Human monocytic U937 cells transfected with human hepatic inducible nitric oxide synthase exhibit leishmanicidal activity Sylvie Bertholet and Jacques Mauël Institute of Biochemistry, University of Lausanne, Switzerland Abstract: In mice, the high inducible synthesis of nitric oxide (NO) resulting from inducible NO synthase (iNOS, NOS2) expression by macrophages (Mf) is considered an essential component of the protective immune response against infection by intracellular pathogens. Conversely, in humans, the question of a role for NO as an antimicrobial defense mechanism has been the subject of much debate. Recently, however, iNOS expression by human Mf and formation of NO or its derivatives have been reported both in vivo and in vitro, strongly suggesting that human Mf are indeed capable of inducible NO synthesis. However, the conditions allowing NO production by human Mf in culture remain poorly defined, rendering more difficult the study of the effector functions of NO in these cells. To alleviate this problem, cells of the U937 monocytoid line were engineered to express iNOS by transfection with human hepatic iNOS (DFGiNOS), leading to production of NO on supplementation with the cofactor tetrahydrobiopterin. We report that U937 cells, when differentiated with 1,25-dihydroxyvitamin D3 and retinoic acid, acquire a phenotype allowing infection by Leishmania parasites and maintain viable intracellular microorganisms up to 72 h post-infection. Leishmania survival in DFGiNOS cells is strongly decreased when the cells are treated with tetrahydrobiopterin. Intracellular killing is evident by 24 h and increases up to 72 h post-infection, and is inhibited by L-N5-(1-iminoethyl)ornithine, an inhibitor of NO synthesis. In contrast, superoxide anion does not appear to play a role in the killing of Leishmania by DGFiNOS U937 cells. The relevance of this model to the study of the mechanisms of intracellular killing by human macrophages is discussed. J. Leukoc. Biol. 67: 34–39; 2000. Key Words: macrophage · Leishmania INTRODUCTION In humans, the contribution of nitric oxide (NO) to host defense and in particular, the capacity of Mf to produce this molecule in amounts sufficient for microbicidal activity, have been debated [reviewed in ref. 1]. Indeed, according to several investigators, human Mf failed to synthesize NO in vitro in 34 Journal of Leukocyte Biology Volume 67, January 2000 response to classical inducers of iNOS commonly used for activation of murine Mf [2–4]. A number of recent studies, however, have documented the presence of NO products and/or iNOS expression in vivo in different infectious conditions [1] and a role for NO in the killing of intracellular Leishmania by human Mf has been proposed [5]. Thus, the emerging view is that human Mf may be capable of inducible NO synthesis, although the mechanisms involved are poorly understood and the conditions allowing reproducible NO production by Mf in culture remain to be defined. Therefore, human cell lines engineered to express iNOS appear as a useful alternative for evaluating the numerous effects of NO on either host cells or pathogens. Leishmania infection was selected as a model because extensive studies performed in mice have stressed the importance of cytokine-dependent induction of iNOS and concomitant production of NO [6–10] as a major immunological mechanism resulting in the control of Leishmania infection both in vitro and in vivo. We thus addressed the question of the sensitivity of intracellular Leishmania parasites to NO produced by human U937 monocytic cells previously transfected with human hepatic iNOS cDNA (DFGiNOS U937) [11]. In these cells, the iNOS transgene is constitutively expressed, and the production of NO can be switched on by simply supplementing the cells with tetrahydrobiopterin (BH4). We describe the conditions required to obtain efficient cellular infection by Leishmania and report on the leishmanicidal effect of NO in this model. The contribution of oxygen radicals in the cytotoxic process was also evaluated. MATERIALS AND METHODS Infection of human cell lines with Leishmania ssp. L. mexicana (WHO reference strain MYNC/BZ/62/M379) and L. major [Liverpool reference strain LV39 (MRHO/SU/59/P)] promastigotes were isolated and propagated as published elsewhere [12, 13]. Promastigotes of both species were used to infect human monocytic cell lines when in the stationary phase of growth. Human monocytic U937 cells (ATCC CRL-1593.2) [14], DFGiNOS U937 transfectant (clone 2), and pBABE U937 vector control transfectant were maintained in culture as previously described [11]. For Correspondence: Sylvie Bertholet, Institut de Biochimie, chemin des Boveresses 155, CH-1066 Epalinges, Switzerland. E-mail: Sylvie. BertholetGirardin@ib.unil.ch Received July 6, 1999; revised September 6, 1999; accepted September 7, 1999. http://www.jleukbio.org infection with Leishmania, these cells were differentiated for 3 days at 5 3 105 cells/mL in Dulbecco’s modified Eagle’s medium (DMEM) 10% fetal calf serum (FCS) containing 10-7 M 1,25-dihydroxy-vitamin D3 (VD3; Calbiochem) and 10-7 M retinoic acid (RA; Sigma), in the absence of geneticin for all cell lines; lipopolysaccharide (LPS; from Escherichia coli 0:55-B.5, Difco Laboratories; 1 µg/mL) was added for the last 12–15 h of incubation. The cells were washed, then seeded in 24-well plates (Costar, Cambridge, MA) at 5 3 105 cells/well in 1 mL fresh culture medium containing or not 10 µM tetrahydrobiopterin (BH4; Sigma) and/or 500 µM L-N5-(1-iminoethyl)ornithine (L-NIO; Alexis Corp.), and were incubated for 1 h at 37°C (this preincubation step did not modify the level of cellular infection by Leishmania, data not shown). Medium was then removed to carry out the infection in one-third of the initial volume for 4 h (or as otherwise indicated) at 37°C with Leishmania promastigotes at a parasite-tocell ratio of 20:1. Non phagocytized parasites were removed by washing the cells, and remaining extracellular parasites were killed by incubation for 1 h at 37°C with 20% normal non-decomplemented human serum. Cells were washed once and cultured for different time periods in DMEM-c containing or not BH4 and/or L-NIO as previously indicated. The rate of cellular infection was determined by microscopic examination of stained preparations. Briefly, 2 3 105 infected cells were centrifuged onto microscope slides for 5 min at 500 rpm in a cytocentrifuge (Cytospin, Shandon Southern, Pittsburgh, PA), fixed in methanol, and stained successively in eosin and May-Grünwald solutions according to the manufacturer’s protocol (DiffQuick, Dade AG, Switzerland). differentiated with 10-7 M VD3/RA, U937 cells acquired macrophage markers such as CD14 and CD68 [S. Bertholet, unpublished data; and ref. 16], and were efficiently infected by L. mexicana with more than 30% of cells containing two to three intracellular parasites. Depending on the experiments, the percentage of infection increased up to 70% of cells. The percentage of VD3/RA-differentiated U937 cells infected with Leishmania was similar to that observed for another pathogen, the bacterium Brucella suis (J. Dornand, personal communication). Kinetic experiments showed that infection was essentially completed during the first 6 h and this was true both for the percentage of infected U937 cells and the number of intracellular parasites (Fig. 1B). Similar results were obtained with L. major promastigotes (not shown). Differentiated DFGiNOS U937 transfectants as host cells for Leishmania We reported recently that U937 cells transfected with human hepatic iNOS cDNA (DFGiNOS U937) were capable of high- Assessment of intracellular killing of Leishmania parasites Intracellular killing was determined by a published technique [7] adapted to nonadherent cells as follows. Leishmania-infected U937, DFGiNOS U937, and pBABE U937 cells were collected in Eppendorf tubes and centrifuged for 5 min at 2000 rpm in a table-top centrifuge. The supernatants were collected for measurement of the NO22 content, and the cell pellets were washed once with phosphate-buffered saline (PBS) to remove FCS. The cells were then lysed by exposure to 0.01% sodium dodecyl sulfate (SDS) in DMEM for 15 min at 37°C to release the surviving parasites. The parasites were then supplemented with HOSMEM II 30% FCS medium, plated in 96-well culture plates in serial dilutions, and incubated at 26°C for 60 h to allow amastigote to promastigote transformation and growth. Parasite proliferation was recorded by measuring [3H]thymidine incorporation ([3H]TdR, 0.5 mCi/well, Amersham, Buckinghamshire, UK) for an additional 12- to 24-h incubation period at 26°C. Measurement of NO22 and O22 Cell culture supernatants were assayed for NO22 by incubating them with Griess reagents as described elsewhere [11]. Absorbance was measured at 550 nm using a 690-nm reference filter. The method used to measure O22 production is based on the superoxide dismutase (SOD)-inhibitable reduction of ferricytochrome c and adapted to a microassay as described previously [15]. Briefly, 2 3 105 cells in Hanks’ balanced salt solution (HBSS) containing ferricytochrome c (from horse heart, type VI, Sigma; 2 mg/mL) were stimulated with phorbol myristate acetate (PMA, Sigma; 1 µg/mL) 6 superoxide dismutase (SOD; Sigma; 30 µg/mL). Absorbance was measured at 550 nm with a 492-nm reference filter. Results are given as the difference in absorbance (DA) between wells containing (1SOD) or not (-SOD) SOD at a given time point (ti) relative to the time when measurements started (t0) according to the following formula: DA 5 [A (ti 2 t0) (-SOD)] 2 [A (ti 2 t0) (1SOD)]. RESULTS Effect of U937 cell differentiation on infection with Leishmania ssp. U937 cells were differentiated or not for 3 days with 10-7 M VD3/RA and further infected for 24 h with L. mexicana promastigotes. The degree of infection was determined by microscopic examination of stained cells. U937 cells were poorly infected by L. mexicana promastigotes with less than 1% of the cells harboring one parasite (Fig. 1A), whereas when Bertholet and Mauël Fig. 1. Effect of VD3/RA differentiation on infection of U937 cells by Leishmania promastigotes. U937 cells differentiated or not with 10-7 M VD3/RA were infected with L. mexicana promastigotes at a parasite to cell ratio of 20:1 and then stained for microscopic examination. The rate of intracellular infection is expressed either as the percentage of infected cells or the number of intracellular parasites per 100 cells. (A) Infection (24 h) of U937 cells by L. mexicana: effect of VD3/RA differentiation. (B) Kinetics of the infection of differentiated U937 cells by L. mexicana. Leishmanicidal activity of iNOS-transfected human macrophages 35 TABLE 1. Infection of DFGiNOS U937 Cells by L. mexicana or L. major Parasites Percentage of infection L. major (pro) L. mexicana (pro) L. mexicana (ama) Number of parasites/100 infected cells DFGiNOS U937 U937 DFGiNOS U937 U937 36 6 6 17 6 5 92 6 1 67 6 5 60 6 3 96 6 2 426 6 135 345 6 163 1107 6 291 533 6 109 581 6 83 1241 6 210 U937 cells and DFGiNOS U937 transfectants differentiated with 1027 M VD3/RA were infected at 37°C for 24 h with L. mexicana or L. major promastigotes (pro), or with freshly isolated L. mexicana amastigotes (ama) at a parasite-to-cell ratio of 20:1. The percentage of cells infected with Leishmania and the number of intracellular parasites per 100 infected cells were determined by microscopic examination. Results are presented as mean 6 SD (n 5 4) from one representative experiment. output NO synthesis when supplemented with BH4 [11]. This unique property allows NO production to be switched on as desired in order to mimic the natural induction process. The capacity of Leishmania to infect the transfected cells in the absence of BH4 was investigated first. VD3/RA-differentiated DFGiNOS U937 cells could be infected by promastigotes of either L. mexicana or L. major species (Table 1), and L. infantum, L. panamensis, and L. enriettii promastigotes (unpublished data). Compared with parental U937 cells, however, the percentage of infected DFGiNOS U937 cells and the number of parasites observed intracellularly was lower. The situation was different when L. mexicana amastigotes were used for the infection. Both DFGiNOS U937 transfectants and parental U937 cells were very efficiently infected with more than 90% of the cells containing 11–12 parasites. Effect of NO on intracellular Leishmania survival DFGiNOS U937 cells were infected with L. mexicana promastigotes and then incubated for 3 days in the presence or absence of exogenous BH4, which was previously shown to be an essential cofactor for iNOS activity and NO production by these transfected cells [11]. The production of NO was assessed by measuring NO22 in the cell culture supernatants. To demonstrate a possible cytotoxicity of NO toward Leishmania in DFGiNOS U937 cells, the viability of the intracellular parasites was determined 72 h post-infection by recovering amastigotes after lysis of the infected cells with 0.01% SDS. The parasites were then cultured at 26°C and proliferation was determined by measuring [3H]TdR uptake. The proliferation of parasites recovered from DFGiNOS U937 cells cultured in BH4-free medium was slightly reduced compared with that of parasites released from U937 cells or pBABE vector control cells. Incubation of infected DFGiNOS U937 cells with BH4 led, however, to a marked decrease in the viability of the intracellular microorganism as determined by [3H]TdR uptake (Fig. 2A), whereas viability of DFGiNOS U937 host cells was not affected, as judged by trypan blue exclusion (results not shown). The production of NO by DFGiNOS U937 cells supplemented with BH4 was confirmed by measuring NO22 accumulating in the cell culture supernatants (Fig. 2B). We demonstrated in a previous study that NO22 represented one-fourth of total NO32 1 NO22 released by DFGiNOS U937 cells cultured with BH4 [11], thus, in Figure 2B NO production can be estimated at 60 µM. The proliferation of parasites recovered from U937 cells or from pBABE U937 cells cultured with BH4 was not decreased, correlating with the absence of production of NO. Similar results were obtained when infection was carried out with L. infantum promastigotes (data not shown). Kinetics of the killing of Leishmania within differentiated DFGiNOS U937 cells The viability of L. mexicana within DFGiNOS U937 cells supplemented or not with BH4 and/or L-NIO was further evaluated at different times post-infection and compared to the production of NO. Intracellular parasites were recovered from the infected cells and their capacity to proliferate at 26°C was measured by [3H]TdR incorporation (Fig. 3). A general decrease of Leishmania proliferation was observed up to 72 h for all conditions tested; however, parasites from cells cultured with BH4 were most strongly affected. At 12 h post-infection, the uptake of [3H]TdR by the recovered parasites was similar for all conditions tested. [3H]TdR incorporation started to decrease at 24 h for parasites from cells cultured with BH4, and was minimal at 72 h. The reduction of [3H]TdR uptake correlated with NO production measured as the accumulation of Fig. 2. Correlation between intracellular killing of Leishmania and NO production. (A) VD3/RAdifferentiated U937, DFGiNOS U937, and pBABE U937 cells were infected for 4 h with L. mexicana promastigotes in the presence or absence of BH4 (10 µM) and/or L-NIO (500 µM). Extracellular parasites were removed as described in Materials and Methods, and cells were further incubated for 72 h with or without BH4 and/or L-NIO. Parasite survival was determined by measuring proliferation, assessed by [3H]TdR incorporation, of parasites recovered from SDS-treated cells. (B) In parallel, NO22 present in the cell culture supernatants was measured as readout for NO production. 36 Journal of Leukocyte Biology Volume 67, January 2000 http://www.jleukbio.org produced by U937 cells after stimulation with PMA but not Leishmania (Fig. 4). In addition, L. mexicana promastigotes did not modify the production of O22 by U937 cells triggered by PMA. DFGiNOS U937 cells, however, did not release detectable amounts of O22 under any condition tested. DISCUSSION The U937 cell line has been successfully used as model for infection with macrophage-specific pathogens as diverse as Leishmania [17–19], Brucella, and Listeria [20] or Salmonella [21] species and thus represents a valuable model to study human macrophage-pathogen interactions. To support infection by Leishmania, U937 cells have been differentiated into Mf-like cells by using phorbol myristate acetate [17, 18, 22], a molecule that triggers a large panel of intracellular events associated with protein kinase C activation. In this study, we applied for U937 cell differentiation a procedure first used to promote infection by bacteria [20]. Using physiological concentrations of 1,25-dihydroxyvitamin D3 and retinoic acid resulted in U937 cell differentiation. These differentiated cells remain poorly adherent but aggregate in culture and are characterized by acquisition of Mf markers such as CD14 and efficient infection by L. mexicana and L. major promastigotes. Depending on the experiment, the percentage of infected U937 cells ranged from 30 to 70%, and the infection rate increased up to a maximum of six parasites per infected cell, probably related to differences in infectivity of individual batches of promastigotes. In our hands, non-differentiated U937 cells essentially failed to become infected by Leishmania promastigotes, unlike the results recently published by Abdullah and collaborators [19]. We then evaluated differentiated DFGiNOS U937 transfectants as host cells for Leishmania ssp. In the absence of BH4, these transfectants did not support the production of NO, behaved like parental U937 cells [11], and could become infected by Fig. 3. Kinetics of the killing of intracellular Leishmania within DFGiNOS U937 cells. VD3/RA-differentiated DFGiNOS U937 cells were infected for 4 h with L. mexicana promastigotes and further incubated for different periods of time in the presence or absence of BH4 (10 µM) and/or L-NIO (500 µM). (A) Parasite survival was determined by measuring proliferation, assessed by [3H]TdR incorporation, of parasites recovered from SDS-treated DFGiNOS U937 cells. (B) In parallel, NO22 present in the cell culture supernatants was measured as readout for NO production by DFGiNOS U937 cells. NO22 in cell culture supernatants. Addition of L-NIO on the DFGiNOS U937 cells cultured with BH4 inhibited NO production and completely restored the uptake of [3H]TdR by parasites recovered from these cells. Production of oxygen radicals in response to infection by Leishmania The production of O22 in response to L. mexicana or PMA stimulation by VD3/RA-differentiated DFGiNOS U937 and U937 cells was investigated by measuring the SOD-dependent reduction of ferricytochrome c over a 120-min period of time. Preliminary results showed that VD3/RA-differentiated U937 cells had to be first stimulated overnight with LPS (1 µg/mL) to further release O22 in response to PMA (not shown). O22 was Bertholet and Mauël Fig. 4. O22-dependent reduction of cytochrome c by DFGiNOS U937 cells in response to Leishmania infection. VD3/RA-differentiated DFGiNOS U937 or U937 cells were exposed overnight to LPS (1 µg/mL), then washed and stimulated by addition of L. mexicana promastigotes (at a 20:1) ratio and/or PMA (1 µg/mL). The reduction of ferricytochrome c was measured at 550 nm over a 120-min period of time. The specific O22 reducing activity at a given time point is expressed as the difference between total and SOD-inhibitable activities. Leishmanicidal activity of iNOS-transfected human macrophages 37 promastigotes of different strains, however, to a somewhat reduced level. Lower infection was also observed for pBABE U937 cells (vector control), suggesting an unknown effect of transfection (possibly promastigote sensitivity to intracellular geneticin, which may remain after culture of the cell transfectants in selective media) rather than an effect of the iNOS transgene. In contrast, no such differences in cellular infection were observed between parental and transfected U937 cells when Leishmania amastigotes were used. Different mechanisms might account for the increased capacity of amastigotes to infect U937 cells such as increased resistance to residual geneticin, enhanced potential to neutralize oxygen metabolites and inhibit Mf respiratory burst, and usage of different ligand(s)/receptor(s) to penetrate U937 cells [reviewed in ref. 23]; however, additional studies are needed to clarify this point. Because DFGiNOS U937 transfectants were successfully infected by Leishmania, we next investigated the effect of NO on the survival of intracellular parasites by adding BH4 for different periods of time to the culture medium. Incubating Leishmania-infected DFGiNOS U937 cells with BH4 led to a marked decrease in the viability of the intracellular parasites as determined by [3H]TdR uptake. Kinetic experiments showed that the intracellular killing of Leishmania was effective in DFGiNOS U937 cells after a 24-h period of culture with BH4, and was maximal at 72 h. The contribution of NO to this effect was demonstrated by using L-NIO, a NOS inhibitor with relative selectivity for iNOS, which protected the intracellular parasites from death in DFGiNOS U937 transfectants. As expected, neither U937 nor pBABE U937 cells displayed the same cytotoxic potential in the presence of BH4, correlating with the absence of production of NO by these cells [11]. It is, however, noteworthy that both cell lines were found positive for iNOS RNA expression by reverse transcriptase-polymerase chain reaction methods [S. Bertholet, unpublished results], suggesting the presence of untranslated iNOS RNA, or, if translated, technical limitations in detecting weak levels of iNOS protein and NO by conventional methods. Nevertheless, these hypothetical small levels of NO were non-cytotoxic toward Leishmania in a 72-h assay. Oxygen radicals (O22, H2O2) did not contribute to the leishmanicidal activity of DFGiNOS U937 cells in our model, since we were unable to demonstrate O22 production by these cells either in response to Leishmania infection or to PMA stimulation. This apparent unresponsiveness to Leishmania might be attributed to a survival strategy of the parasites consisting in either avoiding triggering the host cell respiratory burst or scavenging oxygen radicals through surface molecules (e.g., lipophosphoglycans) as demonstrated in mouse models of infection [reviewed in ref. 23]. Unresponsiveness to PMA was observed for both DFGiNOS U937 cells and pBABE U937 cells (vector control; data not shown) excluding trapping of superoxide by NO to yield peroxynitrite and suggesting an as yet unidentified effect of transfection. A putative increase in SOD activity within these transfected cells is currently under investigation. These observations were confirmed by measurements of oxygen radical-induced chemiluminescence (data not shown). Activation of human Mf with cytokines such as interferon-g and granulocyte-macrophage colony-stimulating factor results 38 Journal of Leukocyte Biology Volume 67, January 2000 in leishmanicidal activity [24–29]; however, the killing mechanisms resulting from such activation remain poorly characterized and appear to rely only partially on Mf oxidative burst. NO might represent another mechanism utilized by human Mf to kill intracellular microorganisms, as extensively demonstrated for murine Mf. The presence of NO metabolites and the expression of Mf iNOS reported to occur in certain infectious states are compatible with the notion that NO also plays a role in host defense in human. However, the precise conditions allowing reproducible NO synthesis by human Mf in culture remain to be defined. 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