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Burkholderia pseudomallei-induced cell fusion in U937 macrophages can be inhibited by monoclonal antibodies against host cell surface molecules
Microbes and Infection 13 (2011) 1006e1011 www.elsevier.com/locate/micinf
Short communication
Burkholderia pseudomallei-induced cell fusion in U937 macrophages can be inhibited by monoclonal antibodies against host cell surface molecules Supaporn Suparak a, Veerachat Muangsombut a, Donporn Riyapa b, Joanne M. Stevens c, Mark P. Stevens c, Ganjana Lertmemongkolchai b, Sunee Korbsrisate a,* a Department of Immunology, Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok 10700, Thailand Centre for Research and Development of Medical Diagnostic Laboratories, Faculty of Associated Medical Sciences, Khon Kaen University, Khon Kaen 40002, Thailand c Roslin Institute and Royal (Dick) School of Veterinary Studies, University of Edinburgh, Bush Farm Road, Roslin, Midlothian, EH25 9RG, United Kingdom b
Received 19 January 2011; accepted 10 June 2011 Available online 6 July 2011
Abstract Burkholderia pseudomallei induces the formation of multinucleated giant cells in cell monolayers. After infection of human macrophage-like U937 cells with B. pseudomallei, addition of monoclonal antibodies against integrin-associated protein (CD47), E-selectin (CD62E), a fusion regulatory protein (CD98), and E-cadherin (CD324) suppressed multinucleated giant cells in a concentration-dependent manner while monoclonal antibodies against other surface molecules did not inhibit fusion despite binding to the cell surface. Flow cytometric analysis showed increased expression of CD47 and CD98, but not CD62E and CD324, upon B. pseudomallei infection. Our data suggest the involvement of specific cellular factors in the process of B. pseudomallei-induced fusion. Ó 2011 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved. Keywords: Burkholderia pseudomallei; U937 cell; Multinucleated giant cell; CD47; CD98
1. Introduction Burkholderia pseudomallei is the causative agent of melioidosis, a severe invasive disease of humans endemic in Southeast Asia, particularly Thailand and Northern Australia [1]. The organism resists phagocytosis and killing by polymorphonuclear cells [2] and internalized bacteria are able to induce actin polymerization at one pole and form membrane protrusions [3]. It has been postulated that such actin-rich membrane protrusions contribute to cell-to-cell spread and multinucleated giant cell (MNGC) formation [3], however other intracellular pathogens capable of actin-based motility do not induce fusion and the mechanism of MNGC formation is unknown. The formation of MNGC has been observed in
* Corresponding author. Tel.: þ66 2 418 0569; fax: þ66 2 418 1636. E-mail address: [email protected] (S. Korbsrisate).
tissue samples from human melioidosis patients [4]. B. pseudomallei has been reported to modify infected macrophagelike cells in a manner analogous to MNGC formation in bone, a process known as osteoclastogenesis [5]. Previously, B. pseudomallei mutants deficient in a structural component of the bsa-encoded a type III secretion system [6] or the RpoS sigma factor [7] were reported to exhibit reduced MNGC formation in macrophage-like cell lines. However, these phenotypes likely result from impairment of earlier stages of intracellular life and the contribution of bacterial and cellular factors to the fusion process remains ill-defined. Several cell surface adhesion molecules have been implicated in macrophage fusion. Members of the immunoglobulin superfamily, the macrophage fusion receptor (also known as signal-regulatory protein a; SIRPa) and its ligand CD47 (integrin-associated protein), participate in fusion of rat alveolar macrophage [8]. The b1 (CD29) and b2 (CD18) integrins influence cell adhesion and multinucleated foreign body giant
1286-4579/$ - see front matter Ó 2011 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved. doi:10.1016/j.micinf.2011.06.007
S. Suparak et al. / Microbes and Infection 13 (2011) 1006e1011
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cell formation by interleukin (IL)-4-induced human monocytederived macrophages [9]. Furthermore, antibodies against LFA-1 and ICAM-1 (CD54) inhibit macrophage fusion [10], as do antibodies against the transmembrane adhesion molecule CD44 [11], E-cadherin [12], and an LPS receptor (CD14) [13]. Potential fusion mediators such as a fusion regulatory protein (FRP-1, CD98) have also been reported to enhance virusinduced cell fusion [14]. Here, we investigated the role of candidate cell surface molecules of human macrophage U937 cells in B. pseudomallei-induced MNGC formation.
the infected cells were fixed and MNGC formation was evaluated as described above. Control experiments were performed with MAbs of the same isotype; Mouse IgG1 for antiCD172a, anti-CD62E, anti-CD47, anti-CD98, anti-CD324, anti-CD11a, anti-CD54, anti-CD18; Mouse IgG2a for antiCD14 and Rat IgG2b for anti-CD44. All antibodies were supplied by Biolegend Inc., San Diego, CA.
2. Materials and methods
Expression of cell surface molecules was assessed by flow cytometry as previously described [2]. Differentiated U937 cells infected with B. pseudomallei K96243 were dislodged and incubated with rat anti-mouse CD16/CD32 (Becton Dickinson, San Diego, CA) for 30 min at 4 C to block the Fc receptor and thus prevent non-specific antibody binding. Individual cell samples were incubated for 30 min at 4 C with each MAb, or isotype controls. The cells were washed and further incubated with fluorescein-isothiocyanate (FITC)labeled anti-mouse IgG antibody (Dako Japan, Kyoto, Japan) for 30 min at 4 C. They were then washed and analyzed by flow cytometry. The percentage of cells that expressed cell surface molecules was determined by comparison with the isotype matched control. To determine the alteration of cell surface molecule expression after infection, the ratio of mean fluorescence intensity (MFI) between infected and uninfected cells at the same time point was calculated. Ratio values greater than 1 indicate an increase in surface molecule expression after infection. Data were analyzed using FACScalibur with CELLQuest software (BD Biosciences, San Jose, CA).
2.1. Bacterial strain, cell line and growth conditions B. pseudomallei K96243 was cultured using LuriaeBertani medium at 37 C. The human leukemic monocyte lymphoma cell line (U937, ATCC CRL-1593.2) was maintained and incubated in RPMI 1640 (Gibco-BRL, Grand Island, NY) at 37 C in a humidified 5% CO2 atmosphere. U937 cells were activated with 20 ng/ml of phorbol 12-myristate 13-acetate (Sigma Chemical Co., St Louis, MO) for 48 h to stimulate differentiation to macrophage-like cells. 2.2. Detection of B. pseudomallei-induced MNGC Stationary phase LB-grown B. pseudomallei K96243 were washed and added to monolayers at a multiplicity of infection (MOI) of one bacterium per two cells and incubated at 37 C with 5% CO2 for 2 h. Thereafter, the extracellular bacteria were killed by the addition of 250 mg/ml kanamycin to the overlay. At various time intervals after infection, cells were stained with Giemsa and MNGC formation assessed by counting the number of nuclei within MNGC (>2 nuclei/cell) from at least 500 nuclei examined, as previously described [6]. The percentage of MNGC reflects the number of nuclei within multinucleated cells/total number of nuclei counted 100. 2.3. Impact of monoclonal antibodies specific to cell surface molecules on B. pseudomallei-induced MNGC formation Differentiated U937 cells were infected with B. pseudomallei K96243 for 2 h as described above. Thereafter, monoclonal antibodies (MAbs) against the following cell surface molecules were separately added to the culture medium at the concentrations specified in Results; LPS receptor (CD14) (clone M5E2), transmembrane adhesion molecule (CD44) (clone IM7), integrin-associated protein (CD47) (clone HCD47), E-selectin (CD62E) (clone HEA-1f), fusion regulatory protein, FRP-1 (CD98) (clone MEM-108), signal-regulatory protein a (CD172a) (clone SE5A5), E-cadherin (CD324) (clone 674A), leukocyte function-associated antigen-1 (LFA-1) composed of CD11a (clone HI111) and CD18 (cloneTS1/18) integrin chain (LFA-1) (clone HI111) and LFA-1 counter-receptor ICAM-1 (intercellular adhesion molecule-1, CD54) (clone HCD54). At various time intervals,
2.4. Flow cytometric determination of cell surface molecule expression
2.5. Statistical analysis All tests for significance were performed using the Student’s t-test. Data were considered significant at a P value of 0.05. 3. Results 3.1. Induction of MNGC formation in B. pseudomalleiinfected human macrophage-like U937 cells The kinetics of B. pseudomallei-induced MNGC formation in human macrophage-like U937 cells were determined following inoculation at a low multiplicity of infection (1 CFU/2 cells). As shown in Fig. 1, MNGC formation could be detected from 15 h post-infection (h.p.i.) with the number of nuclei incorporated into an MNGC gradually increasing over time. Having established a reliable protocol for induction of fusion, we investigated whether culture supernatant collected from differentiated U937 cells infected with B. pseudomallei may induce or enhance cell fusion. Neither the supernatant from uninfected U937 or B. pseudomalleiinfected U937 cells could induce the formation of MNGC on uninfected cells or increase MNGC formation in infected
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S. Suparak et al. / Microbes and Infection 13 (2011) 1006e1011
Fig. 1. Typical Giemsa-stained MNGC formed following infection of differentiated U937 macrophage-like cells by B. pseudomallei K96243. Cells were fixed and observed the development of MNGC at the times indicated. Arrow, MNGC.
monolayers (data not shown). Additionally, no MNGC formation was detected in U937 cell monolayers treated with heat-killed B. pseudomallei (data not shown), indicating that the fusion process requires viable bacteria. Induction of MNGC has been reported in mouse macrophage cell lines (RAW264.7 and J774A.1) [6,7] and human epithelial lines (HeLa and A549 cells) [3]. In all these reports, infected cells were attached to the surface of the culture vessel. We infected a suspension of U937 cells at the monocytic stage with B. pseudomallei at a comparable MOI to the studies using attached cells [3] but no evidence of MNGC formation was
observed (data not shown), supporting earlier data in a macrophage fusion model that cell attachment may be required to reliably detect this phenotype [15]. 3.2. Antibodies specific to selected cell surface molecules can suppress MNGC formation To elucidate the role of host cell surface molecules during B. pseudomallei-induced MNGC formation, MAbs against specific candidates were added to fusion assays at various concentrations. As depicted in Fig. 2A, MAbs against integrin-
S. Suparak et al. / Microbes and Infection 13 (2011) 1006e1011
associated protein (anti-CD47), E-selectin (anti-CD62E), fusion regulatory protein (anti-CD98), and E-cadherin (antiCD324) could each suppress B. pseudomallei-induced MNGC formation at a concentration of 10 mg/ml to an undetectable level whereas 34.7 2.52% of nuclei were in MNGC in nontreated monolayers (P < 0.01, t-test). Under these conditions, the viability of the cells was more than 95% when examined by trypan blue dye and no significant detachment of cells was observed suggesting that the reduced fusion was not due to loss of cells from the monolayer upon addition of the MAbs. No differences in the number of intracellular B. pseudomallei were noted 3, 6, 9, 12 and 24 h.p.i. of U937 cells at an MOI of 0.5 (1 CFU/2 cells) in the presence of 10 mg/ml anti-CD98 (data not shown). This indicates that inhibition of MNGC formation by CD98-specific antibody is unlikely to be a consequence of interference in intracellular replication or survival of B. pseudomallei. Addition of MAbs specific to the signal-regulatory protein a (SIRPa, anti-CD172a; 5.0 2.0%), integrin LFA-1 complex (anti-CD11a and anti-CD18; 16.3 3.75% and 12.5 2.50%, respectively), and LFA-1 counter-receptor ICAM-1 (anti-CD54; 16.9 1.90%) significantly reduced but did not abolish MNGC formation compared to controls (P < 0.05, t-test) (Fig. 2A). Neither the MAbs against LPS receptor (anti-CD14) nor
Fig. 2. Effect of monoclonal antibodies (MAbs) specific to selected cell surface molecules on B. pseudomallei-induced MNGC formation. U937 cells were infected with B. pseudomallei K96243. MAbs were added 2 h.p.i. at the concentrations indicated. MNGC formation was assessed at 18 h.p.i. and is presented as the percentage of nuclei in MNGC relative to the total number of nuclei counted (A) Influence of MAbs against cell surface molecules on MNGC formation at 10 mg/ml. (B) Concentration-dependent inhibition of MNGC formation by selected monoclonal antibodies. Error bars represent standard errors of the means for experiments performed in triplicate. The asterisks indicate statistically significant differences between cultures with and without MAbs (P < 0.05, t-test).
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a transmembrane adhesion molecule CD44 (anti-CD44) could inhibit fusion under the assay conditions (35.7 4.04% and 34.7 3.2%, respectively) (Fig. 2A). Isotype matched control IgGs of the MAbs had no inhibitory effect (Mouse IgG1 ¼ 35.0 3.0%, Mouse IgG2a ¼ 35.3 1.52%, Rat IgG2b ¼ 36 4.0%; P > 0.05, t-test). The suppression of MNGC formation in the presence of 10 mg/ml of either anti-CD47, anti-CD62E, anti-CD98, antiCD172a or anti-CD324 prompted us to investigate the impact of lower antibody concentrations ranging from 2.5 to 10.0 mg/ml. Significant dose-dependent inhibitory effects could be observed from concentration of 4 mg/ml increasing to near-complete inhibition at a concentration 10 mg/ml of each antibody (Fig. 2B). 3.3. Expression of cell surface molecules during MNGC formation To investigate the level of cell surface expression of the molecules under study during B. pseudomallei-induced MNGC formation, flow cytometric analysis was performed. When compared with isotype matched controls, more than 90% of the population of both uninfected and infected cells expressed CD98, CD62E, CD324, and CD172a (Fig. 3A). However, only 66% of uninfected U937 cells expressed CD47 but this percentage increased to more than 90% after interaction with B. pseudomallei. These results indicated that, with the exception of antibody directed against CD44, the antibodies used in this study bound to a comparable proportion of infected cells, even though they differ in their ability to inhibit MNGC formation. When compared with uninfected cells, expression of the cell surface molecules CD47 and CD98 (which were implicated in MNGC formation using specific MAbs) was significantly increased at 18 h.p.i. At this time point, the mean fluorescence intensity (MFI) ratio of CD47 and CD98 was 22.6 and 11.1, respectively (Fig. 3B) and was maintained at this level at 24 h.p.i. When the efficiency of MNGC formation was evaluated over time in the same cultures it was 32.66 6.80% at 18 h.p.i., increasing to 70.33 18.45% at 24 h.p.i. (data not shown). However, the levels of CD62E and CD324 (implicated in MNGC formation), CD172a (partial role) and CD44 (no effect) were not significantly different from the uninfected cells at 18 h.p.i. of U937 cells with B. pseudomallei (average MFI ratio of approximately 1; Fig. 3B). A very low frequency of CD44expressing cells was observed with both infected and uninfected cells (Fig. 3A), and this may partially explain the inability of this particular antibody to interfere with B. pseudomalleiinduced cell fusion. Taken together, our data suggest that fusion is not associated with up-regulation of the expression of CD62E, CD324 or CD172a during B. pseudomallei infection, though levels of other molecules implicated in fusion (CD47 and CD98) are elevated at times when fusion occurs. 4. Discussion The present study shows that binding of antibodies specific to the cell surface molecules integrin-associated protein
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Fig. 3. Analysis of the expression of selected cell surface molecules on B. pseudomallei-infected U937 cells. (A) Histogram at 18 h.p.i. of the expression of cell surface molecules on U937 cells infected with B. pseudomallei compared with the uninfected and isotype controls analyzed by flow cytometry. The percentage of cells that expressed cell surface molecules was determined by comparison with the isotype matched control. (B) Ratio of mean fluorescence intensity (MFI) was calculated from the MFI of infected cells/uninfected cells at the same time point. Ratio values greater than 1 indicate an increase in surface molecule expression after infection.
(CD47), E-selectin (CD62E), a fusion regulatory protein (CD98), and E-cadherin (CD324) could almost completely inhibit MNGC formation whereas MAbs against LFA-1 (composed of CD11a, CD18), ICAM-1 (CD54) and CD172a only partially blocked MNGC formation. It can be hypothesized that MNGC formation may be facilitated by cell surface molecules that may interact with their ligands on adjacent cells. Among 4 different cell surface molecules implicated in MNGC formation, 3 of them (i.e. CD47, CD62E and CD324) are classified as adhesion molecules while CD98 is a fusion mediator [14]. Increased levels of adhesion molecules CD47 and CD98 were detected following B. pseudomallei infection of U937 cells by flow cytometry. Peak levels of CD47 and CD98 were reached at 18 h.p.i and were sustained thereafter. Prolonged high level expression of such molecules may contribute to the high frequency of cell fusion at the later time interval sampled. No significant increases in surface
expression of CD62E or CD324 were detected at 18 h.p.i. relative to uninfected cells, even though MAbs against these surface molecules were shown to inhibit MNGC formation, indicating that the progression of MNGC formation is not dependent on up-regulation of these surface molecules. The same concentration of antibodies that completely inhibited MNGC formation (i.e. anti-CD62E) or partially (i.e. anti-CD172a) were observed to stain the surface of infected U937 cells at comparable levels by flow cytometry, suggesting that antibody-mediated inhibition of fusion is specific to the target protein and the partial inhibition may not be due to simple steric interference. In addition, inhibition of MNGC formation appears not to be due to the proportion of cells expressing these cell surface molecules since CD172a was expressed on a similar proportion of cells, compared to CD47, CD98, CD62E and CD324, whereas antibodies to the latter four molecules almost completely inhibited fusion.
S. Suparak et al. / Microbes and Infection 13 (2011) 1006e1011
Our finding that CD47, CD62E, CD98 and CD324 influence the process of B. pseudomallei-induced MNGC formation has not been reported for other bacteria. The up-regulation of CD47 and CD98 molecules may facilitate the onset of the fusion process. There are several studies reporting the importance of these surface molecules, for example CD47 was found to be involved in macrophage multinucleation by interacting with MFR (also called SIRPa or CD172a) during the macrophage adhesion/fusion process [8]. Human immunodeficiency virus (HIV) infection induces CD98 expression on CD4þ T lymphocyte, which is associated with syncytium formation [16]. Furthermore, the involvement of LFA-1 (composed of CD11a, CD18) and ICAM-1 (CD54) in the B. pseudomallei-induced U937 cell fusion model reported in this study is supported by previous observations in cellecell fusion models and HIV-induced syncytium formation [10,17]. Blocking of ICAM-1 expression could also inhibit concanavalin A-stimulated MNGC formation of mononuclear cells [18]. Furthermore, the expression of ICAM-1 on monocytic cells induced by Mycobacterium tuberculosis is involved in the host response and subsequent formation of granulomas [19]. In the osteoclast model it was suggested that ICAM-1 and E-cadherin (CD324) are involved at an early cell fusion stage such as cell-substrate adhesion whilst SIRPa and FRP-1 may regulate rather than mediate cellecell fusion [20]. To the best of our knowledge, this is the first study to demonstrate that B. pseudomallei-induced fusion of human macrophages in vitro requires cell surface molecules. Contact between cells was reported to be a prerequisite for fusion [15]. We propose that selected surface adhesion molecules of cells undergoing B. pseudomallei-induced fusion may facilitate attachment of cells to each other and bring their membranes into close contact. It is possible that B. pseudomallei may favor this process by modulating the surface expression of such molecules. Acknowledgments This work was supported by the National Science and Technology Development Agency grant BT-B-01-MG-145123. S. Suparak and D. Riyapa were supported by the Office of the Higher Education Commission for postdoctoral and PhD scholarships, respectively. S. Korbsrisate was supported by the Chalermphrakiat Grant, Faculty of Medicine Siriraj Hospital, Mahidol University. We are grateful to G.J. Bancroft, M. Ato and N. Onlamoon for their kind comments, and E. Noulsri and P. Tippayawat for their advice on flow cytometric analysis. Many thanks to V. Sookpatdhee for taking photogragh. References [1] D.A. Dance, Melioidosis as an emerging global problem, Acta Trop. 74 (2000) 115e119. [2] S. Chanchamroen, C. Kewcharoenwong, W. Susaengrat, M. Ato, G. Lertmemongkolchai, Human polymorphonuclear neutrophil responses to Burkholderia pseudomallei in healthy and diabetic subjects, Infect. Immun. 77 (2009) 456e463.
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[3] W. Kespichayawattana, S. Rattanachetkul, T. Wanun, P. Utaisincharoen, S. Sirisinha, Burkholderia pseudomallei induces cell fusion and actinassociated membrane protrusion: a possible mechanism for cell-to-cell spreading, Infect. Immun. 68 (2000) 5377e5384. [4] K.T. Wong, S.D. Puthucheary, J. Vadivelu, The histopathology of human melioidosis, Histopathology 26 (1995) 51e55. [5] J.A. Boddey, C.J. Day, C.P. Flegg, R.L. Ulrich, S.R. Stephens, I.R. Beacham, N.A. Morrison, I.R. Peak, The bacterial gene lfpA influences the potent induction of calcitonin receptor and osteoclast-related genes in Burkholderia pseudomallei-induced TRAP-positive multinucleated giant cells, Cell Microbiol. 9 (2007) 514e531. [6] S. Suparak, W. Kespichayawattana, A. Haque, A. Easton, S. Damnin, G. Lertmemongkolchai, G.J. Bancroft, S. Korbsrisate, Multinucleated giant cell formation and apoptosis in infected host cells is mediated by Burkholderia pseudomallei type III secretion protein BipB, J. Bacteriol. 187 (2005) 6556e6560. [7] P. Utaisincharoen, S. Arjcharoen, K. Limposuwan, S. Tungpradabkul, S. Sirisinha, Burkholderia pseudomallei RpoS regulates multinucleated giant cell formation and inducible nitric oxide synthase expression in mouse macrophage cell line (RAW 264.7), Microb. Pathog. 40 (2006) 184e189. [8] X. Han, H. Sterling, Y. Chen, C. Saginario, E.J. Brown, W.A. Frazier, F. P. Lindberg, A. Vignery, CD47, a ligand for the macrophage fusion receptor, participates in macrophage multinucleation, J. Biol. Chem. 275 (2000) 37984e37992. [9] A.K. McNally, J.M. Anderson, Beta1 and beta2 integrins mediate adhesion during macrophage fusion and multinucleated foreign body giant cell formation, Am. J. Pathol. 160 (2002) 621e630. [10] F. Kazazi, J. Chang, A. Lopez, M. Vadas, A.L. Cunningham, Interleukin 4 and human immunodeficiency virus stimulate LFA-1-ICAM-1mediated aggregation of monocytes and subsequent giant cell formation, J. Gen. Virol. 75 (1994) 2795e2802. [11] H. Sterling, C. Saginario, A. Vignery, CD44 occupancy prevents macrophage multinucleation, J. Cell Biol. 143 (1998) 837e847. [12] G. Mbalaviele, H. Chen, B.F. Boyce, G.R. Mundy, T. Yoneda, The role of cadherin in the generation of multinucleated osteoclasts from mononuclear precursors in murine marrow, J. Clin. Invest. 95 (1995) 2757e2765. [13] P.K. Peterson, G. Gekker, S. Hu, W.B. Anderson, M. Teichert, C.C. Chao, T.W. Molitor, Multinucleated giant cell formation of swine microglia induced by Mycobacterium bovis, J. Infect. Dis. 173 (1996) 1194e1201. [14] N. Tabata, M. Ito, K. Shimokata, S. Suga, S. Ohgimoto, M. Tsurudome, M. Kawano, H. Matsumura, H. Komada, M. Nishio, Y. Ito, Expression of fusion regulatory proteins (FRPs) on human peripheral blood monocytes. Induction of homotypic cell aggregation and formation of multinucleated giant cells by anti-FRP-1 monoclonal antibodies, J. Immunol. 153 (1994) 3256e3266. [15] L. Helming, S. Gordon, The molecular basis of macrophage fusion, Immunobiology 212 (2007) 785e793. [16] S. Suga, M. Tsurudome, M. Ito, S. Ohgimoto, N. Tabata, M. Nishio, M. Kawano, H. Komada, M. Sakurai, Y. Ito, Human immunodeficiency virus type-1 envelope glycoprotein gp120 induces expression of fusion regulatory protein (FRP)-1/CD98 on CD4þ T cells: a possible regulatory mechanism of HIV-induced syncytium formation, Med. Microbiol. Immunol. 185 (1997) 237e243. [17] J.E. Hildreth, R.J. Orentas, Involvement of a leukocyte adhesion receptor (LFA-1) in HIV-induced syncytium formation, Science 244 (1989) 1075e1078. [18] K. Mizuno, H. Okamoto, T. Horio, Inhibitory influences of xanthine oxidase inhibitor and angiotensin I-converting enzyme inhibitor on multinucleated giant cell formation from monocytes by downregulation of adhesion molecules and purinergic receptors, Br. J. Dermatol. 150 (2004) 205e210. [19] G.M. Lopez Ramirez, W.N. Rom, C. Ciotoli, A. Talbot, F. Martiniuk, B. Cronstein, J. Reibman, Mycobacterium tuberculosis alters expression of adhesion molecules on monocytic cells, Infect. Immun. 62 (1994) 2515e2520. [20] A. Vignery, Osteoclasts and giant cells: macrophage-macrophage fusion mechanism, Int. J. Exp. Pathol. 81 (2000) 291e304.
Short communication
Burkholderia pseudomallei-induced cell fusion in U937 macrophages can be inhibited by monoclonal antibodies against host cell surface molecules Supaporn Suparak a, Veerachat Muangsombut a, Donporn Riyapa b, Joanne M. Stevens c, Mark P. Stevens c, Ganjana Lertmemongkolchai b, Sunee Korbsrisate a,* a Department of Immunology, Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok 10700, Thailand Centre for Research and Development of Medical Diagnostic Laboratories, Faculty of Associated Medical Sciences, Khon Kaen University, Khon Kaen 40002, Thailand c Roslin Institute and Royal (Dick) School of Veterinary Studies, University of Edinburgh, Bush Farm Road, Roslin, Midlothian, EH25 9RG, United Kingdom b
Received 19 January 2011; accepted 10 June 2011 Available online 6 July 2011
Abstract Burkholderia pseudomallei induces the formation of multinucleated giant cells in cell monolayers. After infection of human macrophage-like U937 cells with B. pseudomallei, addition of monoclonal antibodies against integrin-associated protein (CD47), E-selectin (CD62E), a fusion regulatory protein (CD98), and E-cadherin (CD324) suppressed multinucleated giant cells in a concentration-dependent manner while monoclonal antibodies against other surface molecules did not inhibit fusion despite binding to the cell surface. Flow cytometric analysis showed increased expression of CD47 and CD98, but not CD62E and CD324, upon B. pseudomallei infection. Our data suggest the involvement of specific cellular factors in the process of B. pseudomallei-induced fusion. Ó 2011 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved. Keywords: Burkholderia pseudomallei; U937 cell; Multinucleated giant cell; CD47; CD98
1. Introduction Burkholderia pseudomallei is the causative agent of melioidosis, a severe invasive disease of humans endemic in Southeast Asia, particularly Thailand and Northern Australia [1]. The organism resists phagocytosis and killing by polymorphonuclear cells [2] and internalized bacteria are able to induce actin polymerization at one pole and form membrane protrusions [3]. It has been postulated that such actin-rich membrane protrusions contribute to cell-to-cell spread and multinucleated giant cell (MNGC) formation [3], however other intracellular pathogens capable of actin-based motility do not induce fusion and the mechanism of MNGC formation is unknown. The formation of MNGC has been observed in
* Corresponding author. Tel.: þ66 2 418 0569; fax: þ66 2 418 1636. E-mail address: [email protected] (S. Korbsrisate).
tissue samples from human melioidosis patients [4]. B. pseudomallei has been reported to modify infected macrophagelike cells in a manner analogous to MNGC formation in bone, a process known as osteoclastogenesis [5]. Previously, B. pseudomallei mutants deficient in a structural component of the bsa-encoded a type III secretion system [6] or the RpoS sigma factor [7] were reported to exhibit reduced MNGC formation in macrophage-like cell lines. However, these phenotypes likely result from impairment of earlier stages of intracellular life and the contribution of bacterial and cellular factors to the fusion process remains ill-defined. Several cell surface adhesion molecules have been implicated in macrophage fusion. Members of the immunoglobulin superfamily, the macrophage fusion receptor (also known as signal-regulatory protein a; SIRPa) and its ligand CD47 (integrin-associated protein), participate in fusion of rat alveolar macrophage [8]. The b1 (CD29) and b2 (CD18) integrins influence cell adhesion and multinucleated foreign body giant
1286-4579/$ - see front matter Ó 2011 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved. doi:10.1016/j.micinf.2011.06.007
S. Suparak et al. / Microbes and Infection 13 (2011) 1006e1011
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cell formation by interleukin (IL)-4-induced human monocytederived macrophages [9]. Furthermore, antibodies against LFA-1 and ICAM-1 (CD54) inhibit macrophage fusion [10], as do antibodies against the transmembrane adhesion molecule CD44 [11], E-cadherin [12], and an LPS receptor (CD14) [13]. Potential fusion mediators such as a fusion regulatory protein (FRP-1, CD98) have also been reported to enhance virusinduced cell fusion [14]. Here, we investigated the role of candidate cell surface molecules of human macrophage U937 cells in B. pseudomallei-induced MNGC formation.
the infected cells were fixed and MNGC formation was evaluated as described above. Control experiments were performed with MAbs of the same isotype; Mouse IgG1 for antiCD172a, anti-CD62E, anti-CD47, anti-CD98, anti-CD324, anti-CD11a, anti-CD54, anti-CD18; Mouse IgG2a for antiCD14 and Rat IgG2b for anti-CD44. All antibodies were supplied by Biolegend Inc., San Diego, CA.
2. Materials and methods
Expression of cell surface molecules was assessed by flow cytometry as previously described [2]. Differentiated U937 cells infected with B. pseudomallei K96243 were dislodged and incubated with rat anti-mouse CD16/CD32 (Becton Dickinson, San Diego, CA) for 30 min at 4 C to block the Fc receptor and thus prevent non-specific antibody binding. Individual cell samples were incubated for 30 min at 4 C with each MAb, or isotype controls. The cells were washed and further incubated with fluorescein-isothiocyanate (FITC)labeled anti-mouse IgG antibody (Dako Japan, Kyoto, Japan) for 30 min at 4 C. They were then washed and analyzed by flow cytometry. The percentage of cells that expressed cell surface molecules was determined by comparison with the isotype matched control. To determine the alteration of cell surface molecule expression after infection, the ratio of mean fluorescence intensity (MFI) between infected and uninfected cells at the same time point was calculated. Ratio values greater than 1 indicate an increase in surface molecule expression after infection. Data were analyzed using FACScalibur with CELLQuest software (BD Biosciences, San Jose, CA).
2.1. Bacterial strain, cell line and growth conditions B. pseudomallei K96243 was cultured using LuriaeBertani medium at 37 C. The human leukemic monocyte lymphoma cell line (U937, ATCC CRL-1593.2) was maintained and incubated in RPMI 1640 (Gibco-BRL, Grand Island, NY) at 37 C in a humidified 5% CO2 atmosphere. U937 cells were activated with 20 ng/ml of phorbol 12-myristate 13-acetate (Sigma Chemical Co., St Louis, MO) for 48 h to stimulate differentiation to macrophage-like cells. 2.2. Detection of B. pseudomallei-induced MNGC Stationary phase LB-grown B. pseudomallei K96243 were washed and added to monolayers at a multiplicity of infection (MOI) of one bacterium per two cells and incubated at 37 C with 5% CO2 for 2 h. Thereafter, the extracellular bacteria were killed by the addition of 250 mg/ml kanamycin to the overlay. At various time intervals after infection, cells were stained with Giemsa and MNGC formation assessed by counting the number of nuclei within MNGC (>2 nuclei/cell) from at least 500 nuclei examined, as previously described [6]. The percentage of MNGC reflects the number of nuclei within multinucleated cells/total number of nuclei counted 100. 2.3. Impact of monoclonal antibodies specific to cell surface molecules on B. pseudomallei-induced MNGC formation Differentiated U937 cells were infected with B. pseudomallei K96243 for 2 h as described above. Thereafter, monoclonal antibodies (MAbs) against the following cell surface molecules were separately added to the culture medium at the concentrations specified in Results; LPS receptor (CD14) (clone M5E2), transmembrane adhesion molecule (CD44) (clone IM7), integrin-associated protein (CD47) (clone HCD47), E-selectin (CD62E) (clone HEA-1f), fusion regulatory protein, FRP-1 (CD98) (clone MEM-108), signal-regulatory protein a (CD172a) (clone SE5A5), E-cadherin (CD324) (clone 674A), leukocyte function-associated antigen-1 (LFA-1) composed of CD11a (clone HI111) and CD18 (cloneTS1/18) integrin chain (LFA-1) (clone HI111) and LFA-1 counter-receptor ICAM-1 (intercellular adhesion molecule-1, CD54) (clone HCD54). At various time intervals,
2.4. Flow cytometric determination of cell surface molecule expression
2.5. Statistical analysis All tests for significance were performed using the Student’s t-test. Data were considered significant at a P value of 0.05. 3. Results 3.1. Induction of MNGC formation in B. pseudomalleiinfected human macrophage-like U937 cells The kinetics of B. pseudomallei-induced MNGC formation in human macrophage-like U937 cells were determined following inoculation at a low multiplicity of infection (1 CFU/2 cells). As shown in Fig. 1, MNGC formation could be detected from 15 h post-infection (h.p.i.) with the number of nuclei incorporated into an MNGC gradually increasing over time. Having established a reliable protocol for induction of fusion, we investigated whether culture supernatant collected from differentiated U937 cells infected with B. pseudomallei may induce or enhance cell fusion. Neither the supernatant from uninfected U937 or B. pseudomalleiinfected U937 cells could induce the formation of MNGC on uninfected cells or increase MNGC formation in infected
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Fig. 1. Typical Giemsa-stained MNGC formed following infection of differentiated U937 macrophage-like cells by B. pseudomallei K96243. Cells were fixed and observed the development of MNGC at the times indicated. Arrow, MNGC.
monolayers (data not shown). Additionally, no MNGC formation was detected in U937 cell monolayers treated with heat-killed B. pseudomallei (data not shown), indicating that the fusion process requires viable bacteria. Induction of MNGC has been reported in mouse macrophage cell lines (RAW264.7 and J774A.1) [6,7] and human epithelial lines (HeLa and A549 cells) [3]. In all these reports, infected cells were attached to the surface of the culture vessel. We infected a suspension of U937 cells at the monocytic stage with B. pseudomallei at a comparable MOI to the studies using attached cells [3] but no evidence of MNGC formation was
observed (data not shown), supporting earlier data in a macrophage fusion model that cell attachment may be required to reliably detect this phenotype [15]. 3.2. Antibodies specific to selected cell surface molecules can suppress MNGC formation To elucidate the role of host cell surface molecules during B. pseudomallei-induced MNGC formation, MAbs against specific candidates were added to fusion assays at various concentrations. As depicted in Fig. 2A, MAbs against integrin-
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associated protein (anti-CD47), E-selectin (anti-CD62E), fusion regulatory protein (anti-CD98), and E-cadherin (antiCD324) could each suppress B. pseudomallei-induced MNGC formation at a concentration of 10 mg/ml to an undetectable level whereas 34.7 2.52% of nuclei were in MNGC in nontreated monolayers (P < 0.01, t-test). Under these conditions, the viability of the cells was more than 95% when examined by trypan blue dye and no significant detachment of cells was observed suggesting that the reduced fusion was not due to loss of cells from the monolayer upon addition of the MAbs. No differences in the number of intracellular B. pseudomallei were noted 3, 6, 9, 12 and 24 h.p.i. of U937 cells at an MOI of 0.5 (1 CFU/2 cells) in the presence of 10 mg/ml anti-CD98 (data not shown). This indicates that inhibition of MNGC formation by CD98-specific antibody is unlikely to be a consequence of interference in intracellular replication or survival of B. pseudomallei. Addition of MAbs specific to the signal-regulatory protein a (SIRPa, anti-CD172a; 5.0 2.0%), integrin LFA-1 complex (anti-CD11a and anti-CD18; 16.3 3.75% and 12.5 2.50%, respectively), and LFA-1 counter-receptor ICAM-1 (anti-CD54; 16.9 1.90%) significantly reduced but did not abolish MNGC formation compared to controls (P < 0.05, t-test) (Fig. 2A). Neither the MAbs against LPS receptor (anti-CD14) nor
Fig. 2. Effect of monoclonal antibodies (MAbs) specific to selected cell surface molecules on B. pseudomallei-induced MNGC formation. U937 cells were infected with B. pseudomallei K96243. MAbs were added 2 h.p.i. at the concentrations indicated. MNGC formation was assessed at 18 h.p.i. and is presented as the percentage of nuclei in MNGC relative to the total number of nuclei counted (A) Influence of MAbs against cell surface molecules on MNGC formation at 10 mg/ml. (B) Concentration-dependent inhibition of MNGC formation by selected monoclonal antibodies. Error bars represent standard errors of the means for experiments performed in triplicate. The asterisks indicate statistically significant differences between cultures with and without MAbs (P < 0.05, t-test).
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a transmembrane adhesion molecule CD44 (anti-CD44) could inhibit fusion under the assay conditions (35.7 4.04% and 34.7 3.2%, respectively) (Fig. 2A). Isotype matched control IgGs of the MAbs had no inhibitory effect (Mouse IgG1 ¼ 35.0 3.0%, Mouse IgG2a ¼ 35.3 1.52%, Rat IgG2b ¼ 36 4.0%; P > 0.05, t-test). The suppression of MNGC formation in the presence of 10 mg/ml of either anti-CD47, anti-CD62E, anti-CD98, antiCD172a or anti-CD324 prompted us to investigate the impact of lower antibody concentrations ranging from 2.5 to 10.0 mg/ml. Significant dose-dependent inhibitory effects could be observed from concentration of 4 mg/ml increasing to near-complete inhibition at a concentration 10 mg/ml of each antibody (Fig. 2B). 3.3. Expression of cell surface molecules during MNGC formation To investigate the level of cell surface expression of the molecules under study during B. pseudomallei-induced MNGC formation, flow cytometric analysis was performed. When compared with isotype matched controls, more than 90% of the population of both uninfected and infected cells expressed CD98, CD62E, CD324, and CD172a (Fig. 3A). However, only 66% of uninfected U937 cells expressed CD47 but this percentage increased to more than 90% after interaction with B. pseudomallei. These results indicated that, with the exception of antibody directed against CD44, the antibodies used in this study bound to a comparable proportion of infected cells, even though they differ in their ability to inhibit MNGC formation. When compared with uninfected cells, expression of the cell surface molecules CD47 and CD98 (which were implicated in MNGC formation using specific MAbs) was significantly increased at 18 h.p.i. At this time point, the mean fluorescence intensity (MFI) ratio of CD47 and CD98 was 22.6 and 11.1, respectively (Fig. 3B) and was maintained at this level at 24 h.p.i. When the efficiency of MNGC formation was evaluated over time in the same cultures it was 32.66 6.80% at 18 h.p.i., increasing to 70.33 18.45% at 24 h.p.i. (data not shown). However, the levels of CD62E and CD324 (implicated in MNGC formation), CD172a (partial role) and CD44 (no effect) were not significantly different from the uninfected cells at 18 h.p.i. of U937 cells with B. pseudomallei (average MFI ratio of approximately 1; Fig. 3B). A very low frequency of CD44expressing cells was observed with both infected and uninfected cells (Fig. 3A), and this may partially explain the inability of this particular antibody to interfere with B. pseudomalleiinduced cell fusion. Taken together, our data suggest that fusion is not associated with up-regulation of the expression of CD62E, CD324 or CD172a during B. pseudomallei infection, though levels of other molecules implicated in fusion (CD47 and CD98) are elevated at times when fusion occurs. 4. Discussion The present study shows that binding of antibodies specific to the cell surface molecules integrin-associated protein
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Fig. 3. Analysis of the expression of selected cell surface molecules on B. pseudomallei-infected U937 cells. (A) Histogram at 18 h.p.i. of the expression of cell surface molecules on U937 cells infected with B. pseudomallei compared with the uninfected and isotype controls analyzed by flow cytometry. The percentage of cells that expressed cell surface molecules was determined by comparison with the isotype matched control. (B) Ratio of mean fluorescence intensity (MFI) was calculated from the MFI of infected cells/uninfected cells at the same time point. Ratio values greater than 1 indicate an increase in surface molecule expression after infection.
(CD47), E-selectin (CD62E), a fusion regulatory protein (CD98), and E-cadherin (CD324) could almost completely inhibit MNGC formation whereas MAbs against LFA-1 (composed of CD11a, CD18), ICAM-1 (CD54) and CD172a only partially blocked MNGC formation. It can be hypothesized that MNGC formation may be facilitated by cell surface molecules that may interact with their ligands on adjacent cells. Among 4 different cell surface molecules implicated in MNGC formation, 3 of them (i.e. CD47, CD62E and CD324) are classified as adhesion molecules while CD98 is a fusion mediator [14]. Increased levels of adhesion molecules CD47 and CD98 were detected following B. pseudomallei infection of U937 cells by flow cytometry. Peak levels of CD47 and CD98 were reached at 18 h.p.i and were sustained thereafter. Prolonged high level expression of such molecules may contribute to the high frequency of cell fusion at the later time interval sampled. No significant increases in surface
expression of CD62E or CD324 were detected at 18 h.p.i. relative to uninfected cells, even though MAbs against these surface molecules were shown to inhibit MNGC formation, indicating that the progression of MNGC formation is not dependent on up-regulation of these surface molecules. The same concentration of antibodies that completely inhibited MNGC formation (i.e. anti-CD62E) or partially (i.e. anti-CD172a) were observed to stain the surface of infected U937 cells at comparable levels by flow cytometry, suggesting that antibody-mediated inhibition of fusion is specific to the target protein and the partial inhibition may not be due to simple steric interference. In addition, inhibition of MNGC formation appears not to be due to the proportion of cells expressing these cell surface molecules since CD172a was expressed on a similar proportion of cells, compared to CD47, CD98, CD62E and CD324, whereas antibodies to the latter four molecules almost completely inhibited fusion.
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Our finding that CD47, CD62E, CD98 and CD324 influence the process of B. pseudomallei-induced MNGC formation has not been reported for other bacteria. The up-regulation of CD47 and CD98 molecules may facilitate the onset of the fusion process. There are several studies reporting the importance of these surface molecules, for example CD47 was found to be involved in macrophage multinucleation by interacting with MFR (also called SIRPa or CD172a) during the macrophage adhesion/fusion process [8]. Human immunodeficiency virus (HIV) infection induces CD98 expression on CD4þ T lymphocyte, which is associated with syncytium formation [16]. Furthermore, the involvement of LFA-1 (composed of CD11a, CD18) and ICAM-1 (CD54) in the B. pseudomallei-induced U937 cell fusion model reported in this study is supported by previous observations in cellecell fusion models and HIV-induced syncytium formation [10,17]. Blocking of ICAM-1 expression could also inhibit concanavalin A-stimulated MNGC formation of mononuclear cells [18]. Furthermore, the expression of ICAM-1 on monocytic cells induced by Mycobacterium tuberculosis is involved in the host response and subsequent formation of granulomas [19]. In the osteoclast model it was suggested that ICAM-1 and E-cadherin (CD324) are involved at an early cell fusion stage such as cell-substrate adhesion whilst SIRPa and FRP-1 may regulate rather than mediate cellecell fusion [20]. To the best of our knowledge, this is the first study to demonstrate that B. pseudomallei-induced fusion of human macrophages in vitro requires cell surface molecules. Contact between cells was reported to be a prerequisite for fusion [15]. We propose that selected surface adhesion molecules of cells undergoing B. pseudomallei-induced fusion may facilitate attachment of cells to each other and bring their membranes into close contact. It is possible that B. pseudomallei may favor this process by modulating the surface expression of such molecules. Acknowledgments This work was supported by the National Science and Technology Development Agency grant BT-B-01-MG-145123. S. Suparak and D. Riyapa were supported by the Office of the Higher Education Commission for postdoctoral and PhD scholarships, respectively. S. Korbsrisate was supported by the Chalermphrakiat Grant, Faculty of Medicine Siriraj Hospital, Mahidol University. We are grateful to G.J. Bancroft, M. Ato and N. Onlamoon for their kind comments, and E. Noulsri and P. Tippayawat for their advice on flow cytometric analysis. Many thanks to V. Sookpatdhee for taking photogragh. References [1] D.A. Dance, Melioidosis as an emerging global problem, Acta Trop. 74 (2000) 115e119. [2] S. Chanchamroen, C. Kewcharoenwong, W. Susaengrat, M. Ato, G. Lertmemongkolchai, Human polymorphonuclear neutrophil responses to Burkholderia pseudomallei in healthy and diabetic subjects, Infect. Immun. 77 (2009) 456e463.
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