and cytoskeletal proteins causing morphological changes during apoptosis [10]. In Shigella-infected murine macrophages, Chen et al. proposed that IpaB protein functions as an activator for procaspase-1 and/or an enhancer for the activity of caspase-1 [6]. Caspase-1 should trigger activation of the apoptotic cascade in infected cells. Virulent Shigella-infected and IpaB-injected J774 cells have been reported to show morphological features typical of apoptosis [6,14]. On the other hand, Fernandez-Prada et al. reported that human monocyte-derived macrophages infected with S. £exneri in vitro underwent rapid cytolytic cell death di¡erent from apoptosis [15]. Shigella-infected macrophages show rupture of the plasma membrane, cell swelling, disintegration of ultrastructure, and generalized karyolysis, which are characteristics of oncosis, a recently proposed form of cell death [16]. In this study, we found that wild-type S. £exneri induces two types of cell death, apoptosis and oncosis, in human monoblastic U937 cells, depending on prior di¡erentiation induced by interferon-Q (IFNQ) or retinoic acid (RA). Our ¢ndings may explain the controversial observations from the two previously reported studies [6,15].
diluted 50-fold in BHI broth (Difco, Detroit, MI) and incubated at 37³C for 2 h until they reached the mid-log phase of growth. Human monoblastic U937 cells, provided by Japanese Cancer Research Resources Bank (JCRB9021), were cultured and treated with di¡erentiation inducers (100 units ml31 IFNQ or 3 WM RA) for 48 h as described previously [17]. 2.2. Infection procedure and cytotoxicity assay Undi¡erentiated, IFNQ-treated, or RA-treated U937 cells (U937UD, U937IFNQ, or U937RA, respectively) were resuspended in fresh medium without antibiotics at a concentration of 106 cells ml31 , 2 h prior to infection. Cells were mixed with bacteria at a multiplicity of infection (MOI) of 100 bacteria per cell, and immediately centrifuged at 700Ug for 10 min. After incubation for 1 h with bacteria at 37³C, cells were washed with medium supplemented with 100 Wg ml31 gentamycin (Gm) and further incubated for 2 h with this antibiotic. Infected cells were assessed by light microscopy and their viabilities were examined by the trypan blue dye exclusion method. 2.3. Detection of apoptosis Cells (3U105 cells) after infection with bacteria (MOI = 100) were cytospun, ¢xed with ethanol and stained with Giemsa's solution (E. Merck, Darmstadt) for morphological analysis. Infected cells (107 cells) at a MOI of 20 after incubation with Gm for 5 h were used for DNA fragmentation assay as described previously [18]. 2.4. Immunoblotting analysis
2. Materials and methods 2.1. Bacterial strains and cell culture S. £exneri YSH6000 (wild-type) and N1411 (a non-virulent mutant lacking ipaBCD) [4] were routinely grown at 30³C overnight in L broth (Difco, Detroit, MI). For infection, overnight cultures were
Infected cells were lysed and treated with 10% (v/v) trichloroacetic acid for 20 min on ice. The precipitated proteins were subjected to 7.5% SDS-polyacrylamide gel electrophoresis, electroblotted, and stained with a polyclonal antibody to the aminoterminal region of the 85 kDa fragment of human poly(ADP-ribose) polymerase (PARP).
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3. Results 3.1. Death of U937 cells infected with virulent S. £exneri Fig. 1 shows the cytotoxicity of wild-type S. £exneri YSH6000 and N1411, a non-virulent mutant lacking ipaBCD, against U937 cells in various di¡erentiation states. The wild-type strain killed U937 cells regardless of di¡erentiation. The highest level of cytotoxicity was seen toward U937RA; nearly twice as many cells were stained by trypan blue in U937RA 2 h after infection as compared with U937UD or U937IFNQ. The cytotoxicity of the wild-type strain to U937 cells was dependent on MOI (data not shown). In contrast, the non-virulent strain N1411 was not cytotoxic to any of these cells as determined by the dye exclusion test. 3.2. Demonstration of apoptosis in infected U937 cells The trypan blue dye exclusion method cannot discriminate between di¡erent types of cell death. Thus, we examined the morphological and biochemical features of Shigella-treated cells. Fig. 2 shows the morphology of U937 cells 2 h after infection with either the wild-type or avirulent mutant strain. Similar de-
Fig. 1. Mortality of U937 cells following infection by S. £exneri. U937UD, U937IFNQ and U937RA cells were infected with wildtype (WT: YSH6000, shaded columns) or N1411 (ipaBCD3 , hatched columns) at MOI of 100. Cytotoxicity was assayed by the trypan blue dye exclusion method. Viable cells were counted after a 2 h incubation in the presence of Gm. The white column indicates dye-stained uninfected U937 cells.
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grees of invasion were observed in U937UD, U937IFNQ and U937RA cells (A^C), but no bacteria were seen in N1411-treated cells (E^G). In Shigellainfected U937IFNQ (arrowheads in panel B), cytoplasmic shrinkage and nuclear fragmentation were observed similarly to the results shown in Fig. 2D, indicating typical apoptotic cells treated with a cytotoxic anti-Fas antibody. However, no changes occurred in the nuclei of U937UD or U937RA cells even when they were invaded by the bacteria (A and C). These infected cells were larger than U937IFNQ cells infected with Shigella, and their plasma membrane seemed to be damaged. On the other hand, no morphological di¡erences were observed in N1411-treated U937 cells (E^G) as compared with control U937UD cells (H). Next, we analyzed DNA fragmentation, a hallmark of apoptosis, in Shigella-infected cells, and compared the DNA electrophoretic patterns with those in Fas-mediated apoptosis. As shown in Fig. 3, internucleosomal cleavage of chromosomal DNA was observed in U937IFNQ cells infected with wildtype bacteria and apoptotic U937IFNQ cells treated with a cytotoxic anti-Fas monoclonal antibody (500 ng ml31 for 5 h) used as a positive control. However, no DNA ladder formation was detected in infected U937UD or U937RA cells. These results indicated that infection with virulent S. £exneri induces apoptosis in U937 cells di¡erentiated with IFNQ but not in undi¡erentiated or RA-treated cells. In apoptotic cells, a series of proteases designated as caspases are activated and cleave various substrate proteins, among which PARP is a target for caspase3, a major apoptosis-executing caspase [10]. Caspase3 cleaves 116 kDa PARP at Asp-214 [19], generating regulatory 30 kDa and catalytic 85 kDa fragments. Such limited proteolysis is seen in many apoptotic cells including U937 cells treated with anti-Fas antibody or tumor necrosis factor K. To determine whether caspase-3 or related caspases were involved in Shigella-induced cell death, we examined the cleavage of PARP during infection by immunoblotting. Whole-cell lysates of infected U937UD, U937IFNQ and U937RA cells were subjected to electrophoresis and immunoblotting analysis using an antibody to the amino-terminal region of the 85 kDa fragment of PARP. Fig. 4 shows the results of immunoblotting analysis of infected cell lysates.
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Fig. 2. Light microscopic analysis of U937 cells infected with S. £exneri strains. Di¡erentiated and undi¡erentiated U937 cells were infected with the wild-type strain YSH6000 (A: U937UD ; B: U937IFNQ; C: U937RA) or avirulent N1411 (E : U937UD ; F: U937IFNQ; G: U937RA) at MOI of 100. Cells were cytospun and stained with Giemsa's solution. D: U937 cells treated with a cytotoxic anti-Fas antibody (100 ng ml31 for 5 h); H: control U937UD cells without infection. Arrowheads represent cells with fragmented nuclei, a typical feature for apoptosis. Magni¢cation, U300.
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Fig. 4. Cleavage of PARP in infected cells. Proteins from U937 cells infected with wild-type Shigella were analyzed by immunoblotting with anti-PARP antibody. Lane 1, U937UD ; lane 2, U937IFNQ; lane 3, U937RA. Lanes 4 and 5 show results of control experiments in which cells were treated with anti-Fas antibody (100 ng ml31 for 5 h) and di¡erentiated with IFNQ (100 units ml31 for 48 h), respectively. Molecular masses of marker proteins are indicated in the left margin.
Fig. 3. DNA fragmentation assay of U937 cells infected with bacteria. U937UD (lane 2), U937IFNQ (lane 3) and U937RA (lane 4) were incubated at 37³C for 5 h after infection with wildtype Shigella at a MOI of 20. Chromosomal DNA was isolated and electrophoresed on 1.5% (w/v) agarose gels. Gels were stained with ethidium bromide, visualized, and photographed under UV illumination. Lane 5 shows DNA extracted from U937IFNQ treated with a cytotoxic anti-Fas antibody (500 ng ml31 for 5 h) used as a positive control for DNA ladder formation. Both lanes 1 and 6 contained 100 base-pair ladder marker (Pharmacia Biotech, Uppsala).
The 85 kDa PARP fragment was seen in infected U937IFNQ and apoptotic cells treated with anti-Fas antibody, but not in infected U937UD or U937RA. No PARP cleavage was seen in lysates from IFNQtreated uninfected U937 cells, indicating that IFNQ itself does not induce the cleavage of PARP. These results suggested that infection-induced cell death is accompanied by the activation of caspase-3 only in U937IFNQ but not in U937UD or U937RA. In fact, a cleavage site sequence-derived tetrapeptidyl inhibitor of caspase-3 and its related enzymes did not protect U937UD or U937RA from infection-induced cytotoxicity (data not shown).
4. Discussion Our report describes for the ¢rst time that human monoblastic U937 cells, potentially di¡erentiated into macrophage-like cells when induced with various reagents, undergo di¡erent types of cell death in response to S. £exneri. IFNQ-treated cells underwent apoptosis upon infection with a virulent strain of S. £exneri as determined both morphologically and biochemically, which is similar to the results reported by Zychlinsky and co-workers using the mouse macrophage cell line J774 [6,14]. They reported that bacterial IpaB protein binds to cellular caspase-1 after infection, and that caspase-1 is a key enzyme for the apoptotic pathway since cells prepared from caspase1-de¢cient mice are insensitive to wild-type S. £exneri [20]. Moreover, procaspase-3 is not activated in such cells [6]. Although the activation of procaspase1 remains to be examined in U937IFNQ, caspase-3, at least in part, catalyzed the cleavage of PARP in our system. Fernandez-Prada et al. reported that infection with virulent S. £exneri induces cell death apparently di¡erent from apoptosis in monocyte-derived macrophages from human peripheral blood [15], which was in contrast to previous reports [6,14]. Our ¢ndings in U937UD and U937RA are
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consistent with their results indicating that Shigellainduced cell death of human macrophages is accompanied by swollen cytoplasm and plasma membrane disruption, but not condensation or fragmentation of the nuclei, or DNA fragmentation as determined by gel electrophoresis. They used macrophage colonystimulating factor as a di¡erentiation inducer, while we employed a leukemic cell line and RA as a di¡erentiation inducer. U937RA may closely resemble normal mature macrophages because RA induces the expression of CD11b on U937 [17]. Biological response modi¢ers such as vitamins and cytokines may regulate death events in cells of the monocyte/ macrophage lineage when infected with virulent bacteria. Macrophages produce IL-1L when infected with S. £exneri [6,15,21]. Other in£ammatory mediators may also be released under such conditions. However, it is unlikely that infection-induced mediators regulate the expression of proteins involved in cell death pathways by the autocrine system, since cytotoxicity was observed as early as 30 min after infection (data not shown). The state of di¡erentiation seems to be important for the response of host cells to bacterial infection. Previously, we reported that di¡erentiated U937 cells show various responses to induction of apoptosis mediated by the death receptors Fas and tumor necrosis factor receptor; IFNQ-treated cells became more sensitive to apoptosis, whereas RA-treated cells acquired resistance to these death factors [17]. As the expression of apoptosis receptors on the cell surface is not changed by di¡erentiation [17], signal transduction downstream of the receptors should be affected in RA-treated cells, the mechanism of which remains to be elucidated. There may be a relationship between death factors and S. £exneri, which failed to induce apoptosis in U937RA, while RA protected the same cells against death factors but caused oncosis-like death triggered by infection. The development of apoptosis-evading machinery during macrophage di¡erentiation should be clari¢ed at the molecular level. It is likely that virulent bacteria are cytotoxic to the host cells, and that the state of di¡erentiation determines the type of cell death, apoptosis or oncosis. Recently, the existence of cell surface receptors for oncosis was proposed, although they have not yet been identi¢ed [22]. Expression of such receptors or of their downstream machinery
may be important events during the di¡erentiation of cells.
Acknowledgments This work was supported in part by grants from the Ministry of Education, Science, Sports and Culture of Japan and from Research Fellowships of the Japan Society for the Promotion of Science for Young Scientists. We thank Shionogi Pharmaceuticals for providing us interferon-Q. We are indebted to Ms. Hitomi Mimuro for the preparation of S. £exneri YSH6000 and N1411.
References [1] Sasakawa, C. (1995) Molecular basis of pathogenicity of Shigella. Rev. Med. Microbiol. 6, 257^266. [2] Baudry, B., Kaczorek, M. and Sansonetti, P.J. (1988) Nucleotide sequence of the invasion plasmid antigen B and C genes (ipaB and ipaC) of Shigella £exneri. Microb. Pathog. 4, 345^ 357. [3] Buysse, J.M., Stover, C.K., Oaks, E.V., Venkatesan, M.M. and Kopecko, D.J. (1987) Molecular cloning of invasion plasmid antigen (ipa) genes from Shigella £exneri: analysis of ipa gene products and genetic mapping. J. Bacteriol. 169, 2561^ 2569. [4] Sasakawa, C., Adler, B., Tobe, T., Okada, N., Nagai, S., Komatsu, K. and Yoshikawa, M. (1989) Functional organization and nucleotide sequence of virulence Region-2 on the large virulence plasmid in Shigella £exneri 2a. Mol. Microbiol. 3, 1191^1201. [5] Venkatesan, M.M., Buysse, J.M. and Kopecko, D.J. (1988) Characterization of invasion plasmid antigen genes (ipaBCD) from Shigella £exneri. Proc. Natl. Acad. Sci. USA 85, 9317^ 9321. [6] Chen, Y., Smith, M.R., Thirumalai, K. and Zychlinsky, A. (1996) A bacterial invasion induces macrophage apoptosis by binding directly to ICE. EMBO J. 15, 3853^3860. [7] Thornberry, N.A., Bull, H.G., Calaycay, J.R., Chapman, K.T., Howard, A.D., Kostura, M.J., Miller, D.K., Molineaux, S.M., Weidner, J.R., Aunins, J., Elliston, K.O., Ayala, J.M., Casano, F.J., Chin, J., Ding, G.J.-F., Egger, L.A., Ga¡ney, E.P., Limjuco, G., Palyha, O.C., Raju, S.M., Rolando, A.M., Salley, J.P., Yamin, T.-T., Lee, T.D., Shively, J.E., MacCross, M., Mumford, R.A., Schmidt, J.A. and Tocci, M.J. (1992) A novel heterodimeric cysteine protease is required for interleukin-1 beta processing in monocytes. Nature 356, 768^774. [8] Alnemri, E.S., Livingston, D.J., Nicholson, D.W., Salvesen, G., Thornberry, N.A., Wong, W.W. and Yuan, J. (1996) Human ICE/CED-3 protease nomenclature. Cell 87, 171. [9] Miura, M., Zhu, H., Rotello, R., Hartwieg, E.A. and Yuan, J.
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[10]
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(1993) Induction of apoptosis in ¢broblasts by IL-1 beta-converting enzyme, a mammalian homolog of the C. elegans cell death gene ced-3. Cell 75, 653^660. Villa, P., Kaufmann, S.H. and Earnshaw, W.C. (1997) Caspases and caspase inhibitors. Trends Biochem. Sci. 22, 388^ 393. Nagata, S. (1997) Apoptosis by death factor. Cell 88, 355^365. Medema, J.P., Sca¤di, C., Kischkel, F.C., Shevchenko, A., Mann, M., Krammer, P.H. and Peter, M.E. (1997) FLICE is activated by association with the CD95 death-inducing signaling complex (DISC). EMBO J. 16, 2794^2804. Enari, M., Sakahira, H., Yokoyama, H., Okawa, K., Iwamatsu, A. and Nagata, S. (1998) A caspase-activated DNase that degrades DNA during apoptosis, and its inhibitor ICAD. Nature 391, 43^50. Zychlinsky, A., Preèvost, M.C. and Sansonetti, P.J. (1992) Shigella £exneri induces apoptosis in infected macrophages. Nature 358, 167^169. Fernandez-Prada, C.M., Hoover, D.L., Tall, B.D. and Venkatesan, M.M. (1997) Human monocyte-derived macrophage infected with virulent Shigella £exneri in vitro undergo a rapid cytolytic event similar to oncosis but not apoptosis. Infect. Immun. 65, 1486^1496. Majno, G. and Joris, I. (1995) Apoptosis, oncosis and necrosis. An overview of cell death. Am. J. Pathol. 146, 3^15.
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[17] Kikuchi, H., Iizuka, R., Sugiyama, S., Gon, G., Mori, H., Arai, M., Mizumoto, K. and Imajoh-Ohmi, S. (1996) Monocytic di¡erentiation modulates apoptotic response to cytotoxic anti-Fas antibody and tumor necrosis factor K in human monoblast U937 cells. J. Leukoc. Biol. 60, 778^783. [18] Kikuchi, H., Sugiyama, S. and Imajoh-Ohmi, S. (1995) A novel anti-apoptosis serum factor that down-regulates Fasmediated apoptosis. J. Biochem. (Tokyo) 117, 936^939. [19] Lazebnik, Y.A., Kaufmann, S.H., Desnoyers, S., Poirier, G.G. and Earnshaw, W.C. (1994) Cleavage of poly(ADP-ribose)polymerase by a proteinase with properties like ICE. Nature 371, 346^347. [20] Hilbi, H., Moss, J.E., Hersh, D., Chen, Y., Arondel, J., Banerjee, S., Flavell, R.A., Yuan, J., Sansonetti, P.J. and Zychlinsky, A. (1998) Shigella-induced apoptosis is dependent on caspase-1 which binds to IpaB. J. Biol. Chem. 273, 32895^ 32900. [21] Hilbi, H., Chen, Y., Thirumalai, K. and Zychlinsky, A. (1997) The interleukin 1L-converting enzyme, Caspase 1, is activated during Shigella £exneri-induced apoptosis in human monocyte-derived macrophages. Infect. Immun. 65, 5165^5170. [22] Zhang, C., Xu, Y., Gu, J. and Schlossman, S.F. (1998) A cell surface receptor de¢ned by a mAb mediates a unique type of cell death similar to oncosis. Proc. Natl. Acad. Sci. USA 95, 6290^6295.
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Shigella £exneri YSH6000 induces two types of cell death, apoptosis and oncosis, in the di¡erentiated human monoblastic cell line U937 Takashi Nonaka
1;a
, Asaomi Kuwae b , Chihiro Sasakawa b , Shinobu Imajoh-Ohmi a *
a
b
Department of Bacterial Infection, The Institute of Medical Science, University of Tokyo, 4-6-1, Shirokanedai, Minato-ku, Tokyo 108-8639, Japan Department of Bacteriology, The Institute of Medical Science, University of Tokyo, 4-6-1, Shirokanedai, Minato-ku, Tokyo 108-8639, Japan Received 21 December 1998 ; received in revised form 22 February 1999; accepted 5 March 1999
Abstract Shigella flexneri, but not a non-invasive mutant derivative, rapidly induced cell death in human monoblastic U937 cells as well as in differentiated cells pretreated with interferon-Q (IFNQ) or retinoic acid (RA). We investigated the morphological and biochemical characteristics of bacterial invasion-induced cell death in these differentiated U937 cells. IFNQ-differentiated cells showed morphological changes typical of apoptosis and their DNA was cleaved giving a ladder-like electrophoretic pattern after infection by Shigellae. In contrast, swelling of the cytoplasm and blebbing of the plasma membrane were observed in RAdifferentiated and undifferentiated cells invaded by the bacteria. No condensation of nuclei was observed in these cells by light microscopy, and no internucleosomal fragmentation of DNA was detected on agarose gels, which resembled the features of oncosis. Furthermore, cleavage of poly(ADP-ribose) polymerase, a substrate for apoptotic caspases, was seen only in IFNQpretreated cells but not in RA-pretreated or undifferentiated cells. These findings suggested that virulent Shigella flexneri induces distinct types of cell death in U937 cells depending on their differentiation state. z 1999 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. Keywords : Apoptosis ; Oncosis ; Shigella £exneri; U937; Di¡erentiation
1. Introduction Shigella £exneri possesses a 230 kb plasmid which encodes virulent proteins [1] including a series of in* Corresponding author. Tel. and Fax: +81 (3) 5449-5311; E-mail: [email protected] 1
Research Fellow of the Japan Society for the Promotion of Science.
vasion plasmid antigens, IpaB, IpaC and IpaD, essential for bacterial invasion into host cells [2^5]. Among these, IpaB is thought to play a key role in induction of apoptosis following infection in the murine macrophage cell line J774 [6]. Microinjection of IpaB induces apoptosis in J774 cells presumably by binding to caspase-1 [7] (ICE; interleukin-1L converting enzyme), a member of the caspase family [8] involved in signal transduction for apoptotic cell
0378-1097 / 99 / $20.00 ß 1999 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. PII: S 0 3 7 8 - 1 0 9 7 ( 9 9 ) 0 0 1 2 5 - 1
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death [9,10]. Sequential activation of procaspases is necessary for signal transduction of apoptosis. For example, Fas, a cell surface death receptor for Fas ligand, interacts with procaspase-8 via an adaptor protein, FADD, when bound with the ligand [11]. The receptor-coupled procaspase-8 is autocatalytically activated, and caspase-8 catalyzes activation of precursors for downstream caspases such as caspase-3 and -7 [12]. Peripheral caspases cleave various proteins: DNase inhibitor inactivated by proteolysis leading to DNA breakdown [13], and cytoskeletal proteins causing morphological changes during apoptosis [10]. In Shigella-infected murine macrophages, Chen et al. proposed that IpaB protein functions as an activator for procaspase-1 and/or an enhancer for the activity of caspase-1 [6]. Caspase-1 should trigger activation of the apoptotic cascade in infected cells. Virulent Shigella-infected and IpaB-injected J774 cells have been reported to show morphological features typical of apoptosis [6,14]. On the other hand, Fernandez-Prada et al. reported that human monocyte-derived macrophages infected with S. £exneri in vitro underwent rapid cytolytic cell death di¡erent from apoptosis [15]. Shigella-infected macrophages show rupture of the plasma membrane, cell swelling, disintegration of ultrastructure, and generalized karyolysis, which are characteristics of oncosis, a recently proposed form of cell death [16]. In this study, we found that wild-type S. £exneri induces two types of cell death, apoptosis and oncosis, in human monoblastic U937 cells, depending on prior di¡erentiation induced by interferon-Q (IFNQ) or retinoic acid (RA). Our ¢ndings may explain the controversial observations from the two previously reported studies [6,15].
diluted 50-fold in BHI broth (Difco, Detroit, MI) and incubated at 37³C for 2 h until they reached the mid-log phase of growth. Human monoblastic U937 cells, provided by Japanese Cancer Research Resources Bank (JCRB9021), were cultured and treated with di¡erentiation inducers (100 units ml31 IFNQ or 3 WM RA) for 48 h as described previously [17]. 2.2. Infection procedure and cytotoxicity assay Undi¡erentiated, IFNQ-treated, or RA-treated U937 cells (U937UD, U937IFNQ, or U937RA, respectively) were resuspended in fresh medium without antibiotics at a concentration of 106 cells ml31 , 2 h prior to infection. Cells were mixed with bacteria at a multiplicity of infection (MOI) of 100 bacteria per cell, and immediately centrifuged at 700Ug for 10 min. After incubation for 1 h with bacteria at 37³C, cells were washed with medium supplemented with 100 Wg ml31 gentamycin (Gm) and further incubated for 2 h with this antibiotic. Infected cells were assessed by light microscopy and their viabilities were examined by the trypan blue dye exclusion method. 2.3. Detection of apoptosis Cells (3U105 cells) after infection with bacteria (MOI = 100) were cytospun, ¢xed with ethanol and stained with Giemsa's solution (E. Merck, Darmstadt) for morphological analysis. Infected cells (107 cells) at a MOI of 20 after incubation with Gm for 5 h were used for DNA fragmentation assay as described previously [18]. 2.4. Immunoblotting analysis
2. Materials and methods 2.1. Bacterial strains and cell culture S. £exneri YSH6000 (wild-type) and N1411 (a non-virulent mutant lacking ipaBCD) [4] were routinely grown at 30³C overnight in L broth (Difco, Detroit, MI). For infection, overnight cultures were
Infected cells were lysed and treated with 10% (v/v) trichloroacetic acid for 20 min on ice. The precipitated proteins were subjected to 7.5% SDS-polyacrylamide gel electrophoresis, electroblotted, and stained with a polyclonal antibody to the aminoterminal region of the 85 kDa fragment of human poly(ADP-ribose) polymerase (PARP).
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3. Results 3.1. Death of U937 cells infected with virulent S. £exneri Fig. 1 shows the cytotoxicity of wild-type S. £exneri YSH6000 and N1411, a non-virulent mutant lacking ipaBCD, against U937 cells in various di¡erentiation states. The wild-type strain killed U937 cells regardless of di¡erentiation. The highest level of cytotoxicity was seen toward U937RA; nearly twice as many cells were stained by trypan blue in U937RA 2 h after infection as compared with U937UD or U937IFNQ. The cytotoxicity of the wild-type strain to U937 cells was dependent on MOI (data not shown). In contrast, the non-virulent strain N1411 was not cytotoxic to any of these cells as determined by the dye exclusion test. 3.2. Demonstration of apoptosis in infected U937 cells The trypan blue dye exclusion method cannot discriminate between di¡erent types of cell death. Thus, we examined the morphological and biochemical features of Shigella-treated cells. Fig. 2 shows the morphology of U937 cells 2 h after infection with either the wild-type or avirulent mutant strain. Similar de-
Fig. 1. Mortality of U937 cells following infection by S. £exneri. U937UD, U937IFNQ and U937RA cells were infected with wildtype (WT: YSH6000, shaded columns) or N1411 (ipaBCD3 , hatched columns) at MOI of 100. Cytotoxicity was assayed by the trypan blue dye exclusion method. Viable cells were counted after a 2 h incubation in the presence of Gm. The white column indicates dye-stained uninfected U937 cells.
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grees of invasion were observed in U937UD, U937IFNQ and U937RA cells (A^C), but no bacteria were seen in N1411-treated cells (E^G). In Shigellainfected U937IFNQ (arrowheads in panel B), cytoplasmic shrinkage and nuclear fragmentation were observed similarly to the results shown in Fig. 2D, indicating typical apoptotic cells treated with a cytotoxic anti-Fas antibody. However, no changes occurred in the nuclei of U937UD or U937RA cells even when they were invaded by the bacteria (A and C). These infected cells were larger than U937IFNQ cells infected with Shigella, and their plasma membrane seemed to be damaged. On the other hand, no morphological di¡erences were observed in N1411-treated U937 cells (E^G) as compared with control U937UD cells (H). Next, we analyzed DNA fragmentation, a hallmark of apoptosis, in Shigella-infected cells, and compared the DNA electrophoretic patterns with those in Fas-mediated apoptosis. As shown in Fig. 3, internucleosomal cleavage of chromosomal DNA was observed in U937IFNQ cells infected with wildtype bacteria and apoptotic U937IFNQ cells treated with a cytotoxic anti-Fas monoclonal antibody (500 ng ml31 for 5 h) used as a positive control. However, no DNA ladder formation was detected in infected U937UD or U937RA cells. These results indicated that infection with virulent S. £exneri induces apoptosis in U937 cells di¡erentiated with IFNQ but not in undi¡erentiated or RA-treated cells. In apoptotic cells, a series of proteases designated as caspases are activated and cleave various substrate proteins, among which PARP is a target for caspase3, a major apoptosis-executing caspase [10]. Caspase3 cleaves 116 kDa PARP at Asp-214 [19], generating regulatory 30 kDa and catalytic 85 kDa fragments. Such limited proteolysis is seen in many apoptotic cells including U937 cells treated with anti-Fas antibody or tumor necrosis factor K. To determine whether caspase-3 or related caspases were involved in Shigella-induced cell death, we examined the cleavage of PARP during infection by immunoblotting. Whole-cell lysates of infected U937UD, U937IFNQ and U937RA cells were subjected to electrophoresis and immunoblotting analysis using an antibody to the amino-terminal region of the 85 kDa fragment of PARP. Fig. 4 shows the results of immunoblotting analysis of infected cell lysates.
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Fig. 2. Light microscopic analysis of U937 cells infected with S. £exneri strains. Di¡erentiated and undi¡erentiated U937 cells were infected with the wild-type strain YSH6000 (A: U937UD ; B: U937IFNQ; C: U937RA) or avirulent N1411 (E : U937UD ; F: U937IFNQ; G: U937RA) at MOI of 100. Cells were cytospun and stained with Giemsa's solution. D: U937 cells treated with a cytotoxic anti-Fas antibody (100 ng ml31 for 5 h); H: control U937UD cells without infection. Arrowheads represent cells with fragmented nuclei, a typical feature for apoptosis. Magni¢cation, U300.
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Fig. 4. Cleavage of PARP in infected cells. Proteins from U937 cells infected with wild-type Shigella were analyzed by immunoblotting with anti-PARP antibody. Lane 1, U937UD ; lane 2, U937IFNQ; lane 3, U937RA. Lanes 4 and 5 show results of control experiments in which cells were treated with anti-Fas antibody (100 ng ml31 for 5 h) and di¡erentiated with IFNQ (100 units ml31 for 48 h), respectively. Molecular masses of marker proteins are indicated in the left margin.
Fig. 3. DNA fragmentation assay of U937 cells infected with bacteria. U937UD (lane 2), U937IFNQ (lane 3) and U937RA (lane 4) were incubated at 37³C for 5 h after infection with wildtype Shigella at a MOI of 20. Chromosomal DNA was isolated and electrophoresed on 1.5% (w/v) agarose gels. Gels were stained with ethidium bromide, visualized, and photographed under UV illumination. Lane 5 shows DNA extracted from U937IFNQ treated with a cytotoxic anti-Fas antibody (500 ng ml31 for 5 h) used as a positive control for DNA ladder formation. Both lanes 1 and 6 contained 100 base-pair ladder marker (Pharmacia Biotech, Uppsala).
The 85 kDa PARP fragment was seen in infected U937IFNQ and apoptotic cells treated with anti-Fas antibody, but not in infected U937UD or U937RA. No PARP cleavage was seen in lysates from IFNQtreated uninfected U937 cells, indicating that IFNQ itself does not induce the cleavage of PARP. These results suggested that infection-induced cell death is accompanied by the activation of caspase-3 only in U937IFNQ but not in U937UD or U937RA. In fact, a cleavage site sequence-derived tetrapeptidyl inhibitor of caspase-3 and its related enzymes did not protect U937UD or U937RA from infection-induced cytotoxicity (data not shown).
4. Discussion Our report describes for the ¢rst time that human monoblastic U937 cells, potentially di¡erentiated into macrophage-like cells when induced with various reagents, undergo di¡erent types of cell death in response to S. £exneri. IFNQ-treated cells underwent apoptosis upon infection with a virulent strain of S. £exneri as determined both morphologically and biochemically, which is similar to the results reported by Zychlinsky and co-workers using the mouse macrophage cell line J774 [6,14]. They reported that bacterial IpaB protein binds to cellular caspase-1 after infection, and that caspase-1 is a key enzyme for the apoptotic pathway since cells prepared from caspase1-de¢cient mice are insensitive to wild-type S. £exneri [20]. Moreover, procaspase-3 is not activated in such cells [6]. Although the activation of procaspase1 remains to be examined in U937IFNQ, caspase-3, at least in part, catalyzed the cleavage of PARP in our system. Fernandez-Prada et al. reported that infection with virulent S. £exneri induces cell death apparently di¡erent from apoptosis in monocyte-derived macrophages from human peripheral blood [15], which was in contrast to previous reports [6,14]. Our ¢ndings in U937UD and U937RA are
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consistent with their results indicating that Shigellainduced cell death of human macrophages is accompanied by swollen cytoplasm and plasma membrane disruption, but not condensation or fragmentation of the nuclei, or DNA fragmentation as determined by gel electrophoresis. They used macrophage colonystimulating factor as a di¡erentiation inducer, while we employed a leukemic cell line and RA as a di¡erentiation inducer. U937RA may closely resemble normal mature macrophages because RA induces the expression of CD11b on U937 [17]. Biological response modi¢ers such as vitamins and cytokines may regulate death events in cells of the monocyte/ macrophage lineage when infected with virulent bacteria. Macrophages produce IL-1L when infected with S. £exneri [6,15,21]. Other in£ammatory mediators may also be released under such conditions. However, it is unlikely that infection-induced mediators regulate the expression of proteins involved in cell death pathways by the autocrine system, since cytotoxicity was observed as early as 30 min after infection (data not shown). The state of di¡erentiation seems to be important for the response of host cells to bacterial infection. Previously, we reported that di¡erentiated U937 cells show various responses to induction of apoptosis mediated by the death receptors Fas and tumor necrosis factor receptor; IFNQ-treated cells became more sensitive to apoptosis, whereas RA-treated cells acquired resistance to these death factors [17]. As the expression of apoptosis receptors on the cell surface is not changed by di¡erentiation [17], signal transduction downstream of the receptors should be affected in RA-treated cells, the mechanism of which remains to be elucidated. There may be a relationship between death factors and S. £exneri, which failed to induce apoptosis in U937RA, while RA protected the same cells against death factors but caused oncosis-like death triggered by infection. The development of apoptosis-evading machinery during macrophage di¡erentiation should be clari¢ed at the molecular level. It is likely that virulent bacteria are cytotoxic to the host cells, and that the state of di¡erentiation determines the type of cell death, apoptosis or oncosis. Recently, the existence of cell surface receptors for oncosis was proposed, although they have not yet been identi¢ed [22]. Expression of such receptors or of their downstream machinery
may be important events during the di¡erentiation of cells.
Acknowledgments This work was supported in part by grants from the Ministry of Education, Science, Sports and Culture of Japan and from Research Fellowships of the Japan Society for the Promotion of Science for Young Scientists. We thank Shionogi Pharmaceuticals for providing us interferon-Q. We are indebted to Ms. Hitomi Mimuro for the preparation of S. £exneri YSH6000 and N1411.
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