From the Institute of Biochemistry, University of
Lausanne, 1066 Epalinges, Switzerland and ¶ Department of
Biophysical Chemistry, Biozentrum, University of Basel, 4056 Basel,
Switzerland
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ABSTRACT |
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Leishmania, a protozoan parasite of
macrophages, has been shown to interfere with host cell signal
transduction pathways including protein kinase C
(PKC)-dependent signaling. Myristoylated alanine-rich C
kinase substrate (MARCKS) and MARCKS-related protein (MRP, MacMARCKS) are PKC substrates in diverse cell types. MARCKS and MRP are thought to
regulate the actin network and thereby participate in cellular responses involving cytoskeletal rearrangement. Because MRP is a major
PKC substrate in macrophages, we examined its expression in response to
infection by Leishmania. Activation of murine macrophages by cytokines increased MRP expression as determined by Western blot
analysis. Infection with Leishmania promastigotes at the time of activation or up to 48 h postactivation strongly decreased MRP levels. Leishmania-dependent MRP depletion
was confirmed by [3H]myristate labeling and by
immunofluorescence microscopy. All species or strains of
Leishmania parasites tested, including
lipophosphoglycan-deficient Leishmania major
L119, decreased MRP levels. MRP depletion was not obtained with other
phagocytic stimuli including zymosan, latex beads, or heat-killed
Streptococcus mitis, a Gram-positive bacterium. Experiments
with [3H]myristate labeled proteins revealed the
appearance of lower molecular weight fragments in
Leishmania-infected cells suggesting that MRP depletion may
be due to proteolytic degradation.
The ability of various intracellular pathogens including
Leishmania to inhibit macrophage effector activities, also
termed "deactivation", is well documented (1, 2). Functional
alterations in Leishmania-infected macrophages include
decreases in cytokine production, oxidative burst activity, antigen
presentation, and expression of major histocompatibility complex class
II genes in response to interferon
(IFN)1- MARCKS and MARCKS-related protein (MRP), also known as MacMARCKS
(Macrophage-MARCKS), are members of a highly acidic myristoylated family of PKC substrates widely distributed in diverse cell types including macrophages (10, 11). Phosphorylation of MARCKS proteins
following activation of PKC has been observed in fibroblasts (12, 13),
macrophages (14) and neutrophils (15). Both proteins are essential for
brain development and survival as shown by mice deficient in the genes
macs or mrp (16, 17).
MARCKS has been shown to cross-link actin filaments in vitro
(18). In macrophages, MARCKS colocalizes with actin, vinculin, and
talin at the site of attachment of the cytoskeleton to the plasma
membrane (19, 20). MRP colocalizes with paxillin at membrane ruffles at
the leading edge of spreading macrophages, suggesting that it also
associates with the actin cytoskeleton (21). Consequently, MARCKS and
MRP are thought to regulate the actin cytoskeleton and thereby
participate in major cellular responses such as phagocytosis,
secretion, motility, mitogenesis, and membrane trafficking.
Expression of MARCKS and MRP is strongly up-regulated in macrophages
stimulated with bacterial lipopolysaccharide (LPS) (22) or zymosan
(23). LPS stimulation increases MRP steady state mRNA levels
30-fold in murine macrophages, and high levels persist for more than
8 h (22). MARCKS mRNA and protein expression can be decreased
in fibroblasts through either PKC-dependent or -independent pathways by a post-transcriptional mechanism (24, 25). MARCKS concentrations may also be regulated by specific proteolytic cleavage of the unphosphorylated protein by a cysteine protease (26, 27), which
has recently been identified as cathepsin B (28). To our knowledge, no
reports concerning the down-regulation of MRP are available. Inasmuch
as MRP is a major PKC substrate in macrophages, we have examined the
expression of MRP in response to infection with Leishmania
promastigotes. Our finding that Leishmania infection
markedly depresses MRP levels may provide an important mechanism for
regulating PKC-dependent effector function in macrophages.
Mice--
CBA/J mice were purchased from Harlan (Horst, The
Netherlands) and were used between 8 and 16 weeks of age.
Reagents--
Recombinant murine IFN- Leishmania--
Leishmania major promastigotes,
strains MRHO/SU/59/P and MRHO/IR/75/ER designated as LV39 and IR75,
respectively, were grown at 26 °C in Dulbecco's minimal essential
medium (DMEM, Life Technologies, Inc., Basel, Switzerland) on blood
agar (29). Promastigotes of Leishmania mexicana strain
MNYC/62/M379, L. donovani strain LV636 and the LPG-deficient
L. major strain L119 (30) were propagated in 10% fetal
bovine serum-supplemented HOSMEM II medium (31). For macrophage
infection, stationary phase parasites were washed and resuspended in
DMEM containing 10% fetal bovine serum.
Macrophage Cultures and Activation--
Bone marrow-derived
macrophages were obtained by in vitro differentiation of
bone marrow precursor cells as described previously (32). Briefly,
cells flushed from mouse tibia and femurs were grown in DMEM with 20%
horse serum (Life Technologies, Inc.) and 30% L cell-conditioned
medium. Day 10-11 macrophages were detached by pipetting, suspended in
DMEM and 10% fetal bovine serum, and distributed in 35-mm tissue
culture dishes (3 × 106 macrophages/dish) or in
24-well cell culture plates (5 × 105
macrophages/well), each well containing a round sterile glass coverslip. After 24 h, macrophages were washed and stimulated with
IFN- Nitrite Determination--
After 24 h of macrophage
activation, 100 µl of supernatants were harvested for nitrite
determination (33). Macrophage supernatants were mixed with an equal
volume of Griess reagent and incubated for 10 min at room temperature.
Absorbance was measured at 550 nm in a micro-enzyme-linked
immunosorbent assay reader (Dynatech MR5000) using a 690-nm reference
filter. NO2 Radiolabeling of MRP--
Aliquots of
[9,10-3H]myristic acid (Amersham, Zurich Switzerland, 53 Ci/mmol) in ethanol were dried under a stream of nitrogen gas,
dissolved in dimethyl sulfoxide (Me2SO) and diluted in DMEM containing 10% fetal bovine serum. Macrophages cultured in 35-mm tissue culture plates (3 × 106 cells/plate) as
described above were washed and stimulated with IFN- Preparation of Macrophage Lysates--
For routine Western blot
analysis, macrophages were washed three times with PBS and detached
with ice-cold PBS containing 5 µg/ml pepstatin, 1 mM
phenylmethylsulfonyl fluoride, 10 µg/ml leupeptin and 10 µg/ml
aprotinin. Samples were sonicated and total cellular protein was
measured by the micro-bicinchoninic acid assay (Pierce). Laemmli sample
buffer was then added, and samples were placed in a 100 °C heat
block for 5 min. For some experiments, parallel samples were prepared
by adding heated SDS sample buffer directly to the tissue culture
plates. Similar results were obtained for lysates prepared by these 2 protocols (not shown). For experiments involving radiolabeled
macrophages, washed cells were lysed in PBS containing the same
protease inhibitors and 0.5% (v/v) Triton X-100. After determination
of total cellular protein, macrophage lysates were heated to 100 °C
for 5 min and centrifuged at 11,000 × g for 5 min at
4 °C to obtain a heat-stable protein fraction (22). Supernatants
were collected, and aliquots for SDS-PAGE were prepared in Laemmli
sample buffer as described above.
SDS-PAGE and Western blot Analysis--
For Western blot
analysis of total cell lysates, equal amounts of protein (30 µg) were
electrophoresed in a 12% polyacrylamide gel, electroblotted to
nitrocellulose, and probed with a polyclonal rabbit antibody
recognizing murine MRP. The anti-MRP antibody was raised against
purified recombinant unmyristoylated MRP (34) by injection of 30 µg
of protein in complete Freund's adjuvant followed by four subsequent
injections of 30 µg in incomplete Freund's adjuvant. The anti-MRP
antibody recognizes both myristoylated and unmyristoylated MRP as well
as MRP phosphorylated in vitro by the catalytic subunit of
PKC (35).2 Anti-MRP was used
as a 1:2000 dilution of serum followed by a 1:5000 dilution of
horseradish peroxidase-conjugated goat anti-rabbit IgG. Immunoreactive
MRP was detected with supersignal chemiluminescent substrate (Pierce).
Films exposed to chemiluminescent blots were scanned on a ScanJet 4c/T
densitometer (Hewlett Packard, Geneva, Switzerland) using the Adobe
Photoshop software package (Adobe Systems, Inc., Mountain View, CA) and
NIH image 1.60 software (NIH Division of Computer Research and
Technology). For experiments with radiolabeled macrophages, aliquots
containing the equivalent of 100 µg of total cellular protein were
electrophoresed in 12% polyacylamide gels. Gels containing
radiolabeled protein were treated with 0.13 M salicylic
acid in 10% v/v methanol, pH 7.0, dried, and exposed to x-ray film at
Immunofluorescence Microscopy--
Immunofluorescence studies
were performed using a polyclonal rabbit antibody (prepared by
Eurogentec, Seraing, Belgium) directed against a synthetic peptide
containing the 15 C-terminal amino acids of murine MRP preceded by the
21 amino acid tetanus toxoid P30 helper epitope (36). Immune serum
recognizing murine MRP in enzyme-linked immunosorbent assay and Western
blot analyses (not shown) was affinity purified by HiTrap
N-hydroxysuccinimide-activated affinity column
chromatography (Pharmacia LKB Biotechnology, Uppsala, Sweden). Control
or Leishmania-infected macrophages were cultured on glass
coverslips with or without IFN- Down-regulation of MRP Expression by Leishmania--
Murine
macrophages activated with IFN-
We then examined whether it was possible to reduce MRP levels by
challenging macrophages with LV39 at various times after addition of
IFN-
As an alternative proof that MRP levels were decreased in
Leishmania-infected cells, the incorporation of
[3H]myristic acid was examined. MRP expression was first
induced for 4 h with IFN- Comparison of Different Species of Leishmania--
Several
additional species of Leishmania were then compared with
LV39 for their effects on MRP levels. Because LPG may be responsible
for certain inhibitory effects of Leishmania, the LPG-deficient L. major strain L119 (30) was also tested. All parasites decreased MRP levels in macrophages stimulated with IFN-
As shown in Table I, infection measured
by microscopic examination of coverslips 24 h after parasite
challenge was lower for both L119 and L. donovani than for
LV39. However, 4 h after infection, parasite loads for L119 and
L. donovani were equal to or greater than for LV39. Both
L119 and L. donovani were rapidly destroyed by host
macrophages even in the absence of cytokine activation (Table I). LV39
persists in the presence or absence of cytokines for up to 24 h
(Table I) (37) but is subsequently eliminated by 48-72 h of culture
(37).
Effect of Other Phagocytic Stimuli--
The effect of other
phagocytic stimuli on MRP levels was also examined. Both zymosan and
latex beads were previously shown to augment
cytokine-dependent NO production similar to
Leishmania (40), and these results were confirmed as shown
in Fig. 5A. As shown in Fig.
5B, neither latex beads (lane 4) nor zymosan (lane 9) markedly reduced the levels of MRP observed in
activated macrophages (lanes 2 and 7) unlike the
strong inhibition obtained with LV39 (lanes 5 and
13). Indeed, zymosan increased MRP levels when added alone
(lane 8) in agreement with a previous report by Aderem
et al. (23). Addition of LV39 together with zymosan (lane 10) resulted in lower levels of MRP induction than
obtained with zymosan alone (lane 8). Like zymosan, the
Gram-positive bacterium S. mitis up-regulated NO production
and MRP expression (lanes 14 and 15).
Immunofluorescence Microscopy Studies of MRP Expression--
MRP
was localized in murine macrophages by indirect immunofluorescence
using an affinity-purified anti-C-terminal peptide antibody, which
recognizes a single 42-kDa doublet in Western blots of macrophage
lysates (data not shown). Strong punctate staining of MRP was observed
in the cytosol of activated macrophages (Fig.
6). In agreement with Western blot
analyses, staining was much less intense in nonactivated macrophages or
in macrophages infected with Leishmania. No staining was
observed with a control rabbit IgG or in the absence of primary
antibody (not shown).
Recently, in vitro studies have demonstrated that
Leishmania is capable of interfering with host macrophage
signal transduction machinery (1, 2) thereby modifying the capacity of
this cell to combat infection. One well studied effect of
Leishmania involves inhibition of macrophage PKC activity
and consequently PKC-dependent cell function. Results
presented here suggest that Leishmania might also regulate
PKC-dependent cell function in a more selective fashion by
decreasing levels of MRP, a major PKC substrate in macrophages.
Addition of Leishmania promastigotes to macrophages strongly
reduced levels of cytokine-induced MRP as early as 4 h after
infection. To date, all species or strains of Leishmania
promastigotes tested were capable of down-regulating MRP levels in
response to IFN- It is highly unlikely that the observed down-regulation of MRP in
Leishmania-infected macrophages reflects an overall
inhibition of protein synthesis or cell function for several reasons.
Nitrocellulose blots stained with Ponceau red showed no significant
differences in lanes containing lysates from infected versus
noninfected macrophages (not shown). Secondly, the expression of an
uncharacterized 50-kDa heat-stable myristoylated protein was unaffected
by Leishmania infection. Third, we have previously shown
that Leishmania increases bone marrow macrophage synthesis
of TNF- We considered the possibility that down-regulation of MRP resulted from
an effect of Leishmania on TNF- As mentioned above, examination of myristic acid incorporation revealed
the presence of a 48-50-kDa protein (designated as p50 in our studies)
in macrophages stimulated by IFN- Although MRP has been shown to be induced at the transcriptional level
by LPS (22), there are no reports concerning factors capable of
down-regulating its expression. Down-regulation of MARCKS in
fibroblasts can occur through a post-transcriptional decrease in MARCKS
mRNA upon incubation with bombesin or platelet-derived growth
factor (24). Down-regulation could be mimicked by short term treatment
with phorbol esters and was inhibited by PKC depletion. Somewhat
paradoxically, Spizz and Blackshear (28) showed that PKC-dependent phosphorylation of MARCKS protects the
protein from another down-regulatory pathway involving proteolysis by
lysosomal cathepsin B. They speculated that targeting of MARCKS to the
lysosomal membrane via a putative LAMP1-specific sequence might permit
the interaction of cytosolic MARCKS and the lysosomal enzyme. That similar mechanisms might be involved in the regulation of MRP levels is
suggested by our observations that the disappearance of radiolabeled
MARCKS proteins in Leishmania-infected macrophages correlates with the appearance of lower molecular weight species. Moreover, we recently demonstrated that rMRP is rapidly cleaved by LV39
lysates or by purified Leishmania surface metalloprotease, leishmanolysin, in a cell-free in vitro
assay.3 It remains to be
determined if this proteolytic event occurs within the macrophage and,
if so, how a Leishmania enzyme, which is presumably
restricted to the phagosomal/phagolysosomal compartment might interact
with a cytosolic protein such as MRP. In this regard, a recent report
by Rittig et al. (44) provided intriguing evidence that some
intracellular promastigotes of L. major are localized in the
cytosol of infected macrophages.
The implications of MRP down-regulation during Leishmania
infection are purely speculative for the time being. It has been proposed that down-regulation of PKC might favor parasite survival (9).
Decreasing the expression of a given PKC substrate could represent an
important mechanism for inhibiting specific PKC-dependent effector functions in the macrophage. Evidence of functional
alterations in fetal cells from animals lacking MARCKS family proteins
or from cell lines expressing incomplete or dominant-negative mutants of MRP or MARCKS is somewhat contradictory (45). In a recent investigation, Underhill et al. (46) reported that MRP is
not essential for phagocytosis by macrophages. However, the authors speculated that due to their high effector domain homology, MRP and
MARCKS might play overlapping roles explaining the normal phagocytic
phenotype of MRP-deficient cells. It is, thus, particularly interesting
that Leishmania infection appears to decrease levels of both
MARCKS proteins in macrophages. Our data, taken together with the
previously documented inhibitory effect of LPG on PKC activity, further
establish the ability of Leishmania parasites to circumvent
normal PKC-dependent function in macrophages.
Finally, we recently showed that peptides corresponding to the effector
domain of MARCKS and MRP induce polymerization of monomeric actin and
bundling of filamentous
actin4 in contrast to
comparatively moderate effects found with the intact MARCKS and MRP
proteins (18).5 We postulated
that in vivo proteolysis might facilitate the interaction between MARCKS proteins and actin by exposing their effector domain. Thus it is interesting to speculate that
Leishmania-dependent degradation of MRP might in
some way modulate the structure and function of the actin cytoskeleton
in infected macrophages.
INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
. One mechanism of
deactivation is indirect, involving induction of autoinhibitory
molecules. In addition, there is evidence for direct interference of
Leishmania with macrophage signal transduction pathways
including inhibition of signaling through Janus kinases and Stat1 (3),
or alterations in stimulus-induced intracellular calcium gradients
related to decreased production of inositol 1,4,5-trisphosphate (4).
Leishmania also inhibits protein kinase C
(PKC)-dependent signaling in host macrophages as evidenced
by alterations in PKC translocation and activity (5) and decreased expression of the transcriptional regulatory protein c-fos
(6). Some of these effects may be ascribed to the properties of
lipophosphoglycan (LPG), the major surface glycoconjugate of
Leishmania, which has been shown to inhibit macrophage
PKC-dependent signaling (7) as well as the activity of
purified PKC in vitro (8). Thus, phagocytosis of LPG-coated
beads inhibited phosphorylation of both a PKC-specific substrate
peptide and myristoylated alanine-rich C kinase substrate (MARCKS), an
endogenous PKC substrate in murine macrophages (9). Furthermore,
depletion of PKC rendered macrophages more permissive for the
proliferation of intracellular Leishmania suggesting that
PKC-dependent events might contribute to parasite destruction (9).
EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
produced by Genentech
Inc. was kindly supplied by Boehringer Ingelheim (Vienna, Austria).
Recombinant human tumor necrosis factor (TNF)-
was a gift of Dr. P. Schneider (Epalinges, Switzerland). LPS (Escherichia coli
055:B5) was purchased from Difco Laboratories, (Detroit, MI). Zymosan
A, latex beads (1.07 µ), pepstatin A, and horseradish
peroxidase-conjugated goat anti-rabbit IgG were purchased from Sigma.
Heat-killed (autoclaved) Streptococcus mitis was a gift of
Dr. D. Le Roy (Lausanne). LPG isolated from Leishmania
donovani was kindly provided by Dr. S. Turco (University of
Kentucky). Aprotinin, leupeptin, and phenylmethylsulfonyl fluoride were
purchased from Roche Molecular Biochemicals GmbH (Rotkreuz, Switzerland).
and/or TNF-
or LPS in the presence or absence of
Leishmania (5 parasites/macrophage unless indicated
otherwise). To quantitate phagocytosis of Leishmania,
coverslips were removed 24 h after infection, rinsed with
phosphate-buffered saline (PBS), fixed and stained with Diff-Quick
(Mertz and Dade, Düdingen, Switzerland) according to the
manufacturer's instructions.
concentration (µM) was
determined using NaNO2 as a standard.
+ TNF-
for
4 h. Medium was then aspirated, and 1 ml of fresh medium
containing IFN-
+ TNF-
and 50 µCi of [3H]myristic
acid in Me2SO (final concentration 0.4%) or
Me2SO alone in the presence or absence of LV39
promastigotes (15 × 106/plate) was added. After
6 h, macrophages were washed 3 times with PBS, and cell lysates
were prepared as described below.
70 °C. Fluorographs were scanned on the ScanJet 4c/T densitometer.
+ TNF-
as described above. After
24 h, macrophages were washed with medium without serum, fixed,
and permeabilized with ice-cold methanol for 1 min, dried, and frozen
at
20 °C. Cells were rehydrated with cold PBS and incubated for 30 min with 1% bovine serum albumin in PBS at room temperaure before
staining. Coverslips were then incubated with affinity purified rabbit
anti-MRP antibody for 1 h at room temperature followed by a 50 min
incubation with fluorescein-conjugated AffiniPure donkey anti-rabbit
IgG (H+L) (Jackson ImmunoResearch Laboratories, Westgrove, PA).
Antibodies were diluted in PBS containing 1% bovine serum albumin.
Coverslips were mounted in Citifluor (Kent Scientific and Industrial
Projects, UK) and stored at 4 °C. Microscopy was performed using a
Zeiss Axioskop microscope fitted with a 100x Plan Neofluar objective.
RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
+ TNF-
produce high levels of
nitric oxide (NO) and are capable of killing intracellular Leishmania (37). We examined the expression of MRP in normal and activated macrophages by Western blot analysis of total cell lysates. Because of its acidic amino acid composition, MRP, whose calculated molecular mass is 20 kDa, exhibits anomalous migration on
SDS gels and is recognized as a 42-kDa doublet in Western blots (38,
39). As shown in panels A and B of Fig.
1, IFN-
+ TNF-
increased the level
of immunoreactive MRP protein after 4 h of culture (lane
2) though much stronger induction was observed after 24 h
(lane 6). A comparison with known amounts of recombinant murine MRP (lanes 9 and 10) indicates that MRP is
present at a concentration of approximately 1 ng/µg total protein in
macrophages activated with IFN-
+ TNF-
. To determine whether
infection by Leishmania modulates MRP expression,
macrophages were challenged with LV39 promastigotes at the same time as
stimulation with IFN-
+ TNF-
. Under these conditions, a strong
decrease in MRP levels was consistently observed either 4 or 24 h
after infection (Fig. 1, A and B, lanes
4 and 8). In many experiments, Leishmania
also decreased the level of MRP in control unstimulated macrophages (Fig. 1B, lane 3; Fig. 4, lane 3,
below and data not shown). As shown in Fig. 1C, a strong
increase in MRP was also observed when macrophages were stimulated with
TNF-
(lane 4) or LPS (lane 5) alone, and LV39
inhibited such induction (lanes 7 and 8). LV39 also inhibited the weak induction of MRP obtained with IFN-
alone (lanes 3 and 6).
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Fig. 1.
Leishmania infection decreases MRP
levels in murine macrophages. Panels A and
B, macrophages were cultured with IFN- (50 units/ml) plus
TNF-
(250 ng/ml) or medium alone in the presence or absence of LV39
promastigotes (5 per macrophage). After 4 or 24 h, total cell
lysates were prepared and MRP levels determined by Western blot
analysis (panel B). Data from the corresponding
densitometric scan is shown for comparison (panel A).
Panel C, macrophages were stimulated with IFN-
(50 units/ml, lanes 3 and 6), TNF-
(250 ng/ml), or
LPS (10 ng/ml) alone or in the presence of LV39 promastigotes (5 per
macrophage). MRP levels present at 24 h were determined by Western
blot analysis of total cell lysates.
+ TNF-
. As shown in Fig. 2,
MRP levels in lysates prepared 24 h after cytokine stimulation
were strongly reduced when LV39 was added either together with the
activating stimuli (lane 4) or when added 8 h after
activation (lane 5). Similarly, addition of LV39 24 or
48 h after activation reduced the amount of MRP in 48-h
(lane 8) or in 72-h (lane 10) lysates,
respectively.
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Fig. 2.
Depletion of MRP by addition of
Leishmania at various times after macrophage
activation. Macrophages were stimulated with IFN- (50 units/ml)
plus TNF-
(250 ng/ml) at time 0 and LV39 promastigotes (5 per
macrophage) were added at 0, 8, 24, or 48 h postactivation. The
amount of MRP present in total cell lysates prepared at 24 h (for
parasites added at 0 or 8 h) or at 48 or 72 h (for parasites
added at 24 or 48 h, respectively) was determined by Western blot
analysis.
+ TNF-
, followed by the addition
of fresh medium containing [3H]myristic acid in the
presence or absence of LV39 promastigotes. After an additional 6 h, heat-stable fractions of total cell lysates were prepared and
subjected to SDS-PAGE. Fluorography revealed three major proteins, a
74-78-kDa protein, most probably MARCKS, an uncharacterized protein of
approximately 48-50 kDa (designated p50), and a broad 42-46-kDa
doublet corresponding to MRP (Fig. 3).
Western blot analyses, performed in parallel on the myristic acid-labeled lysates, confirmed the identity of MARCKS and MRP (data
not shown). In agreement with data presented above, myristoylated MRP
levels were increased upon cytokine activation and decreased in
Leishmania-infected macrophages. Interestingly, MARCKS
levels were also strongly decreased in Leishmania-infected
cells. Although little or no induction of MARCKS expression was
observed in macrophages stimulated with IFN-
+ TNF-
, it should be
pointed out that constitutive levels of MARCKS are generally higher and
induction of MARCKS mRNA and protein is both less pronounced and
occurs with more rapid kinetics when compared with MRP (22, 39).
Expression of the third heat-stable protein, p50, was increased by
cytokine stimulation but, unlike MRP and MARCKS, was unaffected by
Leishmania. In addition to the three major bands discussed
above, additional lower molecular weight bands were observed for the
samples from infected macrophages (lanes 3 and 4)
possibly representing degradation products of MRP and/or MARCKS (see
"Discussion").
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Fig. 3.
Leishmania infection decreases the
levels of [3H]myristate-labeled MRP and MARCKS in murine
macrophages. Macrophages were cultured with IFN- (50 units/ml)
plus TNF-
(250 ng/ml) or medium alone for 4 h. Medium were then
aspirated and fresh medium containing [3H]myristic acid
(50 µCi/ml) plus or minus the initial concentrations of IFN-
+ TNF-
added in the presence or absence of LV39 promastigotes (5 per
macrophage). Incorporation of labeled myristate was demonstrated by
SDS-PAGE and fluorography of heat-stable macrophage proteins.
Panel A, fluorograph of SDS-PAGE exposed for 3 days;
panel B, the fluorograph was scanned and integrated optical
density is presented as relative changes in MARCKS, p50, or MRP with
the DMEM controls considered as 100%.
+ TNF-
albeit to somewhat different degrees. L119 strongly decreased
MRP levels (Fig. 4B) as did
L. mexicana and another L. major strain IR75 (not
shown). However, L. donovani was consistently less potent in
down-regulating MRP in either 4- or 24-h lysates (Fig. 4B).
Because we and others (40-42) have shown that Leishmania strongly up-regulates NO production by murine macrophages,
NO2
release was determined in parallel. A very
similar enhancement of NO production was observed regardless of which
Leishmania was used (Fig. 4A).
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Fig. 4.
Depletion of MRP by different
Leishmania promastigotes. Promastigotes of LV39,
L. donovani (L dono), or L119 were added to
macrophage cultures (5 parasites per macrophage) in the presence or
absence of IFN- (50 units/ml) plus TNF-
(250 ng/ml). NO
production measured as NO2
release in 24-h
supernatants is shown in panel A. MRP levels at 24 or 4 h determined by Western blot analysis of total cell lysates are shown
in panels B and C, respectively. Note because MRP
is found at lower levels 4 h after activation compared with
24 h, the blot in panel C was exposed for a longer
period as evident from the stronger rMRP signal.
Quantitation of macrophage infection
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Fig. 5.
Failure of phagocytic stimuli to deplete
MRP. Macrophages were stimulated with IFN- (50 units/ml) plus
TNF-
(250 ng/ml) alone or together with latex beads (25 µg/ml),
LV39 parasites (5 per macrophage), zymosan (zym, 500 µg/ml) or heat-killed S. mitis (10 µg/ml, approximately
7 organisms per macrophage). Data from three representative experiments
is presented. Panel A, NO production determined as
NO2
release in 24-h supernatants. Panel B,
MRP levels at 24 h determined by Western blot analysis of total
cell lysates.
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Fig. 6.
Subcellular localization of MRP in activated
macrophages and MRP disappearance upon infection with
Leishmania. Macrophages cultured on glass
coverslips were stimulated with medium alone (A) or IFN-
(50 units/ml) plus TNF-
(250 ng/ml) in the absence (B) or
in the presence (C) of LV39 promastigotes (5 per macrophage)
for 24 h. After washing and fixation, the subcellular localization
of MRP was examined by fluorescence microscopy as described under
"Experimental Procedures."
DISCUSSION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
+ TNF-
. This effect did not require viable
parasites as heat-killed (15 min, 56 °C) promastigotes exhibited
comparable activity (data not shown). Other phagocytic stimuli
including yeast cell wall zymosan, latex beads, or heat-killed S. mitis had either no effect or increased MRP levels by themselves. Interestingly, the LPG-deficient strain L119 was as efficient as LV39
suggesting that LPG is not responsible for the effect of
Leishmania infection on MRP. Moreover 10 or 25 µM purified LPG from L. donovani (kind gift of
S. Turco) had no inhibitory effect on macrophage MRP expression in two
independent experiments (data not shown). The reason for the less
pronounced down-regulation of MRP observed with L. donovani
is unknown. However, it appears unlikely that decreased inhibition is
due entirely to a more rapid parasite clearance from macrophage
cultures because L119, which was as effective as LV39 in depleting MRP,
was also efficiently killed by nonactivated macrophages.
and prostaglandin E2 in an identical experimental system
(40). Finally, as shown previously (40-42) and confirmed here,
phagocytosis of Leishmania strongly up-regulates the
synthesis of NO.
receptor expression. However, similar results were obtained with other stimuli capable of
up-regulating MRP levels including LPS and zymosan. Moreover, other
markers of macrophage activation such as NO production or TNF-
synthesis (40) are enhanced under the same conditions. MRP levels in
activated macrophages were also dramatically decreased when parasites
were added 24 or 48 h after stimulation.
+ TNF-
. The identity of this
protein remains unknown though at least two groups (39, 43) have
previously described myristoylated macrophage proteins of comparable
size. Although p50 levels were similar in normal and infected
macrophages, the same studies suggested a profound effect of
Leishmania on the levels of MARCKS, a PKC substrate closely
related to MRP. Further studies are now in progress to examine
Leishmania-dependent modulation of MARCKS expression.
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ACKNOWLEDGEMENTS |
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We are grateful to Dr. Sam Turco for
providing purified LPG and Dr. Pascal Schneider for his generous gift
of rTNF-. We are also grateful to Jeannine Bamat for assistance with
immunofluorescence microscopy and to Dr. Giampietro Corradin for advice
in preparing the MRP peptide construct. We thank Jeannette Holenstein
for excellent technical assistance.
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FOOTNOTES |
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* This work was supported by Grant 3100-050667.97 (to J. M.) and by Grant 3100-042045.94 to G. Schwarz, Biozentrum, University of Basel from the Swiss National Fund for Scientific Research.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§ To whom correspondence should be addressed: Institute of Biochemistry, Chemin des Boveresses 155, 1066 Epalinges, Switzerland. Tel.: 21-6925702; Fax: 21-6925705; E-mail: Sally.CorradinBetz{at}ib.unil.ch.
Present address: ZLB Zentrallaboratorium, Blutspendedienst SRK,
Wankdorfstrasse 10, 3000 Bern 22, Switzerland.
2 G. Vergères, unpublished data.
3 S. Corradin, G. Corradin, J. Mauël, A. Ransijn, M. Rogerro, P. Schneider and G. Vergères, submitted for publication.
4 F. Wohnsland, A. A. P. Schmitz, M. O. Steinmetz, U. Aebi, and G. Vergères, submitted for publication.
5 F. Wohnsland, M. O. Steinmetz, U. Aebi, and G. Vergères, manuscript in preparation.
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ABBREVIATIONS |
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The abbreviations used are: IFN, interferon; DMEM, Dulbecco's minimal essential medium; LPG, lipophosphoglycan; LPS, lipopolysaccharide; MARCKS, myristoylated alanine-rich C kinase substrate; MRP, MARCKS-related protein; PBS, phosphate-buffered saline; PKC, protein kinase C; TNF, tumor necrosis factor; PAGE, polyacrylamide gel electrophoresis; NO, nitric oxide.
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REFERENCES |
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