monitoring of intracellular nitric oxide in leishmaniasis: its applicability in patients with...

Monitoring of Intracellular Nitric Oxide in

Leishmaniasis: Its Applicability in Patients

with Visceral Leishmaniasis

Avijit Sarkar,1 Piu Saha,1 Goutam Mandal,1 Debanjan Mukhopadhyay,1 Susmita Roy,1

Sunny Kumar Singh,2 Sukhen Das,2 R. P. Goswami,2 Bibhuti Saha,2 Deepak Kumar,3 Padma Das,3

Mitali Chatterjee1*

� AbstractNitric oxide (NO) has been demonstrated to be a principal effector molecule respon-

sible for mediating intracellular killing of Leishmania parasites, the causative orga-

nism of leishmaniasis. As measurement of intracellular NO remains a challenge to

biologists, we have developed a flow cytometric approach to perform real time bio-

logical detection of NO within Leishmania parasites and parasitized macrophages

using a membrane permeable derivative of diaminofluorescein [4,5-diaminofluores-

cein diacetate (DAF-2DA)]. Initially, assay optimization was performed in Leishmania

donovani promastigotes, assay specificity being confirmed using both a NO donor

[S-nitroso-N-acetyl-penicillamine (SNAP)] and a NO scavenger [2-(4-carboxy-

phenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide, C-PTIO]. Using 40 lM DAF-

2DA, basal levels of intracellular NO were measured which varied in different Leish-

mania species; addition of conventional anti-leishmanial drugs, antimony and milte-

fosine translated into a dramatic increase in DAF-2T fluorescence. Furthermore, the

assay also measured levels of NO in macrophages, but needed a 20 fold lower concen-

tration of DAF-2DA, being 2 lM. Following parasitization, levels of NO decreased

which was normalized following treatment with anti-leishmanial drugs. Similarly

monocytes of patients with visceral leishmaniasis at disease presentation showed

decreased levels of NO which too reverted on completion of treatment. Taken to-

gether, this study opens new perspectives of research regarding monocyte function

and provides a real time approach for monitoring the effect of anti-leishmanial com-

pounds. ' 2010 International Society for Advancement of Cytometry

� Key termsanti-leishmanial; DAF-2DA; flow cytometry; leishmaniasis; nitric oxide

LEISHMANIA are intracellular protozoan parasites that infect host mononuclear

phagocytes, the resultant complex of diseases being leishmaniasis affecting 12 million

people world wide in 88 countries (1). This disease is characterized by development

of dermal lesions that may be cutaneous leishmaniasis (CL), diffuse cutaneous leish-

maniasis (DCL), or mucocutaneous leishmaniasis (MCL) as also there can be sys-

temic involvement as in the case of visceral leishmaniasis (VL), which is potentially

fatal if left untreated (2).

This obligate intracellular parasite resides and multiplies within macrophages,

and therefore it should be able to evade the cytotoxic mechanisms innately operative

within the macrophages for its survival (3). One of the predominant cytotoxic

mechanisms includes production of reactive nitrogen intermediates, which the Leish-

mania parasite effectively decreases to ensure its survival (4,5).

1Department of Pharmacology, Instituteof Post Graduate Medical Education andResearch, Kolkata 700 020, India2Department of Tropical Medicine,School of Tropical Medicine, Kolkata 700073, India3Department of Cell Biology andPhysiology, Indian Institute of ChemicalBiology, Kolkata 700 032, India

Received 5 July 2010; Revision Received22 September 2010; Accepted 29 October2010

Grant sponsor: Department of Scienceand Technology (DST), Indian Council ofMedical Research (ICMR) and Council forScientific and Industrial Research (CSIR),Government of India.Present address of Goutam Mandal:College of Medicine, Florida InternationalUniversity, Miami, Florida 33199.

*Correspondence to: Mitali Chatterjee,Department of Pharmacology, Instituteof Postgraduate Medical Education andResearch, 244 B Acharya JC Bose Road,Kolkata 700 020, India

Email: [email protected]

Published online 13 December 2010 inWiley Online Library(wileyonlinelibrary.com)

DOI: 10.1002/cyto.a.21001

© 2010 International Society forAdvancement of Cytometry

Original Article

Cytometry Part A � 79A: 35�45, 2011

Considering the biological relevance of NO in leishmani-

asis, its estimation would be very informative, but its direct

measurement to date, remains a challenge, attributable to its

short half-life and low concentration (6). Methods for detect-

ing NO include trapping of NO with hemoglobin, chemilumi-

nescence assays, electron paramagnetic resonance spectros-

copy, or using electrochemical electrodes (7). Other indirect

methods include estimation of nitric oxide synthase (NOS) ac-

tivity, protein and mRNA expression of NOS (8) as also esti-

mation of nitrate/nitrite levels. Unfortunately, these methods

are limited by their inability to define the population of inter-

est and analyze intracellular events at a single cell level. To

address these issues, fluorescent indicators of intracellular NO

have been developed and include aromatic diamino derivatives

(2,3-diaminonaphthalene), 1,2-diaminoanthaquinone, 4,5-

diaminofluorescein (DAF), 4-amino-5-methylamino-20,70-difluorofluorescein, diaminorhodamine-4M. Amongst these,

4,5-diaminofluorescein diacetate (DAF-2DA) is the most sen-

sitive and is widely used (7).

In Leishmania parasites, their antioxidant defenses are rela-

tively compromised owing to the absence of catalase and classi-

cal selenium-dependent glutathione peroxidases (9), thus mak-

ing them overly reliant upon a relatively less efficient, unique

trypanothione dependent antioxidant system (10,11) along

with two iron containing superoxide dismutases, which only

marginally protect them from free radical mediated damage

(12,13). Exploiting the impaired antioxidant capacity of Leish-

mania parasites, triggering of oxidative stress (reactive oxygen

and nitrogen species) seems a logical chemotherapeutic modal-

ity. Indeed, sodium antimony gluconate, the main stay of treat-

ment for leishmaniasis (14), generates free radicals within para-

sites (15). Studies revealed that orthologues of NOS (neuronal

NOS or nNOS) are present in Leishmania (16,17) and its activ-

ity was measured by the NADPH consumption assay (18). In

this study, we have established a flow cytometry based method

to measure production of NO in parasites after treatment with

conventional anti-leishmanial drugs.

As Leishmania parasites reside within macrophages,

survival within this hostile environment hinges on its ability

to modulate various macrophage-related immune functions

(19,20), including alteration of reactive nitrogen species (3).

Balestieri et al. (21) have reported decreased production of

NO in Leishmania infected macrophages following inhibition

of inducible NOS (iNOS). Consequently, it would be interest-

ing to establish a method that can measure alterations in levels

of NO in Leishmania infected macrophages. In view of the

critical role of NO in leishmaniasis, the objectives of this study

were to establish the feasibility of DAF-2DA as a fluorescent

probe for flow cytometric measurement of NO both in Leish-

mania parasites, parasitized murine macrophages, and mono-

cytes from patients with VL.

MATERIALS AND METHODS

Reagents

All chemicals were of analytical grade and obtained from

Sigma Aldrich Chemicals (St Louis, MO) except CD14-PE

(Immunotools, Friesoythe, Germany), 4,5- diaminofluorescein

diacetate (DAF-2DA) and 2-(4-carboxyphenyl)-4,4,5,5-tetra-

methylimidazoline-1-oxyl-3-oxide, (C-PTIO, Cayman Chemi-

cals, Ann Arbor, Michigan). A stock solution of DAF-2DA

(5 mM) was prepared in dimethylsulfoxide (DMSO), stored at

2208C, and diluted immediately before use.

Parasite Culture

Promastigotes representing several Leishmania species

responsible for VL, CL, and MCL from Old and New world

were included (Table 1). Promastigotes were routinely cul-

tured at 248C in Medium 199 (M199) supplemented

with 10% heat inactivated fetal calf serum (FCS), penicillin

G (50 IU/ml), and streptomycin (50 lg/ml), referred to as

Medium A. For experimental purposes, log phase promasti-

gotes were subcultured every 72–96 h, inoculum being 1 3106/ml.

For parasitization of macrophages, 24 h prior to the

experiment, stationary phase promastigotes (4–5 days old cul-

ture) were centrifuged (1,000g 3 10 min) and preconditioned

Table 1. Basal levels of NO as measured in Leishmania spp

SPECIES DESIGNATION CODE DISEASE LOCATION GMFC (DAF-2T)

L. donovani MHOM/IN/1980/DD8 DD8 Visceral Old world 114.21 � 11.32

L. donovani MHOM/IN/83/AG83 AG83 Visceral Old world 29.90 � 3.40

L. infantum MHOM/TN/1980/IPT1 IPT1 Visceral Old world 216.90 � 13.62

L. chagasi MHOM/BZ/1974/PP75 PP75 Visceral New world 207.60 � 21.43

L. major MHOM/SU/1973/5ASKH 5ASKH Cutaneous Old world 187.80 � 13.85

L. tropica MHOM/AZ/1974/SAF-K27 K27 Cutaneous Old world 157.70 � 7.96

L. amazonensis MHOM/BZ/1973/M2269 M2269 Cutaneous/diffuse New world 255.90 � 2.88

L. panamensis MHOM/PA/1971/LS94 LS94 Cutaneous New world 168.90 � 4.91

L. mexicana MHOM/MX/2006/ENCB-MIC MIC Cutaneous New world 346.00 � 24.25

L. braziliensis MHOM/BZ/1975/M2903 M2903 Mucocutaneous New world 40.46 � 5.64

Fluorescence analyzed as described in Materials and methods. Data are expressed as GMFC � SEM of at least three independent

experiments in duplicate.

ORIGINAL ARTICLE

36 Intracellular NO in Leishmaniasis

by resuspending in Schneiders insect medium, pH 5.5 supple-

mented with 20% FCS (22).

Animals

BALB/c mice of either sex (average weight, 25–30 gm)

were maintained at standard temperature (25 � 58C), a 12 h

day/night cycle, fed a standard pellet diet, and provided water

ad libitum. All experimental protocols received prior approval

from the Institutional Animal Ethical Committee.

Isolation of Murine Peritoneal Macrophages

Peritoneal macrophages were lavaged from BALB/c mice

using sterile, chilled phosphate buffered saline (0.02 M, pH

7.2, PBS); following centrifugation (400g 3 10 min), the re-

sultant cell pellet was washed twice with PBS and resuspended

in complete RPMI-1640 phenol red free medium, supplemen-

ted with 10% fetal calf serum (FCS); cell viability ([95%) was

confirmed by Trypan blue dye exclusion.

Study Population

Patients clinically diagnosed with VL (n 5 6) were

admitted to the Dept. of Tropical Medicine, School of Tropical

Medicine, Kolkata. Diagnosis of VL was based on clinical fea-

tures and corroborated by rK39 strip test or ELISA for anti-

leishmanial antibodies (23). Heparinized blood was collected

from age and sex matched healthy volunteers (n 5 5, nonen-

demic for VL) and patients with VL, both at presentation and

after completion of treatment. Treatment comprised a single

dose of liposomal amphotericin B, 7.5 mg/kg body weight, i.v.

followed by miltefosine, 2.5 mg/kg body weight, p.o. for 2

weeks. The study was approved by the Institutional Ethical

Committee (School of Tropical Medicine, Kolkata) and

informed consent was obtained.

Isolation of Peripheral Blood Mononuclear

Cells (PBMC)

PBMC were isolated as previously described (24); briefly,

peripheral blood was carefully layered over Ficoll-Hypaque

(1:1, Histopaque-1077) and after centrifugation (400g 3 30

min), the PBMC-rich interface was washed twice in PBS and

resuspended in RPMI-1640 medium supplemented with peni-

cillin (100 U/ml), streptomycin (100 lg/ml), and 10% FCS;

cell viability was confirmed using trypan blue ([95%).

Measurement of DAF-2DA Based Fluorescence in

Leishmania PromastigotesTo measure NO generated in L. donovani promastigotes

(DD8) by flow cytometry, using DAF-2DAwas used for which,

the following parameters were established:

Optimal concentration of DAF-2DA. Log phase L. donovani

promastigotes were incubated in the dark with increasing con-

centrations of DAF-2DA (0–60 lM) for 45 min at 378C and

resultant fluorescence measured in the FL1 channel and ana-

lyzed on a FACS Calibur (Becton Dickinson).

Optimal temperature and incubation time. Leishmania pro-

mastigotes are routinely cultured at 248C, but as generation of

free radicals is maximal at 378C (15), we evaluated whether

changes in NO are best determined at 24 or 378C, as also

established the optimal incubation time. Accordingly, cells

were incubated in the dark with DAF-2DA (40 lM) at either

24 or 378C and fluorescence was monitored up to 120 min.

Selection of medium. To identify the appropriate incubation

medium, cells were stained with DAF-2DA (40 lM) in serum

free solutions namely (a) PBS, (b) M199, (c) RPMI-1640 with

phenol red (RPMI PR1), (d) RPMI-1640 without phenol red

(RPMI), and (e) DMEM. Cells were then incubated in the

dark at 378C for 45 min and fluorescence was quantified.

Establishment of assay specificity. To confirm that fluores-

cence of DAF-2T is a specific measure of intracellular NO,

parasites (1 3 106/ml) were incubated with a NO donor,

SNAP [250 lM (25)] for 1 h at 378C in PBS followed by label-

ing in the dark with DAF-2DA (40 lM) and fluorescence

measured. Assay specificity was also confirmed using a NO

scavenger, C-PTIO (26) wherein parasites (1 3 106/ml) were

treated with a non toxic concentration of C-PTIO (100 lM,

378C for 3 h) and DAF-2T fluorescence measured; parasite

viability was [95% based upon propidium iodide (PI)

exclusion.

Effect of Anti-Leishmanial Drugs on Intracellular

Levels of NO in Promastigotes

Log phase L. donovani promastigotes (AG83, 1 3 106/ml)

were incubated for 3 h with potassium antimony tartarate

[300 lg/ml, Sb(III)] or miltefosine (0.25 lg/ml) at 378C in se-

rum free medium A. The concentrations of anti-leishmanial

drugs selected did not affect cell viability (as checked by PI

exclusion) and gave maximum generation of NO. The cells

were then washed with PBS, incubated with DAF-2DA and an-

alyzed for DAF-2T fluorescence; parasite viability was con-

firmed based upon PI exclusion. To confirm that NO was

being generated after treatment with antimony and miltefo-

sine, promastigotes were preincubated for 1 h with a NO scav-

enger, C-PTIO at 100 lM (being the optimum concentration

that showed highest scavenging activity yet was not cytotoxic

as measured by PI exclusion) and similarly processed as

described above.

Additionally, these cells were concomitantly observed

under a laser scanning confocal microsope (Leica TCS SP2

system, Leica microsystem, Heidelberg, Germany, objective

1003); briefly, cells were attached on poly-L-lysine coated

slides and mounted in glycerol containing 1,4-diazabicy-

clo[2.2.2]octane; at least 20 microscopic fields were observed.

To substantiate the role of NO in mediating the leishma-

nicidal activity of conventional anti-leishmanial drugs, pro-

mastigotes were incubated with Sb(III) or miltefosine in the

absence or presence of C-PTIO, a NO scavenger and chemo-

sensitivity was determined by a modified 3-(4,5-dimethylthia-

zol-2-yl)-2, 5-diphenyl tetrazolium bromide (MTT) assay

(27). Briefly, log phase L. donovani promastigotes (5 3 104

ORIGINAL ARTICLE

Cytometry Part A � 79A: 35�45, 2011 37

cells/ well) were incubated with Sb(III), 0–200 lg/ml or milte-

fosine (0–12.5 lg/ml) in the presence or absence of a nontoxic

concentration of C-PTIO (100 lM) in a final volume of 50 llof Medium A. After 48 h incubation at 248C, MTT (5 mg/ml

stock in PBS, 50 ll) was added and plates incubated overnight

at 248C. A solution of sodium dodecyl sulphate (10% SDS)

and isopropyl alcohol (50%) was added and kept at 37oC for

6 h. After microscopical confirmation that all crystals had dis-

solved, the optical density at 550 nm (OD550) was determined

using a plate reader. The specific OD550 for each well was cal-

culated by subtracting the OD550 of medium containing anti-

mony � C-PTIO. The mean percent viability was calculated as

Specific OD550 of drug treated cells

Specific OD550 of control cells3100

Specific OD550 of Control Cells

The results were expressed as the IC50, i.e., the concentra-

tion that inhibited 50% cell growth as enumerated by graphi-

cal extrapolation using GraphPad Prism software (version 4).

Determination of Intracellular NO in L. donovaniInfected Macrophages

Murine peritoneal macrophages (1 3 106/ ml of complete

RPMI-1640 phenol red free medium supplemented with 10%

FCS) were seeded in 6 well plates and incubated at 378C, 5%CO2 for 3–4 h to allow macrophages to adhere. The superna-

tants (containing lymphocytes) were gently removed and kept

aside, while the adherent cells were infected with precondi-

tioned, virulent L. donovani promastigotes (AG83) at a macro-

phage: parasite ratio of 1:10 for at least 5 h at 378C, 5% CO2.

After removal of non-internalized parasites, infected macro-

phages were cocultured with supernatants containing lympho-

cytes in the presence of sodium antimony gluconate (SAG,

10 lg/ml). After 24–48 h, the adherent cells were scraped,

washed and resuspended in PBS, labeled with DAF-2DA

(2 lM) for 30 min at 378C and fluorescence was acquired.

Determination of Intracellular NO in Monocytes of

Patients with VL

PBMC (1 3 106) were centrifuged (800g 3 5 min) and

cells resuspended in a minimal volume of PBS containing 2%

FCS and fluorochrome (PE) conjugated anti-human CD14.

Following incubation for 15 min at room temperature, cells

were centrifuged, washed twice, resuspended in PBS, and

stained (1 3 106/ml) with DAF-2DA (2 lM, 28); following a

30 min incubation at 378C, fluorescence was acquired on a

flow cytometer.

Flow Cytometry

Cells (1 3 106/ml) from different experimental groups

were monitored for their intracellular fluorescence on a flow

cytometer (FACS Calibur, Becton Dickinson, CA) equipped

with an argon-ion laser (15 mW) tuned to 488 nm. Fluores-

cence of the triazolic product (DAF-2T) was collected in the

FL1 channel, equipped with a 530/30 nm band pass filter, PI

in the FL2 channel having a 585/42-nm band pass filter. Fluo-

rescence was measured in the log mode using CellQuest Pro

software (BD Biosciences, CA) and expressed as geometrical

mean fluorescence channel, i.e., average or central tendency of

fluorescence of analyzed particles (GMFC). To measure DAF-

2T fluorescence in monocytes, cells were plotted on the basis

of their characteristic linear forward and side scatter, further

gated with CD14 positivity, and then DAF-2T fluorescence

was measured on a logarithmic scale in the FL1 channel. Ac-

quisition was performed on 10,000 gated events. Data analysis

was done using CellQuest Pro software (BD Biosciences, CA).

Statistical Analysis

Each experiment was performed at least thrice in dupli-

cates and results expressed as mean � standard error of the

mean (SEM). Statistical analysis was evaluated by T test or one

way ANOVA followed by Tukey’s multiple comparison test

(wherever applicable), using Graph Pad Prism software, ver-

sion 4 (GraphPad Software Inc, San Diego, CA); P\ 0.05 was

considered as statistically significant.

RESULTS AND DISCUSSION

Standardization of DAF-2DA Labeling in LeishmaniaPromastigotes

DAF-2DA has previously been used for measurement of

NO in a variety of mammalian cells including monocytes, neu-

trophils (28,29) human spermatozoa (30), and endothelial cells

(31). DAF-2DA has also effectively been used in Zebrafish Danio

rerio (32) and the parasite Schistosoma mansoni (33), but the sta-

tus of NO within Leishmania parasites has not been established.

The flow cytometric detection of NO is a promising alternative

and is based on the ability of DAF-2DA to freely penetrate the

cytoplasm, where it is converted into 4,5-diaminofluorescein

(DAF-2), and following its oxidation by NO, leads to formation

of triazolofluorescein (DAF-2T), which by virtue of its green flu-

orescence can be quantified in a flow cytometer (34).

The assay was initially optimized in L. donovani promas-

tigotes (DD8) using increasing concentrations of DAF-2DA

(up to 60 lM). With increasing concentrations, a dose de-

pendent increase in fluorescence was observed up to 40 lM,

GMFC being 4.21 (1 lM), 39.45 (10 lM), 71.43 (20 lM), and

118.66 (40 lM), after which the increase was marginal as

GMFC with 60 lM was 126.66 (Fig. 1A); accordingly, 40 lMwas selected. Importantly, this concentration is at least four-

fold higher than the concentration required to measure levels

of intracellular NO in mammalian cells which ranged from 0.1

to 10 lM (8,28,29,35). This may be attributed to inherently

lower amounts of NO in the parasite or altered esterase activ-

ity or even variations in their membrane permeability.

A similar scenario was evident when reactive oxygen species

were measured with dichlorodihydrofluorescein diacetate

(H2DCFDA) in L. donovani promastigotes that required 50

lM (15) whereas in macrophages, 20 lM was adequate (36).

To establish optimal temperature and time, promastigotes

were incubated with DAF-2DA (40 lM) at 248C and 378C up

to 120 min. At 248C, fluorescence was increased marginally up

ORIGINAL ARTICLE


to 45 min, and remained so up to 120 min; at 378C, the base-line fluorescence was higher in comparison to 248C, increasedup to 45 min, and was maintained up to 120 min (Fig. 1B).

Accordingly, incubation for 45 min was selected as optimal

time. The consistently higher fluorescence obtained at 378C(Fig. 1B) was possibly due to enhanced esteratic cleavage of

DAF-2DA and higher generation of NO. Therefore, promasti-

gotes for all subsequent studies were incubated at 378C for 45

min and no loss in cell viability was confirmed by absence of

PI positivity (data not shown).

To exclude the possibility of culture medium interfering

with fluorescence (37), promastigotes were incubated with

DAF-2DA in serum free PBS, M199, RPMI with or with out

phenol red and DMEM. As cells incubated in PBS gave a 2.8-

to 9.9-fold higher fluorescence (Fig. 1C), it was used subse-

quently for all experiments. These differences may be attribu-

ted to components of the media potentially capable of

quenching the fluorescence.

The specificity of DAF-2DA for NO has previously been

confirmed with different donors of NO and inhibitors of NOS

in mammalian cells (33–35,38) and we corroborated the same

in Leishmania promastigotes. A 4.9-fold increase in base line

fluorescence was observed following incubation with SNAP

(570.86 � 11.58 vs. 114.21 � 11.32, Fig. 1D) indicating that the

assay effectively monitored NO in Leishmania parasites.

Furthermore, incubation of promastigotes with C-PTIO (100

lM) showed an almost twofold depletion of NO (62.30 � 4.00,

P \ 0.01) as compared to control parasites validating that

Figure 1. Assay optimization for flow cytometric measurement of NO using DAF-2DA. A: Log phase L. donovani promastigotes (1 3 106/

ml) were incubated at 378C with increasing concentrations of DAF-2DA (0, 1, 10, 20, 40, and 60 lM) for 45 min and GMFC measured asdescribed in Materials and methods. B: Log phase L. donovani promastigotes (1 3 106/ml) were incubated with DAF-2DA (40 lM) at 248C(n) or 378C (~) up to 120 min and GMFC obtained as described in Materials and methods. C: Log phase L. donovani promastigotes (1 3106/ml) were incubated with DAF-2DA (40 lM) in PBS (1) or different serum free media [M199 (2), RPMI-PR1 (3), RPMI (4), or DMEM (5)] for

45 min and GMFC obtained as described in Materials and methods. Data are expressed as mean GMFC � SEM of at least 2 independent

experiments in duplicate. D: A representative histogram of log phase L. donovani promastigotes [1 3 106/ml (1) unstained] incubated in

the absence (2) or presence (3) of S-Nitroso-N-acetyl-DL-penicillamine (250 lM, SNAP) for 1 h at 378C followed by labeling with DAF-2DA(40 lM) and GMFC measured as described in Materials and methods.

ORIGINAL ARTICLE


DAF-2T fluorescence is contributed primarily by intracellular

NO generated in Leishmania parasites. Evidence suggests that

DAF-based probes can also react with oxidized derivatives of

NO, e.g., peroxynitrite [ONOO2 (29,38)], reductants [dehy-

droascorbic acid, ascorbic acid, dithiothreitol, 2-mercaptoetha-

nol, and glutathione (39,40)] divalent cations [mainly calcium,

(41)], and endogenous nitroso/nitrosyl species [i.e., nitro-

sothiols, nitrosamines, and heme-NO products, (29)], which

could account for the residual DAF-2T fluorescence present af-

ter treatment with C-PTIO.

Basal Levels of Intracellular NO Among

Leishmania spp.NOS enzyme in the native form exists as a dimer in Leish-

mania parasites (17) and has been characterized in both forms

of the parasite (16,18,42). The presence of NOS activity

prompted us to measure levels of intracellular NO in different

Leishmania spp. Results showed that L. mexicana (MIC) and

L. amazonensis (M2269) gave the highest DAF-2T fluores-

cence, GMFC � SEM being 346.00 � 24.25 and 255.90 �2.88, respectively, whereas L. donovani (AG83) and L. brazi-

liensis showed the lowest GMFC being 27.87 � 1.96 and 40.46

� 5.64, respectively, (Table 1). It would be interesting to study

whether this variation in intracellular NO influences their sen-

sitivity to antimony, akin to a study in L. donovani field iso-

lates, where variable responses to hydrogen peroxide and

Sb(III) correlated with their chemosensitivity profiles (15).

Effect of Anti-Leishmanial Drugs upon

Generation of NO

Antioxidant defenses of Leishmania parasites are rela-

tively compromised due to the absence of catalase and classical

Figure 2. Generation of intracellular NO by Sb(III) and miltefosine in L. donovani promastigotes. A: Log phase L. donovani promastigotes(1 3 106/ml) were incubated with Sb(III) (300 lg/ml) or miltefosine (0.25 lg/ml) for 3 h at 378C in the absence (&) or presence (n) of C-PTIO(100 lM) followed by labeling with DAF-2DA (40 lM); fluorescence was measured as described in Materials and methods. Data areexpressed as mean GMFC � SEM of at least 3 independent experiments in duplicate. B: Log phase L. donovani promastigotes [1 3 106/ml,

(1)] were treated (378C, 3 h) with [Sb(III), 300 lg/ml, (2)] or miltefosine [0.25 lg/ml, (3)], labeled with DAF-2DA (40 lM) and fluorescence wasanalyzed by laser scanning confocal microscopy as described in Materials and methods. (a) and (b) represent fluorescence and phase con-

trast panels, respectively. Bar size is 12 lm. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

ORIGINAL ARTICLE


selenium dependent glutathione peroxidases, rendering them

susceptible to free radical damage (9,43). Importantly, any

compound with pro-inflammatory activity could potentially

be a good anti-leishmanial agent, as is the case with SAG, the

conventional anti-leishmanial drug (44). Therefore, as NO is

the main effector molecule for intracellular parasite elimina-

tion, one could extrapolate that compounds capable of gener-

ating NO in Leishmania parasites and/or host macrophages

could contribute towards the leishmanicidal activity. Indeed,

studies have shown that antimony exerts its anti-leishmanial

effect by generating ROS/ RNS and depleting thiols both

within parasites and macrophages (15,37,44,45). As maximal

generation of ROS without affecting cell viability was achieved

with Sb(III), 300 lg/ml for 3 h (15), its addition resulted in a

6.1-fold increase in DAF-2T fluorescence from baseline values,

GMFC � SEM being 182.60 � 3.31 vs. 29.90 � 3.40, P \0.001 (Fig. 2A); pre-incubation with C-PTIO, a known NO

scavenger prevented the Sb(III) mediated increase in fluores-

cence (Fig. 2A).

Miltefosine is an established anti-leishmanial drug that

causes parasite apoptosis, possibly attributable to increased gen-

eration of free radicals. Indeed, addition of miltefosine (0.25

lg/ml) caused a significant increase in NO as the baseline

GMFC (29.90 � 3.40) dramatically increased 10.3 fold to

308.20 � 14.79 (P \ 0.001); pretreatment of miltefosine with

C-PTIO decreased the GMFC by 1.7-fold to 180.80 � 6.13 (P

\ 0.01, Fig. 2A). Although our study confirmed that NO con-

tributes towards the pro-oxidant activity of Sb(III) and miltefo-

sine, it is possibly not the sole determinant. It may also be envi-

saged that other than NO, anti-leishmanial drugs are generating

oxidant derivatives of NO such as peroxynitrite (ONOO-) or

nitroso/nitrosyl species which cannot be scavenged by C- PTIO.

For fluorescence imaging, parasites were treated with

Sb(III) or miltefosine followed by DAF-2DA; in control cells,

no visible fluorescence was observed (Fig. 2B) whereas treat-

ment with Sb(III), 300 lg/ml, 3 h caused a bright green fluo-

rescence (Fig. 2B), which was even more pronounced with

miltefosine (0.25 lg/ml, 3 h, Fig. 2B). This data corroborated

our flow cytometric analysis wherein miltefosine triggered a

greater increase in fluorescence as compared to Sb(III). Speci-

ficity of the signal was confirmed by pretreatment of promasti-

gotes with a NO scavenger (C-PTIO) or a NOS inhibitor [N

(G)-nitro-L-arginine methyl ester, L-NAME, data not shown].

Effect of Removal of NO by C-PTIO upon

Leishmanicidal Activity of Antimony and Miltefosine

To establish whether generation of NO by Sb(III) or mil-

tefosine was pivotal towards its leishmanicidal activity, pro-

mastigotes were incubated with Sb(III) in the presence or ab-

sence of a nontoxic concentration of C-PTIO, an established

NO scavenger. In the absence of C-PTIO, Sb(III) had an IC50

of 79.60 lg/ml, which following co-incubation with C-PTIO

increased 1.9-fold to 152.33 lg/ml. However, with miltefosine,

C-PTIO (100 lM) failed to influence the IC50 as it remained

unchanged at 2.75 lg/ml. Upon increasing the concentration

of C-PTIO to 150 lM, it marginally increased the IC50 by

1.45-fold to 4.0 lg/ml, collectively suggesting that NO within

parasites is one of the many reactive species generated by anti-

leishmanial drugs (Fig. 3A and 3B).

Parasitized Macrophages had Decreased

Levels of NO that was Normalized by SAG

To survive and propagate within the not so conducive envir-

onment of macrophages, the protozoan parasite Leishmania

Figure 3. Role of NO in mediating leishmanicidal activity of Sb(III) and miltefosine in L. donovani promastigotes. Log phase L. donovanipromastigotes (5 3 104 cells in 50 ll/ well) were incubated with [Sb(III), 0—200 lg/ml, -n-, (A)] or miltefosine [0—12.5 lg/ml, -n-, (B)] alongwith C-PTIO (-~-) for 48 h. Cell viability was measured by the MTT assay as described in Materials and methods and each data point repre-sents the mean � SEM value of at least three experiments in duplicate.

ORIGINAL ARTICLE


Figure 4. Measurement of intracellular NO in parasitized macrophages. A: A representative dot plot of uninfected (a,d) and L. donovaniinfected (b,e) murine peritoneal macrophages. Cells were gated on the basis of characteristic linear forward and side scatter features of

macrophages and DAF-2T fluorescence was measured on a logarithmic scale in the FL1 channel at 24 and 48 h. A representative histogram

(c,f) of DAF-2T fluorescence from uninfected (---) and Leishmania infected (��) macrophages. B:Murine peritoneal macrophages (1 3 106/

mL) were infected with L. donovani and incubated with SAG (10 lg/ml) for 24 h (&) and 48 h (n); adherent cells were scraped, washed, andresuspended in PBS containing DAF-2 DA (2.0 lM) for 30 min at 378C and fluorescence measured as described in Materials and methods.Data are expressed as the mean GMFC � SEM of at least three experiments in duplicate.

ORIGINAL ARTICLE


cleverly inhibits several macrophage functions, including produc-

tion of reactive nitrogen intermediates (3,46). Mammalian cells

require only 2.0 lM DAF-2DA for measurement of intracellular

NO (28,29), whereas Leishmania parasites need a 20 fold higher

concentration of DAF-2DA. Importantly, measurable amounts of

fluorescence were detectable only after adding [10 lM DAF-

2DA (Fig. 1A). Therefore, 2.0 lM of DAF-2DA (as used in unin-

fected or Leishmania infected macrophages) would not be able to

detect changes (if any) in levels of NO within intracellular amas-

tigotes. To establish whether DAF-2DA could monitor changes in

levels of NO, the assay was applied in uninfected and Leishmania

infected macrophages. Interestingly, distinct morphological

changes (mainly internal granularity) were observed in Leishma-

nia infected macrophages (Fig. 4A) due to internalization of

parasites and formation of parasitophorous vacuoles. In unin-

fected macrophages, the GMFC was 56.57 � 2.23 which follow-

ing 24 h of parasitization significantly decreased to 32.53 � 3.38

(P \ 0.01); the addition of SAG (10 lg/ml) reverted levels of

NO, GMFC being 55.00 � 1.69 (Fig. 4B).

In uninfected macrophages, the basal levels of NO at

48 h, increased to 72.22 � 1.57. Peritoneal macrophages

when cultured tend to continuously generate a small pro-

portion of reactive species over time, which possibly the

assay was sensitive enough to detect. The degree of parasi-

tization was more prominent at 48 h, as basal levels of

NO decreased 2.78-fold, GMFC being 25.97 � 1.23 (P \0.001); here again, addition of SAG (10 lg/ml) increased

generation of NO, as the GMFC increased to 59.10 � 2.09

(P \ 0.001, Fig. 4B). We confirmed that this fluorescence

was not contributed by internalized parasites, as no meas-

urable fluorescence was obtained when L. donovani pro-

mastigotes (AG83) were treated with Sb(III) using 2 lM

Figure 5. Detection of intracellular NO in CD141monocytes from patients with VL. PBMC (1 3 106/ml) isolated from patients with VL and

healthy controls were initially labeled with anti human CD14-PE, followed by DAF-2DA (2.0 lM, 378C) for 30 min. Cells were gated on thebasis of characteristic linear forward and side scatter features (A) of monocytes. Monocytes were further gated with anti-human CD14-

PE (B), and then DAF-2T fluorescence was measured on a logarithmic scale in the FL1 channel. A representative histogram profile (C) for

DAF-2T fluorescence from CD14-monocytes of a patient with VL at presentation (1), on completion of treatment (2) and a healthy donor

(3). Before and after plots (D) of intracellular NO in patients with VL at presentation (n 5 4, n) and after completion of treatment (n 5 4,

~).

ORIGINAL ARTICLE


DAF-2DA, i.e., the concentration used for staining macro-

phages (data not shown). In uninfected macrophages, no

significant changes were observed in DAF-2T fluorescence

following addition of SAG (Fig. 4B).

Levels of Intracellular NO were Decreased in CD14

Positive Monocytes of Patients with VL and Reverted

with Treatment

All six patients with VL (M:F 5 2:1, aged between 17 and

75 yrs) presented with typical features of the disease, mainly

fever (duration being 2.20 � 0.58 months, range 1–4 months),

weight loss and hepatosplenomegaly along with typical labora-

tory abnormalities including hypergammaglobulinemia and

pancytopenia. These patients tested positive by the rK39 dip-

stick assay and assessment of cure was based on remission of

clinical features.

It is well established that internalization of amastigotes

via phospatidylserine receptors on macrophages induces

secretion of IL-10 and TGF-b, which in turn blocks induc-

tion of iNOS, resulting in decreased production of NO (5).

Studies in the classical hamster model of VL also showed

decreased expression of iNOS mRNA, accounting for defec-

tive parasite elimination, thereby enhancing disease pro-

gression (47). Indeed serum nitrite levels are also decreased

in active VL (48), and our study corroborated that intracel-

lular NO in macrophages of patients with VL (n 5 6) are

significantly decreased as compared to healthy individuals

(n 5 5), GMFC being 38.88 � 25.44 vs. 212.70 � 15.71 (P

\ 0.05, Fig. 5C). After treatment (n 5 4), levels of intracel-

lular NO reverted to near normal values, GMFC being

185.90 � 34.54 and included four matched pairs (i.e.

presentation and post treatment of the same individual,

Fig. 5D).

To the best of our knowledge, this is the first report where

intracellular NO has been directly measured in monocytes of

patients with VL. This method can also be used to monitor

the changes in intracellular NO in macrophage related dis-

eases. Taken together, this study has established DAF-2DA can

serve as a sensitive and specific probe for flow cytometric esti-

mation of NO in Leishmania promastigotes and macrophages,

and could be an effective tool for assessing the effect of anti-

leishmanial drugs.

ACKNOWLEDGMENTS

Dr. Lionel F Schnur, Dept. of Parasitology, Hadassah

Medical School, Jerusalem, Israel kindly provided us with the

Leishmania strains.

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