Down-regulation of MARCKS-related Protein (MRP) in Macrophages Infected with Leishmania*

Sally CorradinDagger §, Jacques MauëlDagger , Adriana RansijnDagger , Christoph Stürzinger, and Guy Vergèresparallel

From the Dagger  Institute of Biochemistry, University of Lausanne, 1066 Epalinges, Switzerland and  Department of Biophysical Chemistry, Biozentrum, University of Basel, 4056 Basel, Switzerland

    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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.

    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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-gamma . 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).

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.

    EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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-gamma produced by Genentech Inc. was kindly supplied by Boehringer Ingelheim (Vienna, Austria). Recombinant human tumor necrosis factor (TNF)-alpha 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).

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-gamma and/or TNF-alpha 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.

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- concentration (µM) was determined using NaNO2 as a standard.

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-gamma  + TNF-alpha for 4 h. Medium was then aspirated, and 1 ml of fresh medium containing IFN-gamma  + TNF-alpha 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.

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 -70 °C. Fluorographs were scanned on the ScanJet 4c/T densitometer.

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-gamma  + TNF-alpha 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
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Down-regulation of MRP Expression by Leishmania-- Murine macrophages activated with IFN-gamma  + TNF-alpha 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-gamma  + TNF-alpha 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-gamma  + TNF-alpha . To determine whether infection by Leishmania modulates MRP expression, macrophages were challenged with LV39 promastigotes at the same time as stimulation with IFN-gamma  + TNF-alpha . 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-alpha (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-gamma alone (lanes 3 and 6).


View larger version (27K):
[in this window]
[in a new window]
 
Fig. 1.   Leishmania infection decreases MRP levels in murine macrophages. Panels A and B, macrophages were cultured with IFN-gamma (50 units/ml) plus TNF-alpha (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-gamma (50 units/ml, lanes 3 and 6), TNF-alpha (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.

We then examined whether it was possible to reduce MRP levels by challenging macrophages with LV39 at various times after addition of IFN-gamma  + TNF-alpha . 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.


View larger version (19K):
[in this window]
[in a new window]
 
Fig. 2.   Depletion of MRP by addition of Leishmania at various times after macrophage activation. Macrophages were stimulated with IFN-gamma (50 units/ml) plus TNF-alpha (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.

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-gamma  + TNF-alpha , 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-gamma  + TNF-alpha , 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").


View larger version (30K):
[in this window]
[in a new window]
 
Fig. 3.   Leishmania infection decreases the levels of [3H]myristate-labeled MRP and MARCKS in murine macrophages. Macrophages were cultured with IFN-gamma (50 units/ml) plus TNF-alpha (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-gamma  + TNF-alpha 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%.

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-gamma  + TNF-alpha 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).


View larger version (35K):
[in this window]
[in a new window]
 
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-gamma (50 units/ml) plus TNF-alpha (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.

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).

                              
View this table:
[in this window]
[in a new window]
 
Table I
Quantitation of macrophage infection
Data is presented from one of two independent experiments.

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).


View larger version (27K):
[in this window]
[in a new window]
 
Fig. 5.   Failure of phagocytic stimuli to deplete MRP. Macrophages were stimulated with IFN-gamma (50 units/ml) plus TNF-alpha (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.

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).


View larger version (42K):
[in this window]
[in a new window]
 
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-gamma (50 units/ml) plus TNF-alpha (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
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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-gamma  + TNF-alpha . 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.

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-alpha 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.

We considered the possibility that down-regulation of MRP resulted from an effect of Leishmania on TNF-alpha 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-alpha 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.

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-gamma  + TNF-alpha . 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.

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.

    ACKNOWLEDGEMENTS

We are grateful to Dr. Sam Turco for providing purified LPG and Dr. Pascal Schneider for his generous gift of rTNF-alpha . 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.

    FOOTNOTES

* 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.

parallel 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.

    ABBREVIATIONS

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.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
  1. Bogdan, C., Gessner, A., Solbach, W., and Rollinghoff, M. (1996) Curr. Opin. Immunol. 8, 517-525[CrossRef][Medline] [Order article via Infotrieve]
  2. Reiner, N. E. (1994) Immunol. Today 15, 374-381[CrossRef][Medline] [Order article via Infotrieve]
  3. Nandan, D., and Reiner, N. E. (1995) Infect. Immun. 63, 4495-4500[Abstract]
  4. Olivier, M., Baimbridge, K. G., and Reiner, N. E. (1992) J. Immunol. 148, 1188-1196[Abstract/Free Full Text]
  5. Olivier, M., Brownsey, R. W., and Reiner, N. E. (1992) Proc. Natl. Acad. Sci. U. S. A. 89, 7481-7485[Abstract]
  6. Moore, K. J., Labrecque, S., and Matlashewski, G. (1993) J. Immunol. 150, 4457-4465[Abstract/Free Full Text]
  7. Frankenburg, S., Leibovici, V., Mansbach, N., Turco, S. J., and Rosen, G. (1990) J. Immunol. 145, 4284-4289[Abstract/Free Full Text]
  8. McNeely, T. B., and Turco, S. J. (1987) Biochem. Biophys. Res. Commun. 148, 653-657[Medline] [Order article via Infotrieve]
  9. Descoteaux, A., Matlashewski, G., and Turco, S. J. (1992) J. Immunol. 149, 3008-3015[Abstract/Free Full Text]
  10. Aderem, A. (1992) Cell 71, 713-716[Medline] [Order article via Infotrieve]
  11. Blackshear, P. J. (1993) J. Biol. Chem. 268, 1501-1504[Free Full Text]
  12. Blackshear, P. J., Witters, L. A., Girard, P. R., Kuo, J. F., and Quamo, S. N. (1985) J. Biol. Chem. 260, 13304-13315[Abstract/Free Full Text]
  13. Rozengurt, E., Rodriguez-Pena, M., and Smith, K. A. (1983) Proc. Natl. Acad. Sci. U. S. A. 80, 7244-7248[Abstract]
  14. Aderem, A. A., Albert, K. A., Keum, M. M., Wang, J. K., Greengard, P., and Cohn, Z. A. (1988) Nature 332, 362-364[CrossRef][Medline] [Order article via Infotrieve]
  15. Thelen, M., Rosen, A., Nairn, A. C., and Aderem, A. (1991) Nature 351, 320-322[CrossRef][Medline] [Order article via Infotrieve]
  16. Wu, M., Chen, D. F., Sasaoka, T., and Tonegawa, S. (1996) Proc. Natl. Acad. Sci. U. S. A. 93, 2110-2115[Abstract/Free Full Text]
  17. Stumpo, D. J., Bock, C. B., Tuttle, J. S., and Blackshear, P. J. (1995) Proc. Natl. Acad. Sci. U. S. A. 92, 944-948[Abstract]
  18. Hartwig, J. H., Thelen, M., Rosen, A., Janmey, P. A., Nairn, A. C., and Aderem, A. (1992) Nature 356, 618-622[CrossRef][Medline] [Order article via Infotrieve]
  19. Allen, L. H., and Aderem, A. (1995) J. Exp. Med. 182, 829-840[Abstract]
  20. Rosen, A., Keenan, K. F., Thelen, M., Nairn, A. C., and Aderem, A. (1990) J. Exp. Med. 172, 1211-1215[Abstract]
  21. Li, J., Zhu, Z., and Bao, Z. (1996) J. Biol. Chem. 271, 12985-12990[Abstract/Free Full Text]
  22. Li, J., and Aderem, A. (1992) Cell 70, 791-801[Medline] [Order article via Infotrieve]
  23. Aderem, A. A., Keum, M. M., Pure, E., and Cohn, Z. A. (1986) Proc. Natl. Acad. Sci. U. S. A. 83, 5817-5821[Abstract]
  24. Brooks, S. F., Herget, T., Broad, S., and Rozengurt, E. (1992) J. Biol. Chem. 267, 14212-14218[Abstract/Free Full Text]
  25. Wolfman, A., Wingrove, T. G., Blackshear, P. J., and Macara, I. G. (1987) J. Biol. Chem. 262, 16546-16552[Abstract/Free Full Text]
  26. Laumas, S., Abdel-Ghany, M., Leister, K., Resnick, R., Kandrach, A., and Racker, E. (1989) Proc. Natl. Acad. Sci. U. S. A. 86, 3021-3025[Abstract]
  27. Spizz, G., and Blackshear, P. J. (1996) J. Biol. Chem. 271, 553-562[Abstract/Free Full Text]
  28. Spizz, G., and Blackshear, P. J. (1997) J. Biol. Chem. 272, 23833-23842[Abstract/Free Full Text]
  29. Behin, R., Mauël, J., and Sordat, B. (1979) Exp. Parasitol. 48, 81[CrossRef][Medline] [Order article via Infotrieve]
  30. Murray, P. J., Handman, E., Glaser, T. A., and Spithill, T. W. (1990) Exp. Parasitol. 71, 294-304[Medline] [Order article via Infotrieve]
  31. Berens, R. L., and Marr, J. J. (1978) J. Parasitol. 64, 160[Medline] [Order article via Infotrieve]
  32. Meerpohl, H.-G., Lohmann-Matters, M. L., and Fischer, H. (1976) Eur. J. Immunol. 6, 213-217[Medline] [Order article via Infotrieve]
  33. Ding, A. H., Nathan, C. F., and Struehr, D. J. (1988) J. Immunol. 141, 2407-2412[Abstract/Free Full Text]
  34. Vergères, G., Manenti, S., Weber, T., and Stürzinger, C. (1995) J. Biol. Chem. 270, 19879-19887[Abstract/Free Full Text]
  35. Schleiff, E., Schmitz, A., McIlhinney, R. A., Manenti, S., and Vergères, G. (1996) J. Biol. Chem. 271, 26794-26802[Abstract/Free Full Text]
  36. Valmori, D., Pessi, A., Bianchi, E., and Corradin, G. (1992) J. Immunol. 149, 717-721[Abstract/Free Full Text]
  37. Mauël, J., Ransijn, A., and Buchmuller-Rouiller, Y. (1991) J. Leukocyte Biol. 49, 73-82[Abstract]
  38. Zhu, Z., Bao, Z., and Li, J. (1995) J. Biol. Chem. 270, 17652-17655[Abstract/Free Full Text]
  39. Rosé, S. D., Byers, D. M., Morash, S. C., Fedoroff, S., and Cook, H. W. (1996) J. Neurosci. Res. 44, 235-242[CrossRef][Medline] [Order article via Infotrieve]
  40. Corradin, S. B., and Mauël, J. (1991) J. Immunol. 146, 279-285[Abstract/Free Full Text]
  41. Corradin, S. B., Buchmüller-Rouiller, Y., and Mauël, J. (1991) Eur. J. Immunol. 21, 2553-2558[Medline] [Order article via Infotrieve]
  42. Green, S. J., Crawford, R. M., Hockmeyer, J. T., Meltzer, M. S., and Nacy, C. A. (1990) J. Immunol. 145, 4290-4297[Abstract/Free Full Text]
  43. Aderem, A. A., Marratta, D. E., and Cohn, Z. A. (1988) Proc. Natl. Acad. Sci. U. S. A. 85, 6310-6313[Abstract]
  44. Rittig, M. G., Schroppel, K., Seack, K. H., Sander, U., N'Diaye, E. N., Maridonneau-Parini, I., Solbach, W., and Bogdan, C. (1998) Infect. Immun. 66, 4331-4339[Abstract/Free Full Text]
  45. Aderem, A. (1992) Curr. Top. Microbiol. Immunol. 181, 189-207[Medline] [Order article via Infotrieve]
  46. Underhill, D. M., Chen, J. M., Allen, L. A. H., and Aderem, A. (1998) J. Biol. Chem. 273, 33619-33623[Abstract/Free Full Text]


Copyright © 1999 by The American Society for Biochemistry and Molecular Biology, Inc.