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J Biol Chem, Vol. 273, Issue 51, 34391-34398, December 18, 1998
From the § Institut Pasteur, Laboratoire des
Mycoplasmes, 25 Rue Docteur Roux, 75724 Paris Cedex 15, France,
Mycoplasma lipoproteins have been demonstrated to
stimulate monocytic cells and induce proinflammatory cytokine
secretion. In this paper, we show that a synthetic analog of the
Mycoplasma fermentans membrane-associated lipopeptide
macrophage-activating lipopeptide-2 (MALP-2) induces mRNA synthesis
and protein secretion of interleukin-1 Mycoplasma fermentans, a human pathogen, is a potent
activator of monocytes/macrophages. Macrophage activation by M. fermentans results in the secretion of numerous cytokines
including interleukin (IL)1-1 Initial partial biochemical characterization of an M. fermentans-derived high molecular weight material that activates
macrophages suggested that it was an amphiphilic lipid-like molecule
(3). Further studies led to the identification of an acylated
S-(2,3-dihydroxypropyl)cysteine, typical for Braun's murein
lipoprotein, in a lipopeptide purified from M. fermentans-derived high molecular weight material (6). This
suggested that this lipopeptide could be the macrophage-activating agent of M. fermentans-derived high molecular weight
material. Very recently, the same group has elucidated the structure of this lipopeptide, named MALP-2 (for
macrophage-activating
lipopeptide) (7). MALP-2 is a 2-kDa free
NH2 terminus lipopeptide with the following structure:
S-(2,3-bisacyloxyprolyl)cysteine-GNNDESNISFKEK. Interestingly, M. fermentans-derived MALP-2 as well as its
synthetic analog induced NO release from murine macrophages and showed
an identical dose-dependent effect (7).
In the present study, we have tested the capacity of synthetic MALP-2
(sMALP-2) to stimulate cytokine production by macrophages and activate
MAPK pathways. Additionally, we have investigated the activation of
NF- Reagents and Antibodies--
PD-98059 and GST-c-Jun-(1-79) were
obtained from Biomol Research Laboratories (Philadelphia, PA). SB203580
was from Calbiochem (Nottingham, United Kingdom). Anti-JNK1 (C17),
anti-ERK (K23), anti-p38 (C20) polyclonal antibodies, A/G-Sepharose
beads, and anti-NF- Synthetic Lipopeptide--
Peptide (CGNNDESNISFKEK) and acylated
peptide corresponding to the previously published MALP-2 sequence (7)
were synthesized as described by Metzger et al. (8, 9).
Synthesis was performed by Synt:em (Nîme, France). Tripalmitoyl
S-glycerylcysteine was kindly provided by Dr. Radolf.
Acylated peptide was resuspended in 25 mM octyl glucoside
in phosphate-buffered saline. The detergent had no effects on cells at
the final used concentration. Lipopeptide preparation was tested for
absence of endotoxin (<60 pg/ml) by Limulus amoebocyte
lysate assay (Hemachem, St. Louis, MO).
Cell Cultures, Stimulation, and Lysate Preparation--
The
murine macrophage cell line RAW 264.7 (American Type Culture
Collection, Rockville, MD) was cultured (37 °C, 5% CO2)
in Dulbecco's modified Eagle's medium culture medium containing 10% fetal calf serum, 2 mM L-glutamine, and
antibiotics. The human monocytic cell line THP-1 (American Type Culture
Collection) was cultured (37 °C, 5% CO2) in RPMI
culture medium containing 10% fetal calf serum, 2 mM
L-glutamine, and antibiotics. Cell lines were tested every
2 weeks by a polymerase chain reaction-based detection assay for
mycoplasma contamination (10). For stimulation experiments, cells were
seeded at 106 cells/ml density and then allowed to
cultivate overnight. Cells were stimulated with sMALP-2 (at 200 nM) for appropriate time intervals. LPS was used in control
experiments at 1 µg/ml to stimulate RAW 264.7 cells and at 100 ng/ml
to stimulate THP-1 cells. For phosphotransferase assays, cells were
washed twice with ice-cold phosphate-buffered saline containing 1 mM Na3VO4. For each 106
cells initially seeded, 100 µl of the following lysis buffer was
added: 20 mM MOPS, pH 7.2, 5 mM EDTA, 1% (w/v)
Nonidet P-40, 1 mM dithiothreitol, 75 mM
Human monocytes/macrophages were prepared from healthy donors'
peripheral blood mononuclear cells by Ficoll/Hypaque density gradient
centrifugation (Amersham Pharmacia Biotech). 50 × 106
peripheral blood mononuclear cells were adhered to six-well tissue culture plates (Costar) for 1 h at 37 °C, 5% CO2,
in RPMI 1640 medium containing 1% human serum (Centre National de
Transfusion Sanguine, Saint Antoine, France), 2 mM
L-glutamine, and antibiotics. To remove nonadherent cells,
wells were washed twice with prewarmed culture medium. Adherent cells
(approximately 5 × 106 cells) were cultured
(37 °C, 5% CO2) in 5 ml of culture medium with or
without stimulus.
Chinese hamster ovary cell lines expressing human CD14 (CHO-CD14) and
harboring the empty expression vector (CHO-RSV) were gifts from Dr.
R. J. Ulevitch (11). CHO-CD14 and CHO-RSV cell lines were grown in
RPMI culture medium containing 10% fetal calf serum, 2 mM
L-glutamine, antibiotics, and G418 (500 µg/ml).
Assay for Cytokines--
Cells were stimulated as indicated
above for a 24-h period and were lysed by two consecutive cycles of
freezing/thawing. Thus, the samples represented the total amount of
cytokines produced (both intracellular and released into the
supernatant). The cytokine concentration from murine or human
macrophages was measured by a corresponding IL-1 RNA Isolation and Analysis--
Cells were stimulated as
described above at the indicated times, and total RNA was extracted
from 107 cells using the total RNA isolation kit from
Bioprobe (Montreuil, France) according to the manufacturer's
instructions. Using Hybond-N+ filters (Amersham Pharmacia
Biotech), slot RNA were performed with 5-10 µg of RNA as described
by Sambrook et al. (12), and slots were UV-cross-linked.
Specific murine IL-1 Immunoprecipitation of ERK, JNK, and p38 from Stimulated Cell
Lysates--
ERK1/2 and JNK were immunoprecipitated by incubating 500 µg of cell lysates with 5 µg of anti-ERK1-CT antibody or 2 µg of anti-JNK1 antibody, respectively, at 4 °C for 4 h with
continuous rotation. Then 30 µl of protein A-Sepharose was added, and
the incubation was extended for an additional 2 h. The mixtures
were then centrifuged (7000 × g for 2 min at room
temperature), and protein A-Sepharose beads were washed three times
with buffer B (12.5 mM MOPS, pH 7.2, 0.5 mM
EGTA, 12.5 mM
For p38 immunoprecipitation, the protocol was slightly modified. 10 µg of anti-p38 antibody was first coupled to A/G-Sepharose beads for
2 h at 4 °C and then washed with buffer B and added to 500 µg
of cell lysates. Immunoprecipitation was allowed overnight at 4 °C
with continuous rotation, and immunoprecipitates were analyzed as
described above.
Measurement of Phosphotransferase Activity--
ERK and p38
activation was determined in immunoprecipitates by means of measuring
radioactively their respective phosphotransferase activities toward a
peptide substrate using p42/44MAPK or p38/Mpk2 detection
kits. Assays were performed according to the manufacturer's
instructions. Activity was expressed as [32P]ATP cpm.
To measure JNK activation, 2 µg of GST-c-Jun was added to
stress-activated protein kinase/JNK immunoprecipitates in the presence 50 µM [ Nuclear Extract Preparation and Electrophoretic Mobility Shift
Assay--
Cells were stimulated with sMALP-2 at indicated time
intervals, nuclear extracts were prepared as described in Ref. 13, and
protein content was determined by micro-BCA assay (Pierce). NF- Plasmids, Cell Transfection, Activation, and Assay for Luciferase
Activity--
The NF- sMALP-2 Induces Transcription and Secretion of IL-1
sMALP-2 preincubation with polymyxin B (1000 units/ml) for 2 h
before its addition to cell cultures had no effect on TNF
As shown in Fig. 1, B and C, sMALP-2 at 200 nM induced the production of a considerable amount of
IL-1
We have further examined the levels of cytokine mRNA in RAW 264.7 cells stimulated with sMALP-2. Data from RNA slot hybridization, presented in Fig. 1D, clearly show that sMALP-2 stimulated
IL-1
Previous reports have strongly suggested that mycoplasma-derived
membrane fractions stimulate monocytes/macrophages by a mechanism distinct from that of LPS and independent of CD14 and/or serum (1, 5,
20). We have therefore investigated whether sMALP-2 was able to induce
NF- sMALP-2 Activates MAPK Pathways and NF-
Transcription factors NF-
As depicted in Fig. 4A, similarly to the MAPK activation,
LPS-mediated NF-
sMALP-2 also activated AP-1 translocation in RAW 264.7 cells, and peak
activity was observed at 4 h of stimulation. Although a basal AP-1
activation could be detected in these cells, PhosphorImager quantification of EMSA gels clearly shows that sMALP-2 induced a
4-5-fold increase in AP-1 DNA binding activity compared with control
level (Fig. 4B). Once again, AP-1 translocation in response to LPS occurred earlier (peak at 2 h) than that induced by sMALP-2 (data not shown).
Pursuing the investigation of AP-1 and NF- Involvement of MAPK Pathways in MALP-2-induced Cytokine Production
and AP-1 and NF-
SB203580 is a bicyclic imidazole compound able to specifically inhibit
p38 (28). This inhibitor selectively blocked the sMALP-2-mediated
activation of p38 in RAW 264.7 cells without significantly affecting
the stimulation of ERK1/2 or JNK (data not shown). Preincubation of RAW
264.7 with SB203580 for 1 h prior to challenging with sMALP-2
blocked in a dose-dependent manner both IL-1
PD-98059 is a synthetic compound that specifically inhibits the
ERK-activating MAPK kinase MEK-1 (27, 29). This compound selectively
inhibited the sMALP-2-mediated activation of ERK1/2 in murine
macrophages without significantly affecting p38 or JNK stimulation
(data not shown). The inhibition of MEK-1 by PD-98059 treatment
partially inhibited, in a dose-dependent manner, cytokine production by RAW 265.7 in response to sMALP-2. Unlike SB203580, PD-98059 was unable to completely block sMALP-2 stimulation, and only
50% inhibition was obtained at 30 µM concentration
(Table I) or at the highest doses (data not shown).
Interestingly, treatment of RAW 264.7 cells with either PD-98059 or
SB203580 yielded a similar inhibition pattern when LPS was used as
stimulating agent (Table I). In addition, ERK1/2 and p38 pathway
inhibitors, respectively, partially or completely inhibited cytokine
production in the human cell line THP-1 stimulated with sMALP-2 or LPS
(data not shown).
We have also assessed the effect of MAPK pathway inhibitors on
sMALP-2-mediated AP-1 and NF- M. fermentans is associated with arthritis in humans
(30, 31) and it has been proposed as a putative co-factor in AIDS progression (30, 32). The ability of mycoplasmas to induce proinflammatory cytokines and to favor Th1 Our results confirmed and extended earlier findings concerning the
macrophage activation by bacterial lipoproteins and lipopeptides. Lipoproteins from the outer membrane of Escherichia coli and
its synthetically prepared NH2-terminal lipopeptide
segments have been demonstrated to stimulate both human and murine
macrophages and to induce the secretion of IL-1, TNF By using both electrophoretic mobility shift and transactivation
assays, we have clearly shown that sMALP-2 stimulates the transcription
factors NF- Findings presented herein indicate that signaling pathways triggered in
response to sMALP-2 and involved in cytokine production include ERK1/2
and p38 MAPKs. These same pathways with identical activation kinetics
have been also implicated in macrophage response to M. fermentans membrane fractions (5). NF- Our data demonstrate that the intracellular events leading to cytokine
synthesis induced by both sMALP-2 or LPS are very similar, suggesting
that common upstream pathways are triggered by these stimuli. In
contrast to LPS, little is known about the mechanism by which bacterial
lipoproteins and lipopeptides activate macrophages. The requirement for
peptide or protein acylation strongly suggest that lipopeptides
interact via the lipid moiety with currently unknown membrane
molecule(s). Two recent papers have reported evidence suggesting that
T. pallidum and B. burdoferi lipoproteins and
synthetic lipopeptides activate monocytes by a
CD14-dependent pathway (48, 49). Actually, mouse anti-human
CD14 antibodies were shown to block the activation of monocytic cells
by these spirochetal products (48) and to significantly decrease the sensitivity of endothelial cells to Borrelia lipoproteins
(49). Very interestingly, Wooten et al. (49) have
demonstrated that OspA and CD14 can form stable complexes. Yet,
spirochetal lipoproteins and lipopeptides were shown to activate
monocytes by a CD14-dependent pathway that fundamentally
differs from that of LPS (48). We and others have previously
demonstrated that M. fermentans lipoproteins induce monocyte
activation by mechanisms distinct from that of LPS (1, 19). In this
way, and in contrast to the results reported with spirochetal products,
anti-CD14 antibodies have been shown to be inefficient in blocking the
effects of mycoplasma lipoproteins on monocytic cells (5, 20). In
addition, human monocytic cell line THP-1 does not require vitamin
D3 treatment, which increases CD14 expression on cell
membrane, to efficiently produce cytokine in response to mycoplasma
lipoproteins.2 Further
experiments are necessary to evaluate whether mycoplasma and
spirochetal derived lipoproteins and/or lipopeptides actually display
some differences in triggering the monocyte activation.
As indicated above, it has been recently reported that MALP-2 derives
from a precursor larger lipoprotein by post-translational processing
(33, 34). Relative amount of MALP-2 and its precursor lipoprotein
varied from one M. fermentans isolate to another. MALP-2 was
initially characterized by selecting for M. fermentans isolates that are strong activators of macrophages (7). Interestingly, the selected strain II-29/1 abundantly expresses MALP-2 on the cell
surface, whereas the precursor lipoprotein was undetectable (33, 34).
To our knowledge, MALP-2 is the first membrane-associated lipopeptide
so far described, and the nature of MALP-2 precursor lipoprotein
processing is presently not understood. It remains to be determined
whether the precursor lipoprotein displays an activity comparable with
that of the lipopeptide. Preliminary data from our laboratory suggest
that the recombinant precursor lipoprotein is capable of activating
macrophages, but unlike MALP-2, acylation is not required. The
confirmation of these results awaits the production of antibodies
against different epitopes of the precursor lipoprotein and MALP-2.
Given that MALP-2 is a surface-associated molecule able to activate
macrophages, a synthetic analog of this lipopeptide constitutes a
valuable tool to address the issue of the contribution of
immunomodulation by mycoplasmas to the pathogenesis.
We thank Dr. I. Saint Girons for stimulating
discussions and critical reading of the manuscript. We thank Dr.
K. S. Wise for helpful discussions related to MALP-2 biology. We
also thank Dr. J. D. Radolf (University of Texas, Southwestern
Medical School, Dallas, TX) for providing acylated cysteine, Dr. R.J.
Ulevitch (The Scripps Research Institute, La Jolla, CA) for providing
the CHO-CD14 cell line, and Dr. O. Acuto (Institut Pasteur, Immunology Moleculaire, Paris, France) for the NF-AT, NF- *
This work was supported by the Pasteur Institute.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.
The abbreviations used are:
IL, interleukin; TNF, tumor necrosis factor; LPS, lipopolysaccharide; LAMP, lipid-associated membrane protein; MAPK, mitogen-activated protein
kinase; MEK, MAPK kinase; ERK, extracellular signal-regulated kinase; JNK, c-Jun NH2-terminal kinase; MALP-2, macrophage-activating lipopeptide-2; sMALP-2, synthetic MALP-2; MOPS, 4-morpholinepropanesulfonic acid; ELISA, enzyme-linked immunosorbent
assay; EMSA, electrophoretic mobility gel shift assay; PMA, phorbol
12-myristate 13-acetate; RSV, Rous sarcoma virus; CHO, Chinese hamster ovary.
2
J. Garcia, B. Lemercier, S. Roman-Roman, and G. Rawadi, unpublished data.
A Mycoplasma fermentans-derived Synthetic Lipopeptide
Induces AP-1 and NF-
B Activity and Cytokine Secretion in Macrophages
via the Activation of Mitogen-activated Protein Kinase Pathways*
,
Université Paris VII, UFR de Biochimie, 2 place
Jussieu, 75251 Paris Cedex 05, France, and
¶ Hoechst-Marion-Roussel, Centre de Recherche Romainville, 111 Route de Noisy, 93230 Romainville, France
ABSTRACT
Top
Abstract
Introduction
Procedures
Results
Discussion
References
and tumor necrosis factor-
in human monocytes/macrophages and the murine macrophage cell line RAW
264.7, whereas the nonlipidated counterpart lacks this effect,
underscoring the importance of protein acylation for cell activation.
Synthetic MALP-2 (sMALP-2) induced the activation of MAPK family
members extracellular signal regulated kinases 1 and 2, c-Jun
NH2-terminal kinase, and p38 and induced NF-
B and AP-1
transactivation in macrophages. Whereas the specific p38 inhibitor
SB203580 abrogated both cytokine synthesis and NF-B and AP-1
transactivation in response to MALP-2, the selective MAPK/extracellular
signal-regulated kinase-1 inhibitor PD-98059 decreased interleukin-1
and tumor necrosis factor- production in response to sMALP-2 without
affecting the transactivation of NF-B or AP-1. These results
indicate that activation of MAPKs by sMALP-2 is a crucial event leading
to the expression of proinflammatory cytokines. Our findings
demonstrate that the synthetic analog of MALP-2 reproduces the
macrophage stimulation activity found in different fractions of
mycoplasmas. Given that MALP-2 has been recently shown to be expressed
at the surface of M. fermentans as a molecular entity,
sMALP-2 constitutes a valuable surrogate for investigating
immunomodulation by these microorganisms and evaluating the role that
this activity plays in the development of inflammatory diseases
associated with mycoplasma infections.
INTRODUCTION
Top
Abstract
Introduction
Procedures
Results
Discussion
References
, IL-6, IL-8,
IL-10, and tumor necrosis factor- (TNF) (1-3). Other
inflammation mediators such as NO have also been shown to be produced
by macrophages in response to mycoplasmas (3). Mycoplasmas, the
smallest self-replicating bacteria, are characterized by a wall-less
envelope (4); thus, they are LPS-free pathogens. We have previously
demonstrated that mycoplasma membrane lipoproteins (lipid-associated
membrane proteins; LAMPs) are responsible for human and murine
macrophage activation (1, 2, 5). Although the signaling pathways
triggered in macrophages by both M. fermentans LAMP and
Gram-negative bacteria LPS are comparable, unlike LPS, M. fermentans LAMP activity does not require binding to CD14 or serum
proteins (5). M. fermentans LAMP activates mitogen-activated protein kinases (MAPKs), including extracellular signal-regulated kinases 1 and 2 (ERK1/2), c-Jun NH2-terminal kinase (JNK),
and p38 (5). MAPKs have been shown to be involved in M. fermentans LAMP-mediated cytokine induction, since selective
inhibitors of these pathways have been shown to impair M. fermentans LAMP-mediated cytokine production by human and murine macrophages.
B and AP-1, two transcription factors playing a role in the
induction of proinflammatory cytokines, in macrophages challenged
with sMALP-2. sMALP-2 stimulatory effects on macrophages were fully
comparable with those previously found with M. fermentans lipoproteins, suggesting that lipopeptides derived from mycoplasmas might constitute excellent tools for better understanding the immunomodulation mediated by mycoplasmas and the role of this property
in the pathogeny of these microorganisms.
EXPERIMENTAL PROCEDURES
Top
Abstract
Introduction
Procedures
Results
Discussion
References
B isoform antibodies were purchased from Santa
Cruz Biotechnology, Inc. (Santa Cruz, CA). A p38/RK/Mpk2 assay kit was
available through Upstate Biotechnology, Inc. (Lake Placid, NY). The
p42/44MAPK enzyme assay and [
-32P]ATP
(3000 Ci/mmol) were commercially available from Amersham Pharmacia
Biotech (Les Ulis, France). Human and murine cytokine ELISAs were
obtained from R & D (Abingdon, UK) and Genzyme (Boston, MA),
respectively. All cell culture media and reagents were obtained from
Life Technologies (Cergy Pantoise, France). Consensus AP-1 and NF-B
oligonucleotides were purchased from Promega (Charbonnieres, France). LPS, PMA, and A23178 were from Sigma (L'Isle d'Abeauchesnes, France).
-glycerol phosphate, 1 mM
Na3VO4, and protease inhibitor mixture
(Boehringer GmbH, Mannheim, Germany). Lysis was performed at 4 °C
for 20 min with continuous shaking. Cell lysates were centrifuged
(10.000 × g for 10 min at 4 °C), and supernatant
was aliquoted and stored at 
80 °C. Protein concentration in cell
lysates was determined by a micro-BCA assay (Pierce).
and TNF ELISA
kit. The assays were performed according to the manufacturer's instructions.
and TNF oligonucleotide probes were from
CLONTECH Laboratories (Heidelberg, Germany). The
amount of RNA in each sample was normalized by probing for -actin.
All probes were labeled by a 5'-DNA labeling system with [-32P]dATP (Amersham Pharmacia Biotech). Slots were
prehybridized in 6× SSC, 0.5% SDS, 5× Denhardt's solution, and 100 µg/ml of denatured salmon sperm DNA for 2 h at 42 °C. An
overnight hybridization was performed at 42 °C in the same buffer
containing the probe at 2.5 × 105 cpm/ml. The
membrane was then washed twice for 10 min in 2× SSC, 0.05% SDS at
room temperature and once in 1× SSC, 0.1% SDS at 42 °C. Slots were
exposed to a PhosphorImager screen and quantitatively assessed by means
of ImageQuant software (Molecular Dynamics, Sunnyvale, CA).
-glycerol phosphate, 7.5 mM
MgCl2, 1 mM dithiothreitol, 1% Nonidet P-40)
containing 250 mM NaCl. The beads were resuspended in 50 µl of buffer B containing 10 mM MgCl2 and 1 mM MnCl2 for phosphotransferase.
-32P]ATP. The reactions were
conducted at 30 °C for 30 min and then terminated by adding SDS
sampler buffer to 1× final concentration. Samples were analyzed by
SDS-PAGE using 12% gels. Gels were fixed in 10% acetic acid and 50%
methanol and then embedded in cellophane sheets, dried, and autoradiographed.
B and
AP-1 binding activity in nuclei of uninduced and induced cells was
determined by an electrophoretic mobility gel shift assay (EMSA) as
described in Ref. 14 using 2-4 µg of nuclear proteins. EMSA gels
were exposed to a PhosphorImager screen and quantitatively assessed by
means of ImageQuant software (Molecular Dynamics). Supershift
experiments were performed by preincubating nuclear extracts with
anti-Rel family antibodies for 1 h at 4 °C before carrying out EMSA.
B, AP-1, and NF-AT (15, 16) luciferase
reporter constructs were kindly provide by Dr. A. Acuto (Institut
Pasteur, Paris, France). RAW 264.7 cells were grown up to 80%
confluence and then transfected with the indicated plasmids by the
electroporation method as described by Stacey et al. (17).
CHO-CD14 and CHO-RSV cells were grown at 80% confluence and
transfected with 15 µg of the indicated reporter plasmid by
electroporation as described in Ref. 18. Transfected cells were
cultured in growth medium for an overnight period and then stimulated
or not stimulated with sMALP-2 (200 nM), LPS (1 µg/ml)
for 6 h. In control experiments, transfected cells were stimulated
with PMA (50 ng/ml) or PMA (50 ng/ml) plus A23178 (2 µg/ml). Cells
were then harvested, and luciferase activity was determined as
described by Brasier (19) using an automated luminometer (Lumat LB
9501, EG1G Berthod, Wilbad, Germany). Cell lysates were analyzed for
protein content using the micro-BCA assay (Pierce), and luminescence
units were normalized for total protein content. Luciferase activities
are reported as means of values from four independent experiments, each
performed in duplicate.
RESULTS
Top
Abstract
Introduction
Procedures
Results
Discussion
References
and TNF
by Macrophages--
M. fermentans sMALP-2 has been
demonstrated to induce NO release from mouse macrophages (7). First, we
have investigated the potential ability of sMALP-2 to stimulate
cytokine production by monocytic cells. The murine macrophage cell line
RAW 264.7 was challenged with increased concentration of sMALP-2, and
TNF production level was determined by ELISA. TNF could be
detected in RAW 264.7 supernatant when cells were stimulated with
sMALP-2 at concentrations higher then 50 nM; the lowest
doses did not stimulate TNF production (Fig.
1A). In a 100-600
nM concentration range of sMALP-2, the TNF level
secreted by RAW 264.7 cells was found to be dose-dependent.
A similar dose-response curve was obtained when measuring IL-1
production by murine macrophages under identical conditions (data not
shown). In following experiments, sMALP-2 was used at 200 nM concentration; however, in some inhibition experiments
the highest concentrations were applied.
View larger version (26K):
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Fig. 1.
Cytokine induction mediated by sMALP-2 in the
murine macrophage cell line RAW 264.7 and in human
monocytes/macrophages. A, RAW 264.7 cells were
stimulated with increasing concentrations of sMALP-2 for 18 h, and
TNF production was measured by ELISA. Results are means ± S.D.
of three independent experiments, each performed in duplicate.
B, adherent human peripheral blood mononuclear cells
(monocytes/macrophages) from healthy donors were cultured during
24 h in the presence of either sMALP-2 (200 nM),
nonlipidated MALP-2 (nlMLAP-2; 200 nM), or
tripalmitoyl S-glycerylcysteine (ACys) (5 µg/ml). Stimulation control experiments were performed using LPS (10 ng/ml). Cytokine levels were determined by ELISA. Data are means ± S.D. of results from three distinct donors, each performed in
duplicate. C, RAW 264.7 cells were stimulated for 18 h
with sMALP-2 (200 nM), nlMALP-2 (200 nM), or
tripalmitoyl S-glycerylcysteine (5 µg/ml). Stimulation
control experiments were performed using LPS (1 µg/ml). Cytokine
levels were determined by ELISA. Results are means ± S.D. of
three independent experiments, each performed in duplicate.
D, RNA slot analysis of cytokine transcripts in RAW 264.7 cells. RAW 264.7 cells were incubated in the presence of sMALP-2 (200 nM), and total RNA was extracted from 107 cells
at the indicated times. RNA from untreated cells was used as control
(CTRL). Samples were analyzed by slot hybridization for
expression of IL-1 and TNF. As a control for equivalent RNA
loading, constitutively expressed -actin transcripts were detected.
Slots were exposed to a PhosphorImager screen for 2-4 h. The slot
shown herein is representative of three different experiments.
production, whereas polymyxin B completely abolished TNF
induction by LPS (data not shown). These data and the results of
the Limulus amoebocyte lysate assay (see "Experimental
procedures") clearly indicate that the stimulatory effect of sMALP-2
preparations are not to be ascribed to endotoxin contamination.
and TNF by murine macrophage RAW 264.7 (Fig. 1B)
and by human monocytes/macrophages as well (Fig. 1C).
Similar results were obtained using the human myelomonocytic cell line
THP-1 (data not shown). When human monocytes/macrophages or murine
macrophages RAW 264.7 were challenged with LPS, TNF, and IL-1
secretion levels were comparable with those obtained in response to
sMALP-2 (Fig. 1, B and C). The nonlipidated form of MALP-2 failed to induce cytokine production by both human
monocytes/macrophages and RAW 264.7 cells. In addition, tripalmitoyl
S-glycerylcysteine was unable to stimulate cytokine
production by these cells (Fig. 1, B and C).
These findings demonstrate that lipid modification of MALP-2 peptide is
required for macrophage stimulation and cytokine production.
and TNF mRNA synthesis, with a peak accumulation found
at about 4 h after challenge.
B and AP-1 transactivation in CHO cells expressing human CD14
(11). CHO cells expressing CD14 (CHO-CD14) were transiently transfected
with NF-B or AP-1 luciferase reporter plasmid, and then cells were
stimulated with LPS and sMALP-2. CHO cells harboring the empty
expression vector (CHO-RSV) were transiently transfected with reporter
plasmids and stimulated as described above. As expected, LPS induced an
important luciferase activity (4-5-fold increases) in CHO-CD14 cells
transfected with either NF-B or AP-1 reporter plasmids, while no
activation could be observed with CHO-RSV under similar conditions
(Fig. 2). On the contrary, sMALP-2
induced neither NF-B nor AP-1 transactivation in transfected
CHO-CD14 cells (Fig. 2), even when it was used at higher concentrations
(data not shown). Control experiments were performed by stimulating
transfected cells with PMA. In both CHO-CD14- and CHO-RSV-transfected
cells, PMA stimulated NF-B and AP-1 transactivation (Fig. 2). These
data suggest that, unlike LPS, sMALP-2 does not require CD14 for cell
stimulation.
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Fig. 2.
sMALP-2 does not activate NF- B or AP-1 in
the CHO-CD14 cell line. CHO cells expressing the human CD14
(CHO-CD14; left panel) or harboring
the empty expression vector (CHO-RSV; right
panel) were transiently transfected with either
NF-B/luciferase or AP-1/luciferase reporter plasmid. Transfected
cells were either left untreated or treated with sMALP-2 (200 nM), LPS (100 ng/ml), or PMA (100 ng/ml). Luciferase
activity was determined in cell lysates after 6-h incubation and
normalized to protein content. Values from four distinct
experiments ± S.D., each performed in duplicate, are expressed as
the -fold increase in luciferase activity seen following treatment
relative to the luciferase activity measured in the absence of any
stimulus.
B and AP-1 Transcription
Factors in Macrophages--
MAPKs are key molecules involved in
macrophage activation by different bacterial products. We have
therefore investigated the activation of the well characterized MAPKs,
ERK1/2, p38, and JNK, in RAW 264.7 cells challenged with sMALP-2. Data
presented in Fig. 3 clearly indicate that
sMALP-2 significantly induced activation of all of the tested kinases.
LPS has also been shown to induce activation of MAPKs in murine
macrophages (21). MAPK activation kinetics in RAW 264.7 stimulated by
LPS and sMALP-2 were compared. As shown in Fig. 3, LPS peak activation
of ERK1/2, p38, and JNK was found at 15 min after stimulation, while
peak activation of these kinases occurred at 30 min after challenge with sMALP-2. The kinetics of MAPK activation by sMALP-2 were comparable with those obtained when cells were stimulated with a crude
extract of M. fermentans membrane lipoproteins (5). A
similar pattern of MAPK activation was observed in human
monocytes/macrophages and the THP-1 cell line stimulated with sMALP-2
(data not shown).
View larger version (47K):
[in a new window]
Fig. 3.
sMALP-2 induces the activation of MAPK
pathways in murine macrophages. RAW 264.7 cells were stimulated
with sMALP-2 (200 nM) or with LPS (1 µg/ml) for different
time intervals (10, 15, 30, and 60 min). Immunoprecipitation
(IP) and phosphotransferase activity (PA)
measurement were performed as described in "Experimental
Procedures." A, ERK1/2 (left panel)
were immunoprecipitated from cell lysates using anti-ERK1-CT antibody,
and their activation was quantified using the p42/44MAPK
detection kit (Amersham Pharmacia Biotech) according to the
manufacturer's instructions. p38 (right panel)
was immunoprecipitated from cell lysates using anti-p38 antibody, and
its activation was quantified using the p38/RK/Mpk2 detection kit
(Upstate Biotechnology, Inc.) according to the manufacturer's
instructions. Untreated cells were used as control (CTRL).
Results are means ± S.D. of three independent assays, each
performed in duplicate. B, JNK was immunoprecipitated from
cell lysates using JNK1 antibody (anti-JNK1 antibody).
Immunoprecipitates were incubated with GST-c-Jun under phosphorylating
conditions, and phosphorylated GST-c-Jun (arrow) was
visualized by gel electrophoresis and autoradiography. Untreated cells
were used as control (0). The presented gels are
representative of three distinct experiments.
B and AP-1 are involved in the
transcription of a variety of genes, including proinflammatory
cytokines, during the immune response (22-24). Thus, we have addressed
the ability of sMALP-2 to activate nuclear translocation of the
transcription factors NF-B and AP-1. Using 32P-labeled
oligonucleotides containing consensus NF-B or AP-1, we have
performed EMSAs on nuclear extracts from RAW 264.7 cells stimulated
with sMALP-2. As demonstrated in the Fig.
4, sMALP-2 was capable of inducing both
NF-B and AP-1 translocation. The specificity of AP-1 and NF-B DNA
biding were verified by competition analysis with an excess of
nonradiolabeled probes. NF-B and AP-1 activation by sMALP-2 was
found to be dose-dependent, and transcription factors were
detectable starting from 100 nM of sMALP-2 concentration, confirming data presented in Fig. 1 (data not shown).
View larger version (81K):
[in a new window]
Fig. 4.
sMALP-2 induces transcription factor NF- B
and AP-1 translocation. RAW 264.7 cells were stimulated with
sMALP-2 (200 nM) or LPS (1 µg/ml) for different time
intervals (1, 2, 4, and 6 h), and untreated cells were used as control (0).
Nuclear extracts and EMSA were performed as indicated under
"Experimental Procedures." A, NF-B EMSA. Nuclear
extracts from cells stimulated with sMALP-2 (left
part) or with LPS (right part) were
probed with 32P-labeled NF-B consensus oligonucleotide.
The specificity of DNA binding was assessed by preincubating extracts
with unlabeled specific (NF-B) or nonspecific (AP-1) competitor
oligonucleotide at a 50-fold molar excess. The arrow
indicates a specific NF-B band. B, AP-1 EMSA. Nuclear
extracts from cells stimulated with sMALP-2 were probed with
32P-labeled AP-1 consensus oligonucleotide. The specificity
of DNA binding was assessed by preincubating extracts with unlabeled
specific (AP-1) or nonspecific (AP-2) competitor oligonucleotide at
50-fold molar excess. The right part presents
AP-1 DNA binding activity from EMSA gel presented herein quantified by
PhosphorImager. EMSA experiments were repeated four times, and gels
presented in the figure are from a representative set.
B translocation was more rapid (with a peak found at
2 h of stimulation) than that observed with sMALP-2 (peak at 4 h). The kinetics of NF-B activation mediated by LPS reported herein were comparable with those previously described by Tebo et
al. (25). We have further investigated by means of EMSA supershift the NF-B isoforms induced by sMALP-2. The NF-B p65 and p50
antibodies supershifted the protein complex, whereas c-Rel and RelB
antibodies did not modify the pattern (data not shown), indicating that
a p65/p50 heterodimer accounts for the NF-B-translocated form.
B induction by sMALP-2, we
have studied the transactivation ability of these factors by means of
luciferase reporter plasmids. RAW 264.7 cells were transiently
transfected with plasmids containing the AP-1- or NF-B-dependent luciferase reporter gene and then
stimulated with sMALP-2, and luciferase activity was determined in cell
lysates. As demonstrated in Fig. 5,
sMALP-2 induced a 3-fold increase in luciferase activity in cells
transfected by either AP-1 or NF-B reporter plasmid, indicating that
the translocated factors can mediate transactivation. sMALP-2 failed to
induce transactivation of the T cell-specific transcription factor
NF-AT-dependent luciferase reporter gene. As a positive
control, stimulation of NF-AT-transfected RAW 264.7 cells with
PMA/A23187 (26) induced a 4-fold increase in specific luciferase
activity (data not shown). Comparable levels of NF-B- and
AP-1-mediated luciferase induction were observed in LPS stimulation
experiments (Fig. 5). sMALP-2 also induced AP-1- and
NF-B-dependent luciferase expression in THP-1 cells (data not shown).
View larger version (41K):
[in a new window]
Fig. 5.
NF- B and AP-1 transactivation mediated by
sMALP-2. RAW 264.7 cells were transiently transfected with either
NF-B- or AP-1-dependent luciferase reporter plasmids.
Transfected cells were stimulated with sMALP-2 (200 nM) or
LPS (1 µg/ml) for 6 h prior to cell lysis. NF-AT luciferase
reporter plasmid was used as a negative control. Luciferase activity
was assessed in stimulated and unstimulated cells (CTRL) and
normalized to protein content. Values from three distinct
experiments ± S.D., each performed in duplicate, are expressed as
the -fold increase in luciferase activity measured following
stimulation relative to the luciferase activity observed with no
stimulation.
B Activation--
To examine the involvement of
MAPK activation in sMALP-2-mediated cytokine production, we have used
the specific inhibitors of MAPK pathways, SB203580 and PD-98059 (27,
28). At the highest used concentration, these two inhibitors, PD-98059
and SB203580, did not induce any cell toxicity as determined by
microscopic observation and trypan blue uptake (data not shown).
and TNF
production (Table I). Comparable
inhibition effects of SB203580 were obtained when cells were stimulated
with higher concentrations of sMALP-2 (data not shown).
Effect of p38 and MEK-1 specific inhibitors on sMALP-2- and
LPS-induced cytokine production
and IL-1 levels were measured 18 h after stimulation. Data
presented are the mean ± S.D. of three independent experiments, each
performed in duplicate. The percentage of cytokine inhibition (p
< 0.01) was determined in inhibitor treated cells versus
Me2SO treatment and indicated in
parentheses.
B transactivation. RAW 264.7 cells were
transiently transfected with either AP-1 or NF-B reporter plasmids,
and then cells were treated with PD-98059 or SB203580 prior to
stimulation with sMALP-2. Whereas the MEK-1 inhibitor, PD-98059, at a
concentration that inhibited 50% of cytokine production (see Table I)
or higher, affected neither AP-1- nor NF-B-dependent luciferase transactivation (Fig. 6),
SB203580 significantly reduced both AP-1- and NF-B-mediated
transactivation (Fig. 6). The effect of SB303580 was
dose-dependent and at 30 µM completely
blocked AP-1 and NF-B activities. Similar inhibitions were obtained
when cells were stimulated with higher sMALP-2 concentrations (600 µM; data not shown). We have additionally assessed the
effect of these inhibitors on LPS-induced NF-B or AP-1
transactivation. In these experiments, inhibition profiles comparable
with that reported with sMALP-2 were observed (data not shown). These
data underscore the involvement of p38 pathway in the nuclear response to bacterial modulin in macrophages, while the ERK1/2 pathway appears
to control distinct events.
View larger version (34K):
[in a new window]
Fig. 6.
Effect of p38- and MEK-1-specific inhibitors
on sMALP-2-induced transcription factor activation in murine
macrophages. RAW 264.7 cells were transiently transfected with
either NF- B- or AP-1-dependent luciferase reporter
plasmids. Transfected cells were treated for 1 h with either
SB203580 (p38 inhibitor) or PD-98059 (MEK-1 inhibitor) at the indicated
concentrations and then stimulated with sMALP-2 (200 nM)
for 6 h prior to cell lysis. Me2SO (DMSO)
(1%) was used as solvent control. Luciferase activity was assessed in
stimulated and unstimulated cells (CTRL) and normalized to
protein content. Values from three distinct experiments ± S.D.,
each performed in duplicate, are expressed as the -fold increase in
luciferase activity measured following stimulation relative to the
luciferase activity observed in unstimulated cells.
DISCUSSION
Top
Abstract
Introduction
Procedures
Results
Discussion
References
Th2 switch might participate in the pathogenesis mechanisms of these bacteria (1, 2).
Previous findings have clearly indicated that membrane lipoprotein
fractions from M. fermentans are responsible of cytokine induction in macrophages. The precise characterization of the biochemical entities involved in such activation is an important step
toward the understanding of the interaction of mycoplasmas and immune
cells and necessary to delineate their contribution to the mycoplasma
pathogenesis and clinical manifestations. MALP-2, a lipopeptide
characterized from M. fermentans, has been shown to strongly
induce NO synthesis by macrophages (7). Very recently, it has been
reported that MALP-2 is a mycoplasma membrane-associated entity
resulting from the processing of a precursor lipoprotein (33, 34). In
the present study, we show that a synthetic analog of MALP-2 is capable
of inducing the activation of NF-B and AP-1 and the cytokine
production by murine macrophages. We have also demonstrated the
involvement of MAPK pathways in the signaling events triggered by
MALP-2.
, and IL-6 (35,
36). Spirochetal lipoproteins have also been shown to stimulate
macrophages (37), and the lipid modification determines the ability to
stimulate the production of cytokines by these cells (38). The
importance of protein acylation to cell activation was further
underscored by the fact that synthetic lipohexapeptides corresponding
to the NH2 termini of the 47-kDa lipoprotein of
Treponema pallidum and the acylated outer surface protein A
(OspA) of Borrelia burdoferi also activated macrophages in
terms of cytokine secretion, whereas the nonlipidated hexapeptides were
without effect (39). In the present report, we also provided evidence
showing that macrophage activation by MALP-2 is dependent upon
acylation. Whereas both native MALP-2 and its synthetic analog have
been previously shown to induce NO release from murine macrophages at a
1-100 pM range of concentrations (7), the effect of
sMALP-2 on cytokine production by macrophages occurs at 100-600
nM concentration. For NO release assay, the simultaneous
stimulation with IFN- and MALP-2 (7) could at least in part account
for the significant high sensitivity of macrophages and the requirement
of lower concentration of lipopeptide to induce NO activity with
respect to cytokine production.
B and AP-1. It has been previously reported that T. pallidum and B. burgdorferi lipoproteins and respective synthetic lipopeptides induce NF-B translocation in different cell
types (40, 41) with kinetics more rapid (maximal stimulation from 1 to
2 h) than that obtained with sMALP-2 (peak at 4 h). Interestingly, the kinetics of NF-B and AP-1 induction by LPS were
similar to that reported for the spirochetal products. It would be
interesting to test whether the observed differences can be ascribed to
the different lipopeptide structures.
B and AP-1 triggering by
sMALP-2 also involved MAPK pathways. In agreement with previously published reports indicating that p38 MAPK is required for the activation of several transcription factors, including cAMP response element-binding protein (42), c-Fos, and c-Jun (30-32, 43-45), we
have shown that p38 pathway activation is required for AP-1 induction
in response to sMALP-2. Interestingly, the p38 pathway was also found
to be required for NF-B response in sMALP-2-stimulated macrophages.
Very recently, Bergmann et al. have shown that p38 inhibitor, SB203580, blocked NF-B-mediated luciferase
transactivation in response to TNF without affecting NF-B
translocation (46). Accordingly, in our cells, SB203580 was unable to
inhibit NF-B translocation in response to sMALP-2 (data not shown).
At present, how p38 MAPK may affect, directly or indirectly, NF-B
function is unclear, and the question remains open; however, our data
underscore the important contribution of this pathway in nuclear
response to bacterial lipopeptide stimuli. ERK1/2 has been clearly
shown to contribute to the cell signaling resulting in cytokine
production, but unlike the p38 MAPK pathway, it is not involved in
sMALP-2-mediated NF-B or AP-1 transactivation. Several other studies
have shown that although the p42/44 MAP kinase pathway is activated in
response to distinct stimuli including anisomycin, fibroblastic growth factor, epidermal growth factor,
12-O-tetradecanoylphorbol-13-acetate, and UV irradiation, it
is not involved in nuclear signaling (42, 44, 47). The signaling events
controlled by ERK1/2 pathway remains to be clearly defined.
ACKNOWLEDGEMENTS
B, and AP-1 reporter plasmids. We acknowledge A. Henry-Dujeancourt and C. Prevost for technical assistance.
FOOTNOTES
To whom correspondence should be addressed. Tel.:
33-1-45688739; Fax: 33-1-40613356; E-mail: rawadi21{at}pasteur.fr.
REFERENCES
Top
Abstract
Introduction
Procedures
Results
Discussion
References
Copyright © 1998 by The American Society for Biochemistry and Molecular Biology, Inc.
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