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. In this respect, the study by Vouldoukis
and collaborators [30] demonstrating human Mf iNOS activation and leishmanicidal activity through CD23 cross-linking
(CD23 mAb or IgE) is of particular interest. Our results clearly
demonstrate that, once iNOS expression is induced and provided that a source of BH4 is available, human Mf possess the
required machinery to exert NO-dependent microbicidal activity. These observations suggest that DFGiNOS U937 transfectants might be of potential interest to study the role played by
NO in Mf-pathogen interactions.
ACKNOWLEDGMENTS
This work was supported by grant 31-50667.97 from the Swiss
National Fund. The authors thank Dr. D. Rivier for providing
the L. mexicana strain, Dr. M. Markert for help with chemiluminescence measurements and stimulating discussions, Dr. S.
Betz-Corradin for critically reviewing this manuscript, and C.
Bodenmann, A. Menetray, and A. Ransijn for technical assistance.
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