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Immunology Program, Departments of
Internal Medicine and
Microbiology, and
Program in Free Radical and Radiation Biology, University of Iowa, and
¶ Veterans Affairs Medical Center, Iowa City, IA 52242; and
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Universidade Federal do Rio Grande do Norte, Natal, Brazil
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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-mediated intracellular killing by both murine and
human macrophages. According to RNase protection assay and
immunohistochemistry, iNOS mRNA and protein were expressed at higher
levels in bone marrow of patients with visceral leishmaniasis than in
controls. The iNOS protein also increased upon infection of human
macrophages with L. chagasi promastigotes in vitro in
the presence of IFN-. These data suggest that
O2- and NO· each contribute to
intracellular killing of L. chagasi in human and murine
macrophages. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The macrophage is armed with antimicrobial mechanisms that intracellular organisms must evade to survive. During leishmaniasis the microbicidal interactions between parasite and host cells occur in two stages. First, during initial phagocytosis of promastigotes the macrophage can undergo an oxidative response stimulated by the phagocytosis event. Second, once infection with amastigotes is established, the quiescent macrophage can be activated to potentially kill intracellular leishmania. Efficient evasion of toxic microbicidal molecules produced at each stage of infection is important for leishmania to be able to initiate and maintain host cell infection.
Two important macrophage-derived oxidants have been identified as critical in controlling leishmania infection. During the first stages of infection superoxide (O2-) is produced as part of the respiratory burst of human and murine macrophages in response to phagocytosis (2, 3). O2- production is catalyzed by the NADPH oxidase, a heme-containing cytochrome that contains cytosolic and membrane-bound components. Once assembled the oxidase transfers an electron from NADPH to molecular oxygen, producing O2-. Leishmania promastigotes have been shown to be susceptible to killing by exposure to O2- and hydroxyl radical (·OH) generated from H2O2 (4, 5).
A second anti-leishmanial oxidant produced by macrophages is
NO· (6, 7, 8). Unlike
O2-, which is a generated during
phagocytosis of the parasite, NO· is generated after macrophage
activation by IFN- and TNF-
and is most relevant to killing
established intracellular amastigotes. Inhibitors such as
NG-monomethyl-L-arginine
(L-NMMA)3
lead to an increase in amastigote survival and replication in murine
macrophages (9). Although there is strong evidence that
NO· plays an important role in murine leishmaniasis, it remains
controversial whether NO· plays a role in the
anti-leishmanial responses of human macrophages (10, 11). NO· was reported to participate in the killing of
L. major by human macrophages that are stimulated through
the low affinity Fc
receptor, CD23, and IFN- (11).
Furthermore, inducible NO synthase (iNOS) is produced in alveolar
macrophages from patients infected with Mycobacterium
tuberculosis, another intracellular micro-organism
(12). Thus, there is emerging evidence suggesting that
NO· is generated and could participate in killing intracellular
microbes by human macrophages.
Macrophages containing intracellular L. donovani amastigotes
have impaired antimicrobial responses, signaling, and cell surface
marker expression (13, 14). This could be due in part to
impaired Ca+2 mobilization and impaired
phosphorylation of protein kinase C, events that are essential for
NADPH oxidase activation and phagocytosis (15, 16).
Intracellular amastigotes also cause impaired IFN- signaling, as
does the lipophosphoglycan (LPG) isolated from promastigote membranes.
It is unclear whether oxidant antimicrobial responses are inhibited by
promastigotes during phagocytosis (17, 18, 19).
Recognizing that there are differences between leishmanial infection of murine vs human macrophages, the work presented in this manuscript contrasts human and murine macrophage responses to L. chagasi, the cause of South American visceral leishmaniasis. Using sensitive techniques for detecting O2- and NO· we documented differences in the amount of oxidants generated after phagocytosis of L. chagasi promastigotes. Despite these differences, both oxidants were found to have functional consequences during leishmanial infection of murine and human macrophages.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Human mononuclear cells were isolated from whole blood of normal healthy human donors by density sedimentation (Histopaque-1077; Sigma, St. Louis, MO). Monocytes were separated by adherence. After 5 days of culture in RPMI 1640 with 10% heat-inactivated FCS, 100 U penicillin/ml, and 50 µg streptomycin/ml (RP-10; University of Iowa Hybridoma facility) at 37°C in 5% CO2, adherent cells assumed characteristics of monocyte-derived macrophages (MDMs). Bone marrow was removed from C3H.HeJ (The Jackson Laboratory, Bar Harbor, ME) mouse long bones and cultured for 5–7 days in RP-10 supplemented with 20% L929 cell supernatant (American Type Culture Collection, Manassas, VA). Bone marrow-derived macrophages (BMMs) were harvested with 0.051 trypsin and 0.1% EDTA, and 1 x 106 cells were allowed to adhere to 24-well plates or on glass coverslips overnight before use.
Parasite culture
A strain of L. chagasi (MHOM/BR/00/1669), originally isolated from a Brazilian patient with visceral leishmaniasis, was maintained by serial passage through hamsters as previously described (20). Amastigotes were recovered from hamster spleens and allowed to convert to promastigotes in hemoflagellate MEM containing 10% heat-inactivated-FCS and 8 µM hemin at 26°C (20). Promastigote cultures were seeded at 3 x 106 cells/ml and grown to stationary phase (5–7 x 107 cells/ml). Promastigotes were opsonized by incubation for 1 h at room temperature in 5% normal human serum (autologous with MDMs) or virus-free murine serum (Harlan, Oxford, MI) in HBSS and washed twice by centrifugation in HBSS. Five percent of parasites were killed by the procedure, presumably via complement-mediated lysis. One milligram of zymosan per milliliter (Sigma) was opsonized in parallel with human or murine serum.
Spin trapping
MDMs or BMMs (1 x 106) were infected by incubation for 30 min with opsonized or unopsonized promastigotes at different parasite:macrophage ratios in HBSS containing 100 µM diethylenetriaminepentacetic acid (DTPA; Sigma) to chelate iron and 100 mM of the spin trap 5,5-dimethyl-1-pyrroline-N-oxide (DMPO; Sigma) at 37°C in 5% CO2. Supernatants were collected, snap-frozen, and stored at -80°C for up to 1 wk. DMPO spin adducts remain stable under these conditions. Electron spin resonance (ESR) spectra were obtained at room temperature using a Bruker ESP 300 spectrometer (Karlsruhe, Germany). Instrument settings were: microwave power, 20 mW; modulation frequency, 100 kHz; modulation amplitude, 0.892 G; sweep time, 0.238 G/min; and response time, 0.655 s. Spectra detected the DMPO-OH spin adduct (AN = AH = 14.9 G). Superoxide dismutase (SOD; Sigma) or catalase was added to some macrophage cultures to differentiate between DMPO-OH generated from O2- and hydroxyl radical, respectively.
NO· detection
BMMs and MDMs (1 x 106) were
cultured in 96-well round-bottom tissue culture plates (Costar,
Corning, NY), and NO· was quantified by the concentration of its
product nitrite (NO2-) in
supernatants, using a NOA 280 NO analyzer (Sievers, Boulder, CO).
Nitrite was reduced to NO· in the presence of 1% KI and glacial
CH2OOH before its detection. During some assays,
nitrate was reduced to NO· in the presence of
0.8%VCl3 in 1 M HCl before its detection. Unless
otherwise indicated, opsonized parasites were added to macrophages at a
ratio of five parasites to one macrophage. Some wells contained 100 U
murine IFN-/ml or 400 U human IFN-/ml (R&D Systems, Minneapolis,
MN). One nanogram of LPS/ml (Sigma) and 10 µg IL-1/ml (R&D Systems)
without promastigotes were added to generate positive control
supernatants. Supernatants collected from murine BMMs and human MDMs
were read 72 h after infection, and the concentration of
NO· was determined.
Infection assays
Human MDMs or murine BMMs (5 x 105) in 10% heat-inactivated FCS/RPMI 1640 (RP-10) were cultured on round glass coverslips (Fisher Scientific, Pittsburgh, PA) in each well of a 24-well plate (Costar, Corning, NY). Opsonized or unopsonized promastigotes at different ratios were added for 2 h and removed by washing. All conditions were performed in duplicate. Slides were collected at different time points and stained with Diff-Quik (Dade-Behring, Newark, DE). The ratio of attached or intracellular parasites to macrophages was quantified microscopically, counting a minimum of 200 cells/coverslip.
The microbicidal effects of O2- or
NO· were inhibited with the
O2- scavenger
4-hydroxy-2,2,6,6-tetramethylpiperidine-N-oxyl (TEMPOL) or
the NOS inhibitor L-NMMA, respectively
(21). To inhibit O2- in
both cell types or to inhibit NO· in murine BMMs, opsonized
promastigotes at a 20:1 ratio to macrophages were added with or without
400 U human IFN-/ml, 100 U murine IFN-/ml (R&D Systems), 0.4 mM
TEMPOL, or 1 mM L-NMMA (Sigma). Compared with ESR
studies, a higher infection ratio was needed to achieve adequate
numbers of intracellular parasites for microscopic assays. After 2
h parasites were removed by washing, and medium with or without IFN-
or L-NMMA was replaced. Different times were
chosen due to the toxic effect of long term TEMPOL incubation on
macrophages. We previously showed attachment/ingestion peaks after
30-min incubation of macrophages with promastigotes
(22).
To inhibit NO· in human macrophages,
5 x 105 MDMs in RP-10 were preinfected with
20:1 opsonized promastigotes-MDMs similar to microscopic assays. After
5 h promastigotes were removed, and MDMs containing intracellular
parasites were incubated for an additional 48 h. MDMs were washed
and incubated with or without 1 mM L-NMMA and IFN- for 4
and 48 h.
RNase protection assay
RNA was extracted from human bone marrow aspirates using RNA-STAT (Tel-Test, Friendswood, TX) following the protocol provided by the manufacturer. Human bone marrow aspirates were obtained from Brazilians hospitalized with active visceral leishmaniasis prior to or within 48 h after initiating therapy. Normal Iowan bone marrow donors served as a source of control samples (provided by Roger Gingrich, University of Iowa, Ames, IA). The custom template, in vitro transcription kit, and RNase protection kit were obtained from BD PharMingen (San Diego, CA). Two micrograms of RNA were hybridized with [32P]UTP-labeled probes, RNase digested, and separated on a 5% polyacrylamide-urea gel using procedures recommended by the manufacturer. The intensity of the bands was quantified with a phosphorimager (Packard-Canberra, Meriden, CT) and calculated from cpm.
Immunohistochemistry
Bone marrow smears from Brazilian patients or normal donors were air-dried and fixed with 2% paraformaldehyde in PBS (20 min, 4°C). Human MDMs were infected with opsonized L. chagasi promastigotes at a 5:1 ratio and similarly fixed. Samples were blocked in 2% heat-inactivated FCS in Earle’s balanced salt solution, pH 7.4 (Sigma). After permeabilizing with 0.1% saponin buffer in Earle’s balanced salt solution, slides were incubated for 30 min with rabbit polyclonal IgG recognizing human iNOS (4 µg/ml saponin buffer; Santa Cruz Biotechnology, Santa Cruz, CA) in a humidified chamber. Inducible NOS was detected by incubating in alkaline phosphatase-labeled anti-rabbit IgG followed by alkaline phosphatase substrate plus levamisole according to the manufacturer’s protocol (Vector Laboratories, Burlingame, CA). Slides were counterstained with Nuclear Fast Red (Vector Laboratories), dehydrated, and visualized with light microscopy.
MTT assay
Promastigotes (2 x 106) in 100 µl HBSS were exposed to increasing doses of menadione (Sigma) or Sulfo-NONOate (Alexis, San Diego, CA) in 96-well plates at 26°C. All conditions were performed in triplicate. After 1 h 10% heat-inactivated FCS was added. Parasite viability was measured by incubation in 0.5 mg/ml MTT (Sigma) for 3 h, followed by addition of 100 µl 0.04 N HCl in isopropanol. Living mitochondria convert MTT to dark blue formazan that is soluble in acid-isopropanol and detectable on a microplate reader at 570 nm. The percentage of viability was calculated from OD readings in wells with menadione or Sulfo-NONOate compared with those in wells without these.
2',7'-Dichlorodihydrofluorescein diacetate (DCF) assay
The fluorescent compound DCF was used to measure the production of reactive oxygen intermediates (ROI), including O2-, during promastigote phagocytosis. Five x 106 human MDMs or murine BMMs were suspended in HBSS containing 25 µM DCF (Molecular Probes, Eugene, OR). After 45 min at room temperature, a stimulus was added. This consisted of unopsonized or opsonized L. chagasi promastigotes at a 5:1 promastigotes-macrophages ratio, 40 µg opsonized zymosan/ml, or buffer. Macrophages were added to a 96-well microtiter plate at 4°C, and the volume was adjusted to 200 µl with 25 µM DCF in HBSS. Fluorescence due to the generation of reactive oxygen species was detected at 485 nm excitation and 538 nm emission. Emissions were monitored every 30 s at 37°C for 50 min on a BMG FLUOstar 403 microplate spectrofluorometer (BMG Lab Technologies, Durham, NC) in the Cell Fluorescence Core Facility at the Iowa City Veterans Affairs Medical Center.
Measurement of O2- by ferricytochrome c reduction
O2- generated after addition of opsonized zymosan to macrophages was measured in the absence or the presence of TEMPOL. Macrophages (5 x 106) were incubated with 60 µM ferricytochrome c (Sigma). After addition of stimulus the A550 was monitored continuously (23). The reference cuvette contained the same reagents with 62.5 µg/ml SOD.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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L. chagasi promastigotes were incubated with increasing
concentrations of menadione, a redox-cycling compound that generates
O2- and
H2O2 (24, 25).
Other promastigotes were incubated with Sulfo-NONOate, which generates
NO· and has a half-life of 24 min at 25°C (26).
Both compounds had a dose-dependent toxic effect on promastigotes (Fig. 1
), indicating that L. chagasi promastigotes are
susceptible to killing by
O2-/H2O2
and NO·. The increased metabolism of MTT at low concentrations
of menadione is a consistent observation after exposure to either
menadione or H2O2 and could
reflect increased metabolic activity after oxidant exposure.
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To determine whether human and murine macrophages each produce O2- in response to L. chagasi promastigote phagocytosis, BMMs or MDMs were allowed to phagocytose serum-opsonized or unopsonized promastigotes in the presence of the spin trap DMPO. O2- produced during phagocytosis reacts with DMPO to generate DMPO-OOH, which spontaneously decays to DMPO-OH and is detectable by ESR. Negative controls included medium with DMPO alone, macrophages alone plus DMPO, or parasites alone plus DMPO. Positive control macrophages were incubated with opsonized zymosan, a potent stimulus of the respiratory burst. DTPA was included with all conditions to chelate iron contaminants that might catalyze hydroxyl radical formation (27).
Supernatants of either BMM or MDM cultures incubated with
serum-opsonized promastigotes or with opsonized zymosan showed the
characteristic 1:2:2:1 spectrum of DMPO-OH generated during the 30-min
incubation (Fig. 2). DMPO alone generated insignificant peaks and macrophages without
stimulus spontaneously generated only small amounts of
O2-. Opsonized promastigotes alone
also generated small peaks, presumably due to aerobic respiration.
Phagocytosis of opsonized promastigotes by murine BMMs consistently
resulted in a lower amplitude of DMPO-OH peaks than MDMs, reflecting a
lower amount of DMPO-OH generated. To appreciate all spectral peaks on
the same figure, the left panel of Fig. 2 shows spectra from
BMMs at a scale that was two-thirds of the MDM-derived spectra. If they
were shown in the same scale, either the MDM peak values would have
been cut off or the BMM peaks would diminish and be difficult to
visualize. A greater amount of DMPO-OH was formed by macrophages
incubated with zymosan than macrophages incubated with promastigotes
(BMM data in Fig. 2; MDM data not shown).
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Opsonization presumably alters the mechanism of receptor-mediated entry
through which promastigotes undergo phagocytosis (22, 28, 29). We questioned whether the difference between
O2- generated
during phagocytosis of opsonized or unopsonized promastigotes simply
reflected a fewer number of parasites taken up. Intracellular parasites
were quantified microscopically 30 min after addition of different
promastigote-BMM or MDM ratios. These data are plotted on the
x-axis of Fig. 3
as parasites phagocytosed per 100 macrophages. Data for the highest
parasite-macrophage ratio tested for each condition are marked.
Macrophages consistently took up more opsonized than untreated
organisms at the same parasite-macrophage ratio, and this difference
was greatest in MDMs. Plotted on the y-axis are the heights
of the second low field DMPO-OH peaks on ESR spectra, normalized so
that the arbitrary unit scales are the same in the upper and
lower panels. Two observations can be made from the figure.
First, spectral peaks generated by MDMs were consistently higher than
peaks generated by BMMs, reflecting a greater amount of
O2- generated per cell by MDMs.
Second, the amount of O2- did not
continue to rise with increasing numbers of parasites phagocytosed, but
instead peaked at a 5:1 or 10:1 ratio. These data suggest that
differences in the amount of O2-
generated after phagocytosis of opsonized vs unopsonized parasites is
not merely due to differences in the number of parasites
internalized.
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NO· detection in human and murine samples
To determine whether detectable NO· is produced during
infection of murine or human macrophages with L. chagasi, we
measured NO2- produced from
NO· released into culture supernatants of infected cultures.
IFN- is necessary for leishmanicidal activity and transcription of
iNOS. Therefore, 100 U recombinant murine IFN-/ml or 400 U
recombinant human IFN-/ml were added before measuring NO·
(9). To maximally stimulate NO· production by
murine BMMs, IFN-, IL-1, and LPS were added to some conditions.
BMMs infected with L. chagasi promastigotes plus IFN- for
72 h showed increased NO· production over the IFN-
treatment alone (Fig. 4). NO· was also detected at 48 h (Fig. 4), but not at 4
h (data not shown), after infection. Consistent with some prior
studies, nitrite resulting from NO· generation was not detected
in human MDMs infected with L. chagasi (10)
even in the presence of stimulatory cytokines. Furthermore, after fully
reducing supernatants to detect any NO· that was converted to
NO3, NO· was still not detected in human
cells stimulated with IFN-, LPS, or in the presence of IFN- and
parasites. The amounts of NO· generated by BMMs infected with
opsonized vs unopsonized promastigotes were similar.
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To document the importance of
O2- and NO· in leishmanial
killing by human and murine macrophages in vitro, infection assays were
conducted in the presence of inhibitors of both oxidant species. Human
MDMs and murine BMMs were infected with opsonized promastigotes in
the presence of TEMPOL, an O2-
scavenger that crosses cell membranes and therefore can be used to
scavenge O2- in living phagocytes
(21). Although the respiratory burst occurs within 30 min
of phagocytosis (30), intracellular parasites were
quantified after 24 h, so that viable and dead parasites could be
clearly distinguished microscopically. Incubation in 0.4 mM TEMPOL
during phagocytosis significantly increased the level of intracellular
infection of human MDMs (p < 0.05; Fig. 5A). TEMPOL did not appear to enhance phagocytosis, because
the number of parasites ingested in the presence or the absence of
TEMPOL was approximately equal 1 h after infection (data not
shown). Similarly, murine cells showed increased infection when
incubated with TEMPOL, although the differences did not reach
statistical significance (p = 0.08;
n = 4 experiments; Fig. 5A). This could be
due to the fact that murine macrophages produce less
O2- than human cells upon
phagocytosis of opsonized parasites (Figs. 2
and 3). IFN-
significantly augmented parasite killing by murine and human cells
48 h after its addition (Fig. 5B). The difference was
not apparent at 24 h (Fig. 5A), probably because an
IFN--mediated increase in microbicidal activity was not evident
at this early time point.
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The iNOS inhibitor L-NMMA has been shown to increase
leishmania survival in murine macrophages, but it has been reported
that L-NMMA does not affect parasite survival in human
macrophages (10, 31, 32). To test the importance of
NO· for intracellular killing of L. chagasi in murine
BMMs vs human MDMs, we quantified intracellular parasites after
L-NMMA addition. Parasites in murine BMMs were
quantified after infection in the presence of IFN- with or without
L-NMMA. Infected macrophages with
L-NMMA resulted in a significantly higher
parasite burden compared with untreated control BMMs at 48 h after
infection (Fig. 5B). When MDMs were preinfected with
L. chagasi for 48 h to allow their conversion to
amastigote forms, treatment with L-NMMA for a
subsequent 48 h significantly inhibited intracellular killing of
parasites by human macrophages. L-NMMA inhibited
NO· detected in BMM cultures stimulated with IFN- and LPS by
74.8 ± 2.7%, indicating that the inhibitor was active.
These data suggest that although NO· production by human
cells after L. chagasi infection is not detectable according
to the current methods available, NO· is nonetheless helping to
control L. chagasi infection.
Expression of iNOS mRNA in bone marrow aspirates from Brazilian patients with visceral leishmaniasis
To determine whether humans could produce NO· during
L. chagasi infection, we studied the expression of iNOS in
mRNA extracted from bone marrow aspirates of Brazilian patients with
visceral leishmaniasis using an RNase protection assay (Fig. 6). The two visceral leishmaniasis patients represented had their
aspirates before (lane 1) or after 1 day
(lane 2) of therapy. Aspirates from healthy Iowan
bone marrow transplant donors served as controls (lanes
3 and 4). The ratio of iNOS to GAPDH mRNA was
0.245 ± 0.007 (mean ± SD) in visceral leishmaniasis
patients vs 0.125 ± 0.035 in control samples.
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Immunohistochemistry was used to document the presence of iNOS
protein in bone marrow smears from visceral leishmaniasis patients and
in infected MDMs (Fig. 7). Inducible NOS was found in nucleated cells in the bone marrow of
patients with visceral leishmaniasis (see a representative marrow smear
in Fig. 7B; n = 4). This appeared as
discrete cytoplasmic staining in large cells from bone marrow of
infected individuals. In contrast, bone marrow smears from normal
controls displayed substantially less or no staining for iNOS compared
with patients’ smears. Shown as a positive control for the staining
procedure, the same bone marrow samples were stained for a protein that
is irrelevant to this study but is expressed in both samples, IL-12 p35
(Fig. 7, C and D).
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also stained positively
for iNOS protein. Using the same infection protocol that resulted in
L-NMMA inhibition of parasite killing (Fig. 5B),
human MDMs were cultured on slides with IFN- with or without
parasites and stained for iNOS. Staining of nuclei with the
counterstain Nuclear Fast Red is evident in Fig. 7E. MDMs
that had been exposed to parasites for 96 h and to IFN- for
48 h showed increased cytoplasmic staining for Ab to iNOS compared
with those treated with IFN- alone (Fig. 7F). Together
the RNase protection and immunohistochemistry data indicate that iNOS
is expressed by human macrophages both in vitro and in vivo during
L. chagasi infection. |
Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The kinetics of O2- vs NO·
production by macrophages in response to L. chagasi
infection are different. Prior studies using luminol-enhanced
chemiluminescence showed that a respiratory burst occurs within 1
h of initial phagocytosis of serum-opsonized L. donovani
promastigotes by human MDMs (30).
O2- has also been detected in murine
peritoneal macrophages after phagocytosis of L. donovani
(2). During the current study we detected
O2- production and an effect of
O2- scavenging early after
promastigote phagocytosis, whereas the effect of NO· occurred
later (48–72 h after infection). These observations are consistent
with a requirement for iNOS transcription and translation before
NO· generation. IFN- enhanced macrophage-mediated killing of
leishmania by either murine or human cells (see Fig. 5B, 4
vs 48 h control samples), and this effect was dramatically
reversed by inhibiting NO·-mediated parasite killing with
L-NMMA. These observations are logical, in that
IFN- is necessary for transcriptional activation of the iNOS
promoter in both human and murine cells (33).
Macrophage infection with L. donovani amastigotes has been
shown to have a profound effect on several key macrophage functions.
The presence of intracellular L. donovani amastigotes leads
to diminished PMA-stimulated respiratory burst activity, PKC
translocation and activation, and mobilization of intracellular calcium
stores (15, 16). During established amastigote infection,
the expression of MHC class I and II Ags is down-modulated, as is the
expression of the costimulatory molecule B7.2 (34). These
events could interfere with the presentation of parasite Ags to T
cells. Several sites on the NADPH oxidase
p47phox subunit are phosphorylated by protein
kinase C (35), a necessary step before oxidase activation.
Thus, amastigote infection is expected to impair both phagocytosis and
the oxidative burst. Human macrophages containing L.
donovani amastigotes are also unresponsive to IFN- stimulation
due to impaired tyrosine phosphorylation of Jak1, Jak2, and the
transcription factor Stat1 (17). Macrophage phosphatase
activity and the expression of SHP-1 are increased (36, 37). Many of the signaling events that are attenuated by
intracellular amastigote infection can also be mediated by treatment
with isolated LPG from the promastigote surface membrane (18, 19, 38). LPG can also directly scavenge oxygen-derived radicals
(39). However, because amastigotes express low to
undetectable levels of LPG, this molecule probably could not explain
all of the above observations (40). The extent to which
LPG or other parasite molecules can partially or totally abrogate
macrophage responses during initial infection with promastigotes or
during established amastigote infection is not entirely clear.
Previous work demonstrated a role for NO· in controlling
infection of murine macrophages with Leishmania sp.
(6, 8, 32, 41, 42, 43, 44). IFN--induced intracellular killing
proceeds through the intermediate formation of TNF-. The roles of
both NO· and O2- in murine
L. donovani infection have been addressed by the use of the
iNOS knockout and X-CGD mice. Mice lacking iNOS could not control
infection, whereas mice lacking gp91phox (X-CGD
mice), and therefore unable to generate a respiratory burst, exhibited
a delayed inflammatory response, but eventually controlled infection
(45). The authors concluded that reactive nitrogen
intermediates were sufficient to clear L. donovani infection
in this model, although O2-
contributes to the efficiency of parasite clearance. Our data document
the role of both oxidants in killing intracellular L.
chagasi in vitro, complementing these previous in vivo findings in
murine models. NO· is an important signal transduction
intermediate, and it is not clear whether the accelerated infection of
knockout mice was due to defects in macrophage microbicidal activity or
aberrant signaling (7).
It is of particular importance that L. chagasi survival in
human macrophages was enhanced by L-NMMA, because
there has been considerable controversy over the importance of
NO· in human macrophage microbicidal functions. Consistent with
our results, other laboratories have also had difficulty detecting
NO· in cultured human cells. This finding could be explained
either by low levels of NO· produced or by efficient
intracellular scavenging of the molecule before its release as
NO2-. Nonetheless, our ability to
detect a biological consequence of NO· formation, a finding that
contrasts with some prior reports (10), supports this
pathway as being important for the leishmanicidal activity of human
cells. The difference between the results presented here and literature
reports could be due to our use of different kinetics in infection
studies or to differences in susceptibility of different
Leishmania sp. to NO· killing. The presence of iNOS
in human MDMs cultured in the presence of IFN- and parasites
enhances the functional data found with the NO· inhibitor
L-NMMA.
Vouldoukis et al. reported that ligation of human macrophage CD23
(FcRII) with IgE elicited detectable NO· production in the
presence of L. major or L. infantum and
contributed to parasite killing, either independently or in the
presence of IFN- (11). This result could be explained
by the action of iNOS or endothelial NOS, which is known to be
activated by ligation of CD23 via up-regulation of CD11b and CD11c
(46). Although we could not repeat the finding of
NO· during L. chagasi infection of MDMs, we also
found a biological effect of iNOS inhibition on parasite survival in
the absence of CD23 ligation. As we did not use the same
Leishmania sp. as Vouldoukis, our results cannot be directly
compared. However, it is possible that the lack of effect of CD23
ligation in our system could in part be attributed to the probability
that L. chagasi infection caused a loss of surface
expression of CD23 on human macrophages, an observation made using B
cells and a macrophage cell line (47).
Both iNOS mRNA and protein were expressed in bone marrow aspirates from
Brazilians with symptomatic visceral leishmaniasis, and the level of
iNOS mRNA was greater than that in normal controls. Consistent with our
data, a prior report showed iNOS protein in a human splenic granuloma
from a patient infected with L. donovani (48).
Thus, despite the immunosuppression that occurs during human visceral
leishmaniasis, the expression of this molecule with potential
microbicidal activity was not suppressed. This observation probably
reflects the balance between factors enhancing leishmanicidal activity
(e.g., iNOS and IFN-) and factors inhibiting macrophage activation
(e.g., TGF-
and IL-10) during human infection. There is evidence in
the literature that macrophage iNOS expression is also enhanced in the
presence of other intracellular organisms. Importantly, Nicholson et
al. previously demonstrated that iNOS is expressed in alveolar
macrophages of human tuberculosis patients (12), as we
report here for visceral leishmaniasis.
It is becoming clear that oxidants act not only as anti-microbial
effectors, but also through effects on signaling and recruitment of
inflammatory cells (49). Thus, NO· has been shown
to be important not only for direct antimicrobial activity but also as
a signaling molecule promoting IL-12- and IFN-/-mediated
activation of NK cells (7). The methods used in this study
would not differentiate between a role of NO· in direct
microbicidal activity vs a role as a signaling intermediate. The same
applies to O2-, whose role in
signaling has been documented in a few situations (50, 51). Furthermore, O2- has been
implicated as a sink for NO·, potentially explaining why it is
not detected in human macrophages that produce more
O2- (44). The exact
molecular mechanisms through which these radicals promote intracellular
killing of leishmania have yet to be fully defined.
Our data support a model in which intracellular leishmania killing is
effected initially through complement opsonization and stimulation of
O2- generation during the initial
phagocytosis event in both murine and human macrophages. Once
intracellular, the macrophage becomes quiescent, and amastigotes
replicate. Late intracellular killing can occur through activation of
macrophages by lymphocyte-derived factors, including IFN-, invoking
a mechanism that requires NO· in both murine and human cells.
Among other possibilities, the greater amount of NO· detected in
supernatants of murine cells could reflect a greater importance of this
effector molecule in leishmanicidal activity of murine as opposed to
human cells. Nonetheless, our data support a model in which both human
and murine macrophages use similar pathways, albeit to differing
extents, to effect killing of this obligate intracellular parasite.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Mary E. Wilson, Department of Internal Medicine, University of Iowa, SW34-GH, 200 Hawkins Drive, Iowa City, IA 52242. E-mail address: mary-wilson{at}uiowa.edu
3 Abbreviations used in this paper: L-NMMA, NG-monomethyl-L-arginine; TEMPOL, 4-hydroxy-2,2,6,6-tetramethylpiperidine-N-oxyl; MDM, monocyte-derived macrophage; BMM, bone marrow-derived macrophage; DCF, 2',7'-dichlorodihydrofluorescein diacetate; LPG, lipophosphoglycan; DTPA, diethylenetriaminepentacetic acid; ROI, reactive oxygen intermediates; iNOS, inducible NO synthase; SOD, superoxide dismutase; ESR, electron spin resonance.
Received for publication December 18, 2000. Accepted for publication May 11, 2001.
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) or human MDMs (
) were pretreated
with medium alone or medium containing 400 U (human) or 100 U (murine)
IFN-
) or with (
