Modulation of Interferon- Phosphotyrosine Phosphatases Inhibition -induced Macrophage Activation by

Modulation of Interferon- Phosphotyrosine Phosphatases Inhibition -induced Macrophage Activation by
THE JOURNAL OF BIOLOGICAL CHEMISTRY
© 1998 by The American Society for Biochemistry and Molecular Biology, Inc.
Vol. 273, No. 22, Issue of May 29, pp. 13944 –13949, 1998
Printed in U.S.A.
Modulation of Interferon-g-induced Macrophage Activation by
Phosphotyrosine Phosphatases Inhibition
EFFECT ON MURINE LEISHMANIASIS PROGRESSION*
(Received for publication, September 10, 1997, and in revised form, February 22, 1998)
Martin Olivier,a,b,c,d,e Bertha-Judith Romero-Gallo,a,b,f Claudine Matte,a Julie Blanchette,a
Barry I. Posner,g,h Michel J. Tremblay,a,d,e,g and Robert Fauree,i,j
From the aCentre de Recherche en Infectiologie and Département de Biologie Médicale, iUnité de Recherche en
Neuroscience and Département de Médecine, Centre Hospitalier Universitaire de Québec, Pavillon CHUL, Faculté de
Médecine, Université Laval, Ste-Foy (Québec) Canada G1V 4G2, and hDepartment of Medicine, McGill University,
Montréal,Canada H3A 1A1
Phagocyte functions are markedly inhibited after infection with the intracellular protozoan parasite Leishmania. This situation strongly favors the installation
and propagation of this pathogen within its mammalian
host. Previous findings by us and others have established that alteration of several signaling pathways
(protein kinase C-, Ca21- and protein-tyrosine kinasesdependent signaling events) were directly responsible
for Leishmania-induced macrophage (MØ) dysfunctions. Here we report that modulation of phosphotyrosine-dependent events with a protein tyrosine phosphatases (PTP) inhibitor, the peroxovanadium (pV)
compound bpV(phen) (potassium bisperoxo(1,10phenanthroline)oxovanadate(Vi)), can control hostpathogen interactions by different mechanisms. We observed that the inhibition of parasite PTP resulted in an
arrest of proliferation and death of the latter in coincidence with cyclin-dependent kinase (CDK1) tyrosine 15
phosphorylation. Moreover the treatment of MØ with
bpV(phen) resulted in an increased sensitivity to interferon-g stimulation, which was reflected by enhanced
nitric oxide (NO) production. This enhanced IFN-g-induced NO generation was accompanied by a marked
increase of inducible nitric oxide synthase (iNOS)
mRNA gene and protein expression. Finally we have
verified the in vivo potency of bpV(phen) over a 6-week
period of daily administration of a sub-toxic dose. The
results revealed its effectiveness in controlling the progression of visceral and cutaneous leishmaniasis. Therefore PTP inhibition of Leishmania and MØ by the pV
compound bpV(phen) can differentially affect these eukaryotic cells. This strongly suggests that PTP plays an
* The costs of publication of this article were defrayed in part by the
payment of page charges. This article must therefore be hereby marked
“advertisement” in accordance with 18 U.S.C. Section 1734 solely to
indicate this fact.
b
Contributed equally to this work.
c
Supported by funds from the Medical Research Council of Canada.
To whom correspondence should be addressed: Centre de Recherche en
Infectiologie, RC-709, Centre Hospitalier Universitaire de Québec,
Pavillon CHUL, 2705 boul. Laurier, Ste-Foy (Québec), Canada G1V 4G2.
Tel.: 418-654-2705; Fax: 418-654-2715; E-mail: martin.olivier@
crchul.ulaval.ca.
d
Members of a Medical Research Council group grant in infectious
diseases.
e
Hold Junior 2 scholarship award from the Fonds de la Recherche en
Santé du Québec.
f
Recipient of a studentship from the Ministère de la Science et de
l’Éducation du Québec.
g
Supported by Medical Research Council grants.
j
Holds a grant from the Natural Sciences and Engineering Research
Council of Canada.
important role in the progression of Leishmania infection and pathogenesis. The apparent potency of pV compounds along with their relatively simple and versatile
structure render them attractive pharmacological
agents for the management of parasitic infections.
Parasitic protozoa of the order Kinetoplastidae are the causative agents of several subtropical and tropical diseases including leishmaniasis. This infection is estimated to affect more
than 15 million people around the world with 400,000 new
cases/year (1). Leishmania donovani, the causative agent of
visceral leishmaniasis, is often fatal if left untreated, whereas
other Leishmania species are mainly responsible for cutaneous
and mucocutaneous afflictions. The incidence of leishmaniasis
is rising because of increased traveling, the lack of vaccines,
difficulty in controlling vectors, and an increase in resistance to
chemotherapy (2). In addition to Leishmania (3–7), numerous
potentially deadly intracellular pathogens, such as Yersinia (8),
human immunodeficiency virus (9, 10), and others can promote
mononuclear phagocyte dysfunctions that inhibit the ability of
these cells to elicit an effective immune response, which may
favor persistent infection. We previously reported that several
of these Leishmania-induced macrophage (MØ)1 dysfunctions
were related in part to the alteration of Ca21- and protein
kinase C-dependent signaling pathways (3, 4). More recently, it
has been demonstrated that dysregulation of protein-tyrosine
kinase (PTK)-dependent signaling events in L. donovani-infected MØ (11)2 could also account for the inhibition of several
PTK-regulated MØ functions (i.e.. IFN-g-inducible MØ major
histocompatability complex class II expression) (3, 4, 7, 12–18).
It is necessary for cells that both the protein tyrosine phosphatases (PTP) and PTK maintain their physiological balance
to sustain a normal regulation of their Tyr(P)-dependent
events. It was thus of interest to determine if changes in the
PTP/PTK homeostatic balance could lead to protection against
leishmaniasis.
Peroxide of vanadium (pV, a mixture of vanadate and H2O2)
is an insulinomimetic agent and potent inhibitor of PTP (re-
1
The abbreviations used are: MO
y , macrophage; iNOS, inducible nitric oxide synthase; pV, peroxovanadium; phen, phenanthroline; bpV(phen), potassium bisperoxo(1,10-phenanthroline)oxovanadate; PTK,
protein-tyrosine kinase; PTP, protein tyrosine phosphatase; Vi, sodiumorthovanadate; L-NMMA, L-NG-monomethylarginine; IFN, interferon;
PBS, phosphate-buffered saline; PI, propidium iodide; Tyr(P),
phosphotyrosine.
2
M. Olivier, J. Blanchette, R. Faure, N. Racette, and K. Siminovitch,
unpublished data.
13944
This paper is available on line at http://www.jbc.org
Protein Tyrosine Phosphatases and Leishmania Pathogenesis
13945
FIG. 1. Proliferation of L. donovani
grown in presence of pV derivatives.
A, parasites were grown in the presence
or absence of Vi, bpV(phen), bpV(bipy),
bpV(pic), bpV(HOpic) over a 6-day period.
Results shown are representative of a
minimum of four experiments. 0 (close
square), 0.1 mM (open diamond), 1 mM
(close circle), 5 mM (open circle), 10 mM
(close triangle). Effects obtained with
bpV(HOpic) and bpV(bipy) (not shown)
were similar to those of bpV(pic). B, L.
donovani promastigotes were grown in
the presence or absence of bpV(phen) (10
mM) and vanadate (10 mM) for 48 –72 h,
and their cell cycle distribution was analyzed by flow cytometry. Results are representative of a minimum of three experiments. Treatment with pV compounds
did not lead to DNA nick formation as
evaluated by a K1-SDS assay (37) (data
not shown). C, inhibition of the PTP activity in L. donovani treated with PTP
inhibitors. Results are the mean 6 S.E. of
three experiments performed in quadruplicate. Similar levels of inhibition were
observed from 1 to 24 h post-treatment.
D, CDK1 hyperphosphorylation in L. donovani treated with bpV(phen). Upper
panel, log phase promastigotes were
treated with Vi or bpV(phen) at a concentration of 10 mM for 0, 2, 4, and 6 h. CDK1
(P34) hyperphosphorylation was assessed
by immunoblotting of cell lysates using a
phosphospecific CDK1 antibody. Increase
in CDK1 tyrosyl phosphorylation was
120 –150% for bpV(phen) over untreated
cells and 30 – 40% over untreated cells for
vanadate-treated parasites at 6 h as determined by scanning densitometry of the
autoradiographs. Lower panel, Leishmania CDK1 tyrosyl phosphorylation in response to bpV(phen) was followed over a
48-h period to correlate with the cell cycle.
Experiments were performed at least two
times. Co, unstimulated cells.
viewed in Ref. 19). It was demonstrated that a number of
chemically defined pV derivatives, each containing an oxo ligand, one or two peroxo anions in the inner coordination sphere
of vanadium, and an ancillary ligand, were equally potent PTP
inhibitors stable in aqueous solution (20) that can activate the
insulin receptor kinase and mimic insulin biological action in
vivo (21). Moreover, they have the capacity to inhibit the proliferation of nervous cell lines in vitro (22) and activate the
response of immune cells (23).
Protein tyrosine phosphorylation events are also playing an
important role in the regulation of Kinetoplastidae growth (24,
25), and PTP activities were previously detected in L. donovani
promastigote extracts (26). Several findings also support the
pivotal role of PTK-dependent signaling in agonist-induced MØ
functions including cytokine-induced nitric oxide (NO) generation (12–16). PTP could also play a pivotal role in Leishmania
pathogenesis since several important immune functions necessary for the development of a protection against leishmaniasis
are PTK-regulated, and their Leishmania-induced inhibitions
are correlated with host signaling alterations (3–7, 11). Thus,
we have evaluated the effectiveness of bpV(phen) in vivo in
controlling the development of the cutaneous lesions and inflammation of the hind footpad in BALB/c mice induced by
Leishmania major infection. The effect of similar in vivo PTP
inhibition on the development of visceral leishmaniasis has
also been evaluated. The present study firmly establishes that
the pV compound bpV(phen) can modulate both Leishmania
and MØ cellular physiology to effect protection against
leishmaniasis.
EXPERIMENTAL PROCEDURES
Materials—Isotopes were obtained from ICN Pharmaceuticals Canada Ltd. (Montréal, QC, Canada). Recombinant murine IFN-g (2 3 105
units/ml) was purchased from Life Technologies, Inc. Phosphospecific
CDK1 antibody was purchased from New England Biolabs (Beverly,
MA). The antiphosphotyrosine antibody (clone 4G10) was purchased
from Upstate Biotechnology Inc. (UBI, Lake Placid, NY), and the inducible nitric oxide synthase (iNOS) antibody was from Cedarlane
(Hornby, ON, Canada). The iNOS inhibitor L-NG-monomethylarginine
(L-NMMA) was purchased from BioMol (Plymouth Meeting, PA). The
peroxovanadium complexes (PTP inhibitors) used in this study are
K(VO(O2)2phen)z3H2O, bpV(phen); K(VO(O2)2bipy)z5H2O, bpV(bipy);
K2(VO(O2)2pic)zH2O, bpV(pic); K2(VO(O2)23-OHpic)zH2O, bpV(OHpic)
were synthesized as we previously described (20). Sodium orthovanadate (Vi) was purchased from Sigma. BALB/c and C57BL/6 (6 – 8-weeksold female, 20 –30 body weight) were purchased from Charles River
(St-Constant, QC, Canada).
Cell Culture—Leishmania promastigotes were grown at room temperature and maintained in the laboratory by weekly transfers in
SDM-79 culture medium as described previously (17, 27). For specific
experiments, parasites were transferred (5 3 106 log phase promastigotes in 100 ml) into 10 ml of fresh SDM-79 culture medium in the
presence or absence of PTP inhibitors. The growth of the parasites was
followed over 6 days by measuring the absorbance at 610 nm using an
automated microplate reader (Organon Teknika). The murine macro-
13946
Protein Tyrosine Phosphatases and Leishmania Pathogenesis
FIG. 2. Modulation of murine MØ functions by bpV(phen). A, NO production by the J774 cell line (5 3 105cell/well) cultured in the presence
or absence of Vi (V) or bpV(phen) (1 h) before IFN-g (100 units/ml). Results are the mean 6 S.E. of three separate experiments. *, NO production
was significantly increased (p , 0.05) in comparison to control (cells solely stimulated with IFN-g). L-NMMA (5 mM) treatment reduced NO
generation by more than 75%. bpV(phen) treatment per se (10 mM) induced NO generation (.15 mM). B, effects of bpV(phen) in vitro on
Leishmania-infected murine MØ. Parasitic load was significantly reduced (p , 0.05; mean 6 S.E. of four experiments) by 5 (;45% reduction) and
10 (;70% reduction) mM treatments after 24 h in culture in comparison to untreated and vanadate-treated cells. L-NMMA (5 mM) and bpV(phen)
were added to cell cultures simultaneously. C, pattern of Tyr(P) proteins in MØ after treatment with bpV(phen). Cells were incubated in 24 well
dishes (106 cells/well) for 1 h with 10 mM of bpV(phen) or Vi and subjected to Western blotting. MØ Tyr(P) protein levels were revealed using an
anti-phosphotyrosine antibody. Phosphorylation levels were maximal 1–2 h post-stimulation and were dose-dependent (data not shown). Results
are representative of four experiments similarly performed. MW, Mr. D, tyrosine kinase activity in Vi- and bpV(phen)-treated-murine MØ. J774
cells (5 3 105) were incubated in 24-well dishes with Vi (●) or bpV(phen) (E) (10 mM) over a 1-h period at 37 °C. PTK activity in cell lysates was
assessed by their capacity to phosphorylate poly(Glu-Tyr). The capacity of bpV(phen) to inhibit MØ PTP activities by ;90% (data not shown) was
similar to that attained in bpV(phen)-treated parasites (Fig. 1C). E, IFN-g-induced intracellular tyrosyl phosphorylation in bpV(phen)-treated
phagocytes. Cells were treated as above and further treated with IFN-g (100 units (U)/ml) for 30 – 60 min. Levels of Tyr(P) (Y) proteins were
monitored by flow cytometry in permeabilized MØ incubated with an anti-phosphotyrosine antibody. Results represent the mean 6 S.E. of three
experiments. F, effect of bpV(phen) treatment on MØ iNOS gene expression in response to IFN-g stimulation. Cells were treated as above and
further stimulated with IFN-g for an 8-h period. RNA isolated from cells was subjected to Northern analysis to evaluate iNOS gene expression.
Expression of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene was also evaluated as the control for equal RNA loading. The level of
iNOS protein in cells treated for 24 h was monitored by Western blotting (i.b.) using an anti-iNOS antibody (a-iNOS). Results are representative
of three experiments independently performed.
phage cell line J774 was maintained in Dubelcco’s modified Eagle’s
medium (Life Technologies, Inc.) supplemented with 10% fetal calf
serum 1 streptomycin (100 mg/ml) and 2 mM L-glutamine at 37 °C and
5% CO2. All cells mentioned above and used in this study were obtained
from the American Type Culture Collection (Manassas, VA).
Flow Cytometric Cell Cycle Analysis—107 pre-stationary parasites
were transferred into 10 ml of fresh SDM-79 culture medium in the
presence or absence of Vi (10 mM) and bpV(phen) (10 mM). After 48 –72 h,
106 promastigotes were collected, washed with phosphate-buffered saline (PBS) (pH 7.4), and fixed for 1 h in 1 ml of 70% methanol, PBS. The
cells were resuspended in PBS containing 10 mg/ml of RNase A (20 min
at 37 °C), labeled with propidium iodide (PI, 50 mg/ml; Sigma), and
analyzed using a Coulter EPICS 753 pulse cytometer (Hialeah, FL) to
estimate the DNA content of each cell.
Western Blotting—Cells were collected (106–107), lysed in buffer containing 20 mM Tris-HCl (pH 8.0), 0.14 M NaCl, 10% glycerol (v/v), 1%
Nonidet P-40 (v/v), 25 mM nitrophenyl guanidinobenzoate, 10 mM NaF, 1
mM Vi, 25 mg/ml leupeptin and aprotinin. The lysates (20 mg/lane) were
submitted to SDS-polyacrylamide gel electrophoresis, the separated
proteins were transferred on a polyvinylidene difluoride membrane
(Millipore). Membranes blocked overnight in Tris-buffered saline/
Tween containing 1% gelatin were washed and incubated with an
Protein Tyrosine Phosphatases and Leishmania Pathogenesis
FIG. 3. Protection by bpV(phen) treatment of mice against
Leishmania infections. A, L. major-infected BALB/c mice were subjected to daily saline (Control), Vi (oV), bpV(phen), and phenanthroline
(phen) intraperitoneal injections over a period of 6 weeks. Infection is
expressed as the net increase in hind footpad volume (mm3). Treatments performed at 0.5 mmol/30 g (100 nM) of body weight were ineffective to control the progression of the L. major infection (data not
shown). Doses $5 mmol/30 g (1 mM) were toxic for the animals. B, this
photograph represents typical L. major cutaneous lesions illustrating
the reduced foot pad inflammation and the total inhibition of lesion
development observable in animals treated with bpV(phen) (2.5
mmol/30 g; 500 nM). C, L. donovani infection in C57BL/6 mice receiving
saline (control) or bpV(phen) treatments. At 1 and 2 weeks post-infection, the parasitic load (L. donovani units) in the infected organ was
monitored as described previously (38). In all in vivo experiments, each
time point represents the mean value obtained for five animals and are
representative of two experiments separately performed. *, reductions
of footpad inflammation, lesion development (L. major), or liver parasitic load (L. donovani) from bpV(phen)-treated animals were significantly different (p , 0.05) compared with controls.
13947
anti-phosphotyrosine antibody (clone 4G10; UBI), washed with Trisbuffered saline/Tween, incubated with anti-mouse horseradish peroxidase-conjugated antibody (Life Technologies, Inc.), and developed using
ECL Western blotting detection system (Amersham Pharmacia Biotech). In addition, iNOS antibody has been used to reveal the level of
expression of MØ iNOS protein in cells treated or not with PTP inhibitors and IFN-g. Leishmania CDK1 hyperphosphorylation was assessed
using a phosphospecific CDK1 antibody recognizing the yeast and human CDK1 (P34) Tyr-15 phosphorylated residue. The doublet signal
was revealed according to the manufacturer’s protocol (New England
Biolabs).
Nitric Oxide (NO) Production—Macrophages were seeded in 24-well
dishes (5 3 105cell/well) and cultured in the presence or absence of Vi or
bpV(phen) for 1 h, IFN-g (100 units/ml) was then added, and the cells
were further incubated for 24 h. The iNOS inhibitor L-NMMA (BioMol)
has been used in some experiments at a concentration of 5 mM. The NO
production was evaluated by measuring the accumulation of nitrite in
the culture medium as described previously (28).
PTP Activity Determination—Cells were grown in SDM-79 culture
medium in the presence or absence of Vi or bpV(phen) (10 mM). After 6 h,
107 cells were collected, rinsed 3 times in serum-free medium, resuspended, and disrupted in a buffer containing 50 mM Tris-HCl (pH 7.0 at
25 °C), 0.1 mM EDTA, 0.1 mM EGTA, 0.1% b-mercaptoethanol (v/v), 25
mg/ml aprotinin, and 25 mg/ml leupeptin. The PTP activity was determined in total cell preparation by measuring the dephosphorylation of
32
P-labeled poly(Glu-Tyr) (Glu/Tyr ratio, 4:1). In some experiments,
PTP activity was also monitored over a 24-h period (data not shown).
Poly(Glu-Tyr) was phosphorylated by partially purified insulin receptor
kinase from rat hepatic endosomes as described previously (20).
PTK Activity Measurement—J774 cells (5 3 105) incubated in 24-well
dishes in the presence of Vi or bpV(phen) (10 mM) over a 1-h period at
37 °C were lysed in buffer containing 1 M Tris-HCl (pH 8.0), 3 M NaCl,
100% glycerol, 10% Nonidet P-40, 0.5 M NaF, 50 mM nitrophenyl guanidinobenzoate, and protease inhibitors (5 mM aprotinin and leupeptin).
After 1 h on ice with gentle mixing, the lysate was spun at 15,000 3 g
for 30 min at 4 °C in a microfuge. The phosphorylation reaction was
initiated by the addition of a reaction mixture (25 mM ATP in 50 mM
Hepes (pH 7.4), 40 mM MgCl2, 2.5 mg/ml synthetic substrate, poly(GluTyr) (4:1) (Sigma), and 5 mM ([g-32P])ATP (New England Biolabs) to a
total volume of 100 ml]. After incubation (10 min at 22 °C), the reaction
was terminated by spotting 50 ml of reaction solution onto Whatman
No. 3MM square paper (2.5 3 2.5 cm). The paper was extensively
washed with 10% trichloroacetic acid containing 10 mM sodium pyrophosphate with anhydrous ethanol for 10 min, air-dried, and counted
(LKB rackbeta) using universol (ICN).
Northern Blot Analysis—Expression of gene iNOS in Vi- (10 mM)- and
bpV(phen)-treated (10 mM, 1 h) and -untreated J774 cells in response to
IFN-g-stimulation (100 units/ml, 8 h) was evaluated by a Northern blot
of total mRNA, as we described previously with some modifications (29).
Briefly, after incubation under appropriate conditions, cells were
washed twice with Hepes-buffered saline solution, and total RNA was
extracted using TRIzol reagent (Life Technologies, Inc.). Ten to 20
micrograms of RNA were loaded onto 1% agarose gels, and equal loading and RNA integrity were confirmed by ethidium bromide staining.
RNA was then transferred onto Hybond-N filter paper and hybridized
with random primer-labeled cDNA probes. Equal loading of RNA was
also confirmed by hybridization with glyceraldehyde-3-phosphate dehydrogenase cDNA probe. All washes were performed under stringent
conditions. The mRNA hybridizing with the cDNA probe was visualized
by autoradiography. Probes have been kindly provided by Dr. Danuta
Radzioch from the Montreal General Hospital Research Center (McGill
University, Montréal, Québec, Canada).
Determination of Total MØ Phosphotyrosyl Phosphorylation by Intracellular Flow Cytometry—Evaluation of IFN-g-induced MØ intracellular phosphotyrosine contents was performed by flow cytometry as we
previously described (23). Briefly, J774 cells (5 3 105) were treated with
PTP inhibitors (10 mM, 1–2 h) and further stimulated or not with IFN-g
(100 units/ml, 10 min). Next, MØ were washed in PBS (pH 7.4), pelleted, and fixed with 25 ml of reagent A (Fix & Perm cell permeabilization kit from CALTAG Laboratories, South San Francisco, CA) for 15
min at room temperature. After further PBS washing, cells were resuspended in reagent B (25 ml) in the presence of the 4G10 anti-phosphotyrosine monoclonal antibody (UBI) and incubated at room temperature
for 15 min. Cells were subsequently washed with PBS and 1% NaN3
and resuspended with 100 ml of PBS containing a fluorescein isothiocyanate-labeled goat anti-mouse (1 mg total) and further incubated for
15 min at room temperature. Finally, cells were centrifuged and resus-
13948
Protein Tyrosine Phosphatases and Leishmania Pathogenesis
TABLE I
Liver and popliteal lymph node parasitic loads of L. major-infected mice
Popliteal lymph nodes and livers from BALB/c mice infected with L. major (6 weeks post-infection) were collected. Organ impression smears,
giemsa staining, and parasite enumeration have been performed as previously described (Olivier and Tanner (38)).
Treatment
Popliteal lymph node
Liver
nM
Leishmania unitsa
Leishmania unitsa
Control
Vanadate, 100
Vanadate, 500
bpV(phen), 100b
bpV(phen), 500b
Phenanthroline, 100
Phenanthroline, 500
9315 6 1875
6164 6 1627
4041 6 1350
1500 6 175
550 6 43
5140 6 1600
3560 6 2100
8479 6 1700
3014 6 1800
10960 6 2500
1917 6 300
060
8150 6 800
3630 6 1800
% of
visceralization
66
66
100
33
0
66
66
Number of amastigotes/1000 cells 3 by organ weight (mg).
Level of infection was significantly reduced in comparison with values obtained for control (P , 0.05 as determined by student’s t test). Data
are expressed as the mean 6 S.D. and are representative of values obtained for 3– 6 mice/group.
a
b
pended in 1% paraformaldehyde in PBS before being analyzed by flow
cytometry (EPICS XL, Coulter Corporation, Miami, FL).
In Vivo Protozoan Infections and pV Treatments—The mice were
injected daily with Vi, bpV(phen), or phenanthroline (2.5 mmol/30 g (500
nM) of body weight, intraperitoneal injection) 2 days before their infection with parasites and further injected daily for a period of 2– 6 weeks
post-infection. In different experimental groups, animals were inoculated with L. major (106–5 3 106 stationary phase promastigotes, subcutaneous injection in hind footpad) and L. donovani (107 stationary
phase promastigotes, injected intravenous in tail vein). For the in vitro
infection, MØ cultures were incubated for 6 h with L. major and L.
donovani stationary phase promastigotes at a 20:1 parasite to cell ratio
as described previously (17). After several washes, infected cells were
further cultured for 24 h in the presence or not of Vi or bpV(phen) at a
concentration of 5–10 mM. Pretreatment of cells before infection did not
further increase the reduction of parasitic load at time point 24 h (data
not shown).
Statistical Analyses—Statistically significant differences between
groups were performed with the analysis of variance (ANOVA) module
of SAS software (version 6.07, SAS Institute, Cary, NC) using the
Fisher least significant difference test. P values ,0.05 were considered
to be statistically significant (P values are given in the figure legends).
All data are presented as mean 6 S.E.
RESULTS AND DISCUSSION
Attenuation of MØ and other host immune functions by
Leishmania infection has been correlated with alterations of
several signaling pathways including PTK activation (3–7, 11–
18). Involvement of PTK-dependent events in the regulation of
cell proliferation, including that of protozoan parasites, and in
the modulation of immune cell functions has been increasingly
recognized (14, 24, 25, 30). Modulation of these cellular processes using PTP inhibitors has been widely studied in a range
of in vitro systems including activation of T lymphocytes (23).
With the exception of experiments in animal diabetes, few
studies have tested the in vivo effects of PTP inhibitors, including the recently characterized pV compounds (20), on the modulation of PTK-dependent cellular events. In this study, we
have thus evaluated the role of PTP in the development of
murine leishmaniasis using the pV compound bpV(phen) to
inhibit both the parasite and host cell PTP activities.
We measured the effects of vanadate and different pV compounds on the growth of L. donovani promastigotes (Fig. 1A).
Only bpV(phen) was found to inhibit L. donovani growth in a
dose- and time-dependent manner. No inhibition by vanadate
or other pV compounds tested (bpV(pic), bpV(OHpic),
bpV(bipy)) was observed, showing that the nature of the ancillary ligand is important. Similar effects have been observed on
the growth of several Leishmania species including L. major
(data not shown). The inhibitory effect of bpV(phen) on L.
donovani was observed at a dose of 10 mM and was characterized by an increase in the number of cells at the SG2/M phase
of the cell cycle (Fig. 1B) when compared with vanadate and
untreated parasites. In parallel, PTP activity was measured
(20) in whole Leishmania extracts using 32P-labeled poly GluTyr as a substrate. As shown in Fig. 1C, incubation of L.
donovani for 6 h with 10 mM bpV(phen) reduced parasite PTP
activity by more than 90%. Similar levels of inhibition were
measurable from 1 to 24 h after treatments (data not shown).
One direct effect of this inhibition was further documented by
the Tyr-15 hyperphosphorylation of CDK1 observed upon incubation with bpV(phen) (Fig. 1D). This showed that CDK1 is an
endogenous target. Vanadate, albeit less efficient, was also able
to inhibit PTP activity in these conditions and induced a slight
increase in CDK1 phosphorylation. This observation may in
part explain the absence of Leishmania growth inhibition in
response to vanadate treatment in vitro. These results are in
accordance with the report of Morla et al. (31) concerning the
hyperphosphorylation of CDK1 by vanadate (50 mM) in 3T3
cells and the observation of Faure et al. (22) on the effect of
bpV(phen) to promote the inhibition of mitosis by blocking
progression at the SG2/M interphase in coincidence with CDK1
hyperphosphorylation and loss of catalytic activity. These observations suggest that, as in mammalian cells (22, 31), the
protein tyrosine phosphatase Cdc25 is an important endogenous target in protozoan cells.
Leishmania can inhibit several MØ functions to improve its
survival and propagation. It is also well established that phagocytes play a key role in controlling Leishmania infection in vivo
by secreting molecules such as NO (32), which are regulated by
Tyr(P)-dependent events (14). We thus evaluated the effect of
bpV(phen) on MØ functions since an inhibition of MØ PTP
activities may contribute to increased responsiveness toward
cytokine stimulation. The effects of bpV(phen) treatment on
murine MØ responsiveness to IFN-g are shown in Fig. 2A. NO
production was significantly increased in bpV(phen)-treated
cells over untreated or vanadate-treated MØs at various doses
and inhibitable by the iNOS inhibitor L-NMMA. Similarly,
IFN-g-stimulated bpV(pic)-treated MØ has generated NO levels comparable to that of bpV(phen)-treated cells (data not
shown). In addition, the inhibition of MØ PTK by genistein led
to an almost complete abrogation of inducible NO production as
previously reported by others (14). These results established
that PTP inhibition by bpV(phen) can modulate events in cells
as different as the pathogen Leishmania and its host cell, the
macrophage. We then assessed whether this bpV(phen) dual
effect may attenuate parasite persistence within its host cells.
As shown in Fig. 2B, bpV(phen) at a dose of 10 mM can effectively reduce the parasitic load of Leishmania-infected MØ by
more than 75% in comparison to control cells. In addition, we
observed that the NO inhibitor L-NMMA almost completely
Protein Tyrosine Phosphatases and Leishmania Pathogenesis
reversed this bpV(phen)-mediated protection against Leishmania infection, suggesting that NO is a key player in pV-mediated leishmanicidal activity. MØ PTP activities, as for Leishmania, were substantially inhibited by bpV(phen) (data not
shown). This was paralleled by augmented levels of Tyr(P)
proteins in MØ (Fig. 2C), which may result from enhanced MØ
PTK activity (Fig. 2D). Total MØ Tyr(P) protein levels were
further induced by IFN-g stimulation as revealed by intracellular flow cytometry determinations (Fig. 2E). In addition,
bpV(phen) treatment enhanced iNOS gene mRNA and iNOS
protein expressions both in the basal state and in response to
IFN-g stimulation in comparison to their respective controls
(Fig. 2F). Altogether, these observations document the capacity
of bpV(phen) to favor PTK-dependent signaling and to prime
MØ for enhanced responsiveness toward stimulants. Regulation of iNOS has been previously reported to involve the participation of the transcription factor NF-kB (33). Our recent
observations that several pV compounds can strongly induce
NF-kB nuclear translocation in lymphoid and monocytoid cells
(23) support the hypothesis that nuclear translocation of
NF-kB is involved in the effect of bpV(phen) to increase iNOS
mRNA expression. Furthermore, the activity of iNOS may be
directly influenced by tyrosine phosphorylation (34).
In view of this, experiments were done to determine whether
bpV(phen) (21) could modulate the course of the infection in a
murine leishmaniasis model (32). Infection by L. major, the
causative agent of cutaneous leishmaniasis, was inhibited by
60% (p , 0.05) in bpV(phen)-treated BALB/c mice (Fig. 3, A and
B). As shown in Fig. 3A, mice treated daily with bpV(phen) (2.5
mmol/30 g (500 nM) of body weigh, intraperitoneal injection)
over a 6 week period showed significantly reduced footpad
inflammation compared with control, vanadate, and phenanthroline-treated groups. The bpV(phen) treatment not only reduced the inflammation of the footpads but also completely
blocked the development of the cutaneous lesion (Fig. 3B). The
remarkable reduction of these typical features of cutaneous
leishmaniasis was further evidenced by the almost complete
disappearance of parasite from the popliteal lymph node and
complete absence of hepatic involvement (Table I). The effect of
bpV(phen) on the course of murine visceral leishmaniasis was
also tested and found to be even more striking. As noted in Fig.
3C, bpV(phen) was capable of completely reducing the liver
parasitic load at two weeks post-infection, whereas hepatic
infestation of control C57BL/6 was still at peak levels. These
data strongly suggest that in vivo modulation of PTK/PTP
balance by pV compounds (35) can restrict and favor regression
of leishmaniasis in mammals.
Various pathogens, including viruses (36) and bacteria (8),
exploit signaling systems modulated by PTP for their growth
and pathogenesis. Furthermore, cellular defenses utilize killing strategies modulated by PTP (14, 32). It is thus plausible
that the inhibition of PTP could influence the course of various
infections (e.g. Yersinia). Presently, we cannot say with certainty the extent to which the in vivo protective and curative
effects of bpV(phen) are due to direct actions on the growth of
the parasite or on the potentiation of MØ functions. Nevertheless our in vitro experiments strongly suggest the pivotal role
played by NO in the control of Leishmania infection (Fig. 2, A
and B). Indeed, we have evidence that NO is effectively the key
molecule that restrains the progression of infection in pVtreated animals, since bpV(pic) was similarly capable as bpV(phen) to significantly abolish L. major-induced footpad inflam-
13949
mation and lesion development.3
In conclusion, the results of the present study emphasize the
important role that modulation of PTP plays in the development of Leishmania infection. Our findings highlight the fact
that the apparent potency of pV compounds, along with their
relatively simple and versatile structure, may represent a new
avenue for the development of novel therapeutic agents against
parasitic infections.
Acknowledgments—We thank André Marette for its critical review of
this paper and Danuta Radzioch for iNOS and glyceraldehyde-3-phosphate dehydrogenase cDNA probes.
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M. Olivier, C. Matte, J. F. Marquis, P. L. Janvier, and P. Gros,
manuscript in preparation.
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