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• W2049977846 abstract "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 ofLeishmania parasites tested, including lipophosphoglycan-deficient Leishmania majorL119, decreased MRP levels. MRP depletion was not obtained with other phagocytic stimuli including zymosan, latex beads, or heat-killedStreptococcus mitis, a Gram-positive bacterium. Experiments with [3H]myristate labeled proteins revealed the appearance of lower molecular weight fragments inLeishmania-infected cells suggesting that MRP depletion may be due to proteolytic degradation. 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 ofLeishmania parasites tested, including lipophosphoglycan-deficient Leishmania majorL119, decreased MRP levels. MRP depletion was not obtained with other phagocytic stimuli including zymosan, latex beads, or heat-killedStreptococcus mitis, a Gram-positive bacterium. Experiments with [3H]myristate labeled proteins revealed the appearance of lower molecular weight fragments inLeishmania-infected cells suggesting that MRP depletion may be due to proteolytic degradation. The ability of various intracellular pathogens includingLeishmania to inhibit macrophage effector activities, also termed “deactivation”, is well documented (1Bogdan C. Gessner A. Solbach W. Rollinghoff M. Curr. Opin. Immunol. 1996; 8: 517-525Crossref PubMed Scopus (168) Google Scholar, 2Reiner N.E. Immunol. Today. 1994; 15: 374-381Abstract Full Text PDF PubMed Scopus (149) Google Scholar). 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) 1The 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 -γ. One mechanism of deactivation is indirect, involving induction of autoinhibitory molecules. In addition, there is evidence for direct interference ofLeishmania with macrophage signal transduction pathways including inhibition of signaling through Janus kinases and Stat1 (3Nandan D. Reiner N.E. Infect. Immun. 1995; 63: 4495-4500Crossref PubMed Google Scholar), or alterations in stimulus-induced intracellular calcium gradients related to decreased production of inositol 1,4,5-trisphosphate (4Olivier M. Baimbridge K.G. Reiner N.E. J. Immunol. 1992; 148: 1188-1196PubMed Google Scholar).Leishmania also inhibits protein kinase C (PKC)-dependent signaling in host macrophages as evidenced by alterations in PKC translocation and activity (5Olivier M. Brownsey R.W. Reiner N.E. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 7481-7485Crossref PubMed Scopus (116) Google Scholar) and decreased expression of the transcriptional regulatory protein c-fos(6Moore K.J. Labrecque S. Matlashewski G. J. Immunol. 1993; 150: 4457-4465PubMed Google Scholar). Some of these effects may be ascribed to the properties of lipophosphoglycan (LPG), the major surface glycoconjugate ofLeishmania, which has been shown to inhibit macrophage PKC-dependent signaling (7Frankenburg S. Leibovici V. Mansbach N. Turco S.J. Rosen G. J. Immunol. 1990; 145: 4284-4289PubMed Google Scholar) as well as the activity of purified PKC in vitro (8McNeely T.B. Turco S.J. Biochem. Biophys. Res. Commun. 1987; 148: 653-657Crossref PubMed Scopus (71) Google Scholar). 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 (9Descoteaux A. Matlashewski G. Turco S.J. J. Immunol. 1992; 149: 3008-3015PubMed Google Scholar). Furthermore, depletion of PKC rendered macrophages more permissive for the proliferation of intracellular Leishmania suggesting that PKC-dependent events might contribute to parasite destruction (9Descoteaux A. Matlashewski G. Turco S.J. J. Immunol. 1992; 149: 3008-3015PubMed Google Scholar). 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 (10Aderem A. Cell. 1992; 71: 713-716Abstract Full Text PDF PubMed Scopus (431) Google Scholar, 11Blackshear P.J. J. Biol. Chem. 1993; 268: 1501-1504Abstract Full Text PDF PubMed Google Scholar). Phosphorylation of MARCKS proteins following activation of PKC has been observed in fibroblasts (12Blackshear P.J. Witters L.A. Girard P.R. Kuo J.F. Quamo S.N. J. Biol. Chem. 1985; 260: 13304-13315Abstract Full Text PDF PubMed Google Scholar, 13Rozengurt E. Rodriguez-Pena M. Smith K.A. Proc. Natl. Acad. Sci. U. S. A. 1983; 80: 7244-7248Crossref PubMed Scopus (278) Google Scholar), macrophages (14Aderem A.A. Albert K.A. Keum M.M. Wang J.K. Greengard P. Cohn Z.A. Nature. 1988; 332: 362-364Crossref PubMed Scopus (153) Google Scholar) and neutrophils (15Thelen M. Rosen A. Nairn A.C. Aderem A. Nature. 1991; 351: 320-322Crossref PubMed Scopus (308) Google Scholar). Both proteins are essential for brain development and survival as shown by mice deficient in the genesmacs or mrp (16Wu M. Chen D.F. Sasaoka T. Tonegawa S. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 2110-2115Crossref PubMed Scopus (85) Google Scholar, 17Stumpo D.J. Bock C.B. Tuttle J.S. Blackshear P.J. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 944-948Crossref PubMed Scopus (266) Google Scholar). MARCKS has been shown to cross-link actin filaments in vitro(18Hartwig J.H. Thelen M. Rosen A. Janmey P.A. Nairn A.C. Aderem A. Nature. 1992; 356: 618-622Crossref PubMed Scopus (620) Google Scholar). In macrophages, MARCKS colocalizes with actin, vinculin, and talin at the site of attachment of the cytoskeleton to the plasma membrane (19Allen L.H. Aderem A. J. Exp. Med. 1995; 182: 829-840Crossref PubMed Scopus (265) Google Scholar, 20Rosen A. Keenan K.F. Thelen M. Nairn A.C. Aderem A. J. Exp. Med. 1990; 172: 1211-1215Crossref PubMed Scopus (165) Google Scholar). MRP colocalizes with paxillin at membrane ruffles at the leading edge of spreading macrophages, suggesting that it also associates with the actin cytoskeleton (21Li J. Zhu Z. Bao Z. J. Biol. Chem. 1996; 271: 12985-12990Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar). 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) (22Li J. Aderem A. Cell. 1992; 70: 791-801Abstract Full Text PDF PubMed Scopus (110) Google Scholar) or zymosan (23Aderem A.A. Keum M.M. Pure E. Cohn Z.A. Proc. Natl. Acad. Sci. U. S. A. 1986; 83: 5817-5821Crossref PubMed Scopus (62) Google Scholar). LPS stimulation increases MRP steady state mRNA levels 30-fold in murine macrophages, and high levels persist for more than 8 h (22Li J. Aderem A. Cell. 1992; 70: 791-801Abstract Full Text PDF PubMed Scopus (110) Google Scholar). MARCKS mRNA and protein expression can be decreased in fibroblasts through either PKC-dependent or -independent pathways by a post-transcriptional mechanism (24Brooks S.F. Herget T. Broad S. Rozengurt E. J. Biol. Chem. 1992; 267: 14212-14218Abstract Full Text PDF PubMed Google Scholar, 25Wolfman A. Wingrove T.G. Blackshear P.J. Macara I.G. J. Biol. Chem. 1987; 262: 16546-16552Abstract Full Text PDF PubMed Google Scholar). MARCKS concentrations may also be regulated by specific proteolytic cleavage of the unphosphorylated protein by a cysteine protease (26Laumas S. Abdel-Ghany M. Leister K. Resnick R. Kandrach A. Racker E. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 3021-3025Crossref PubMed Scopus (9) Google Scholar, 27Spizz G. Blackshear P.J. J. Biol. Chem. 1996; 271: 553-562Abstract Full Text Full Text PDF PubMed Scopus (27) Google Scholar), which has recently been identified as cathepsin B (28Spizz G. Blackshear P.J. J. Biol. Chem. 1997; 272: 23833-23842Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar). 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 Leishmaniapromastigotes. Our finding that Leishmania infection markedly depresses MRP levels may provide an important mechanism for regulating PKC-dependent effector function in macrophages. CBA/J mice were purchased from Harlan (Horst, The Netherlands) and were used between 8 and 16 weeks of age. Recombinant murine IFN-γ produced by Genentech Inc. was kindly supplied by Boehringer Ingelheim (Vienna, Austria). Recombinant human tumor necrosis factor (TNF)-α was a gift of Dr. P. Schneider (Epalinges, Switzerland). LPS (Escherichia coli055: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 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 (29Behin R. Mauël J. Sordat B. Exp. Parasitol. 1979; 48: 81Crossref PubMed Scopus (94) Google Scholar). Promastigotes of Leishmania mexicana strain MNYC/62/M379, L. donovani strain LV636 and the LPG-deficientL. major strain L119 (30Murray P.J. Handman E. Glaser T.A. Spithill T.W. Exp. Parasitol. 1990; 71: 294-304Crossref PubMed Scopus (32) Google Scholar) were propagated in 10% fetal bovine serum-supplemented HOSMEM II medium (31Berens R.L. Marr J.J. J. Parasitol. 1978; 64: 160Crossref PubMed Scopus (95) Google Scholar). For macrophage infection, stationary phase parasites were washed and resuspended in DMEM containing 10% fetal bovine serum. Bone marrow-derived macrophages were obtained by in vitro differentiation of bone marrow precursor cells as described previously (32Meerpohl H.-G. Lohmann-Matters M.L. Fischer H. Eur. J. Immunol. 1976; 6: 213-217Crossref PubMed Scopus (103) Google Scholar). 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 × 105macrophages/well), each well containing a round sterile glass coverslip. After 24 h, macrophages were washed and stimulated with IFN-γ and/or TNF-α or LPS in the presence or absence ofLeishmania (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. After 24 h of macrophage activation, 100 μl of supernatants were harvested for nitrite determination (33Ding A.H. Nathan C.F. Struehr D.J. J. Immunol. 1988; 141: 2407-2412PubMed Google Scholar). 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. 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-γ + TNF-α for 4 h. Medium was then aspirated, and 1 ml of fresh medium containing IFN-γ + TNF-α 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. For routine Western blot analysis, macrophages were washed three times with PBS and detached with ice-cold PBS containing 5 μg/ml pepstatin, 1 mmphenylmethylsulfonyl 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 (22Li J. Aderem A. Cell. 1992; 70: 791-801Abstract Full Text PDF PubMed Scopus (110) Google Scholar). Supernatants were collected, and aliquots for SDS-PAGE were prepared in Laemmli sample buffer as described above. 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 (34Vergères G. Manenti S. Weber T. Stürzinger C. J. Biol. Chem. 1995; 270: 19879-19887Abstract Full Text Full Text PDF PubMed Scopus (79) Google Scholar) 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 (35Schleiff E. Schmitz A. McIlhinney R.A. Manenti S. Vergères G. J. Biol. Chem. 1996; 271: 26794-26802Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar). 2G. Vergères, unpublished data. 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 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 (36Valmori D. Pessi A. Bianchi E. Corradin G. J. Immunol. 1992; 149: 717-721PubMed Google Scholar). Immune serum recognizing murine MRP in enzyme-linked immunosorbent assay and Western blot analyses (not shown) was affinity purified by HiTrapN-hydroxysuccinimide-activated affinity column chromatography (Pharmacia LKB Biotechnology, Uppsala, Sweden). Control or Leishmania-infected macrophages were cultured on glass coverslips with or without IFN-γ + TNF-α 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. Murine macrophages activated with IFN-γ + TNF-α produce high levels of nitric oxide (NO) and are capable of killing intracellularLeishmania (37Mauël J. Ransijn A. Buchmuller-Rouiller Y. J. Leukocyte Biol. 1991; 49: 73-82Crossref PubMed Scopus (142) Google Scholar). 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 (38Zhu Z. Bao Z. Li J. J. Biol. Chem. 1995; 270: 17652-17655Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar,39Rosé S.D. Byers D.M. Morash S.C. Fedoroff S. Cook H.W. J. Neurosci. Res. 1996; 44: 235-242Crossref PubMed Google Scholar). As shown in panels A and B of Fig.1, IFN-γ + TNF-α 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-γ + TNF-α. To determine whether infection by Leishmania modulates MRP expression, macrophages were challenged with LV39 promastigotes at the same time as stimulation with IFN-γ + TNF-α. 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, Leishmaniaalso decreased the level of MRP in control unstimulated macrophages (Fig. 1 B, lane 3; Fig. 4, lane 3, below and data not shown). As shown in Fig. 1 C, a strong increase in MRP was also observed when macrophages were stimulated with TNF-α (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-γ alone (lanes 3 and 6).Figure 4Depletion of MRP by differentLeishmania 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-γ (50 units/ml) plus TNF-α (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.View Large Image Figure ViewerDownload (PPT) We then examined whether it was possible to reduce MRP levels by challenging macrophages with LV39 at various times after addition of IFN-γ + TNF-α. 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. As an alternative proof that MRP levels were decreased inLeishmania-infected cells, the incorporation of [3H]myristic acid was examined. MRP expression was first induced for 4 h with IFN-γ + TNF-α, 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 inLeishmania-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-γ + TNF-α, 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 (22Li J. Aderem A. Cell. 1992; 70: 791-801Abstract Full Text PDF PubMed Scopus (110) Google Scholar, 39Rosé S.D. Byers D.M. Morash S.C. Fedoroff S. Cook H.W. J. Neurosci. Res. 1996; 44: 235-242Crossref PubMed Google Scholar). Expression of the third heat-stable protein, p50, was increased by cytokine stimulation but, unlike MRP and MARCKS, was unaffected byLeishmania. 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”). 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 (30Murray P.J. Handman E. Glaser T.A. Spithill T.W. Exp. Parasitol. 1990; 71: 294-304Crossref PubMed Scopus (32) Google Scholar) was also tested. All parasites decreased MRP levels in macrophages stimulated with IFN-γ + TNF-α albeit to somewhat different degrees. L119 strongly decreased MRP levels (Fig. 4 B) as didL. 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. 4 B). Because we and others (40Corradin S.B. Mauël J. J. Immunol. 1991; 146: 279-285PubMed Google Scholar, 41Corradin S.B. Buchmüller-Rouiller Y. Mauël J. Eur. J. Immunol. 1991; 21: 2553-2558Crossref PubMed Scopus (46) Google Scholar, 42Green S.J. Crawford R.M. Hockmeyer J.T. Meltzer M.S. Nacy C.A. J. Immunol. 1990; 145: 4290-4297PubMed Google Scholar) have shown that Leishmaniastrongly up-regulates NO production by murine macrophages, NO2− release was determined in parallel. A very similar enhancement of NO production was observed regardless of whichLeishmania was used (Fig. 4 A). 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 andL. 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) (37Mauël J. Ransijn A. Buchmuller-Rouiller Y. J. Leukocyte Biol. 1991; 49: 73-82Crossref PubMed Scopus (142) Google Scholar) but is subsequently eliminated by 48–72 h of culture (37Mauël J. Ransijn A. Buchmuller-Rouiller Y. J. Leukocyte Biol. 1991; 49: 73-82Crossref PubMed Scopus (142) Google Scholar).Table IQuantitation of macrophage infectionParasites/100 macrophages4 h24 hLV395480+IFN-γ/TNF-α6844L. donovani685+IFN-γ/TNF-α681L119848+IFN-γ/TNF-α1189Data is presented from one of two independent experiments. Open table in a new tab Data is presented from one of two independent experiments. 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 toLeishmania (40Corradin S.B. Mauël J. J. Immunol. 1991; 146: 279-285PubMed Google Scholar), and these results were confirmed as shown in Fig. 5 A. As shown in Fig.5 B, 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 and13). Indeed, zymosan increased MRP levels when added alone (lane 8) in agreement with a previous report by Aderemet al. (23Aderem A.A. Keum M.M. Pure E. Cohn Z.A. Proc. Natl. Acad. Sci. U. S. A. 1986; 83: 5817-5821Crossref PubMed Scopus (62) Google Scholar). 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). 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 thatLeishmania is capable of interfering with host macrophage signal transduction machinery (1Bogdan C. Gessner A. Solbach W. Rollinghoff M. Curr. Opin. Immunol. 1996; 8: 517-525Crossref PubMed Scopus (168) Google Scholar, 2Reiner N.E. Immunol. Today. 1994; 15: 374-381Abstract Full Text PDF PubMed Scopus (149) Google Scholar) thereby modifying the capacity of this cell to combat infection. One well studied effect ofLeishmania 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 Leishmaniapromastigotes tested were capable of down-regulating MRP levels in response to IFN-γ + TNF-α. 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 ofLeishmania 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. donovaniis 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 inLeishmania-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 versusnoninfected 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-α and prostaglandin E2 in an identical experimental system (40Corradin S.B. Mauël J. J. Immunol. 1991; 146: 279-285PubMed Google Scholar). Finally, as shown previously (40Corradin S.B. Mauël J. J. Immunol. 1991; 146: 279-285PubMed Google Scholar, 41Corradin S.B. Buchmüller-Rouiller Y. Mauël J. Eur. J. Immunol. 1991; 21: 2553-2558Crossref PubMed Scopus (46) Google Scholar, 42Green S.J. Crawford R.M. Hockmeyer J.T. Meltzer M.S. Nacy C.A. J. Immunol. 1990; 145: 4290-4297PubMed Google Scholar) 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-α 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-α synthesis (40Corradin S.B. Mauël J. J. Immunol. 1991; 146: 279-285PubMed Google Scholar) 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-γ + TNF-α. The identity of this protein remains unknown though at least two groups (39Rosé S.D. Byers D.M. Morash S.C. Fedoroff S. Cook H.W. J. Neurosci. Res. 1996; 44: 235-242Crossref PubMed Google Scholar, 43Aderem A.A. Marratta D.E. Cohn Z.A. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 6310-6313Crossref PubMed Scopus (15) Google Scholar) 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 ofLeishmania on the levels of MARCKS, a PKC substrate closely related to MRP. Further studies are now in progress to examineLeishmania-dependent modulation of MARCKS expression. Although MRP has been shown to be induced at the transcriptional level by LPS (22Li J. Aderem A. Cell. 1992; 70: 791-801Abstract Full Text PDF PubMed Scopus (110) Google Scholar), 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 (24Brooks S.F. Herget T. Broad S. Rozengurt E. J. Biol. Chem. 1992; 267: 14212-14218Abstract Full Text PDF PubMed Google Scholar). Down-regulation could be mimicked by short term treatment with phorbol esters and was inhibited by PKC depletion. Somewhat paradoxically, Spizz and Blackshear (28Spizz G. Blackshear P.J. J. Biol. Chem. 1997; 272: 23833-23842Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar) 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 vitroassay. 3S. Corradin, G. Corradin, J. Mauël, A. Ransijn, M. Rogerro, P. Schneider and G. Vergères, submitted for publication. 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. (44Rittig M.G. Schroppel K. Seack K.H. Sander U. N′Diaye E.N. Maridonneau-Parini I. Solbach W. Bogdan C. Infect. Immun. 1998; 66: 4331-4339Crossref PubMed Google Scholar) 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 Leishmaniainfection are purely speculative for the time being. It has been proposed that down-regulation of PKC might favor parasite survival (9Descoteaux A. Matlashewski G. Turco S.J. J. Immunol. 1992; 149: 3008-3015PubMed Google Scholar). 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 (45Aderem A. Curr. Top. Microbiol. Immunol. 1992; 181: 189-207PubMed Google Scholar). In a recent investigation, Underhill et al. (46Underhill D.M. Chen J.M. Allen L.A.H. Aderem A. J. Biol. Chem. 1998; 273: 33619-33623Abstract Full Text Full Text PDF PubMed Scopus (32) Google Scholar) 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 actin 4F. Wohnsland, A. A. P. Schmitz, M. O. Steinmetz, U. Aebi, and G. Vergères, submitted for publication. in contrast to comparatively moderate effects found with the intact MARCKS and MRP proteins (18Hartwig J.H. Thelen M. Rosen A. Janmey P.A. Nairn A.C. Aderem A. Nature. 1992; 356: 618-622Crossref PubMed Scopus (620) Google Scholar). 5F. Wohnsland, M. O. Steinmetz, U. Aebi, and G. Vergères, manuscript in preparation. 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 thatLeishmania-dependent degradation of MRP might in some way modulate the structure and function of the actin cytoskeleton in infected macrophages. We are grateful to Dr. Sam Turco for providing purified LPG and Dr. Pascal Schneider for his generous gift of rTNF-α. 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." @default.
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• W2049977846 title "Down-regulation of MARCKS-related Protein (MRP) in Macrophages Infected with Leishmania" @default.
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