| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
*
Institut Pasteur, Laboratoire des Mycoplasmes, Paris; and
Hoechst-Marion-Roussel, Centre de Recherche Romainville, Romainville, France
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
synthesis. The selective MAPK/extracellular signal-regulated
kinase 1 (MEK-1) inhibitor PD-98059 blocked both IL-1ß and TNF-
but not IL-6 production by RAW 264.7 cells in response to LAMPf.
Additionally, transfection of murine macrophages with a JNK dominant
negative mutant significantly reduced only IL-6 production. These data
underscore the role of MAPKs as signal transduction molecules
controlling the expression of cytokines upon mycoplasma stimulation. |
Introduction |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Stimulation of monocytes and resident macrophages by mycoplasmas
induces the production of numerous cytokines (i.e., IL-1ß, TNF-,
IL-6, IL-10, IFN-
, and granulocyte/mcrophage-CSF) (4, 5, 6). This
immunomodulatory effect seems to reside in the lipid-associated
membrane protein
(LAMP)2
fraction of mycoplasmas (4). We have previously reported that protein
tyrosine phosphorylation is an early event in macrophage activation by
LAMP and that blocking of protein tyrosine kinases (PTKs) inhibits
downstream pathways leading to cytokine production in response to LAMP
(4). However, the biochemical events involved in the mycoplasma-induced
cytokine synthesis by macrophages are poorly understood.
Mitogen-activated protein kinases (MAPKs) are a group of serine/threonine-specific, proline-directed protein kinases that are activated by a wide spectrum of extracellular stimuli. They are important mediators involved in the intracellular network of interacting proteins that transduce extracellular signals to intracellular responses. To date, several distinct MAPKs expressed in vertebrates have been identified, including extracellular signal-regulated kinase 1 and 2 (ERK1/2), c-Jun NH2-terminal kinase (JNK)/stress-activated protein kinases (SAPK), and p38/RK/Mpk2 (7). Each of these effectors is regulated by other upstream kinases. MAPK/ERK kinase 1 (MEK-1) is responsible of ERK1/2 activation, and Raf1 is the kinase upstream of MEK-1. Likewise, SEK1 (stress-activated protein kinase/ERK kinase) is responsible of SAPK/JNK phosphorylation, and MEKK1 (mitogen-activated protein kinase/ERK kinase kinase) is the upstream effector of SEK1. MKK3 (mitogen-activated protein kinase kinase homologue) activates p38, which in turn phosphorylates a MAPKAPK2 (MAPK-activated protein kinases 2) (7).
Bacterial Gram-negative LPS, a very potent inducer of cytokine
synthesis by monocytic cells, has recently been demonstrated to
activate multiple MAPK-related pathways (8). To examine further aspects
of signaling pathways involved in macrophage activation by mycoplasmas,
we tested the ability of M. fermentans-derived LAMP (LAMPf)
to induce the activation of the different MAPKs and the involvement of
these pathways in cytokine synthesis. The present study demonstrates
that treatment of murine macrophage cells with LAMPf results in the
activation of ERK, JNK, and p38 kinases. Furthermore, our results show
that MAPK pathways are involved in LAMPf-induced IL-1ß, TNF-, and
IL-6 production by murine macrophages. Unlike LPS, LAMPf do not require
serum proteins or cell surface CD14 to exert these effects.
|
Materials and Methods |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
LPS from Escherichia coli O55:B5, myelin basic
protein (MBP), and polymyxin B were purchased from Sigma (Saint
Quentin, Fallavier, France). Genistein, PD-98059, SB203580, and
GST-c-Jun(1–79) were from Biomol Research Laboratories (Philadelphia,
PA). Triton X-114 (TX-114) was obtained from Merck (Nogent sur Marne,
France). Anti-JNK1 (C17), anti-ERK (K23), and anti-p38 (C20)
polyclonal Abs were from Santa Cruz Biotechnology (Santa Cruz, CA).
Anti-phosphotyrosine and agarose-coupled anti-phosphotyrosine mAbs
(anti-PY 4G10 Ab), anti-rat MAPK R2 (anti-ERK1-CT),
GST-MAPKAPK2, and p38/RK/Mpk2 assay kits were obtained through Upstate
Biotechnology (Lake Placid, NY). Peroxidase-coupled
anti-phosphotyrosine mAb (anti-PY RC20 Ab) was from
Transduction Laboratories (Lexington, KY). Enhanced chemiluminescence
kit (ECL), p42/44MAPK enzyme assay, and
[-32P]ATP (3000 Ci/mmol) were commercially available
from Amersham (Les Ulis, France). MY4, an anti-human CD14 mAb, was
purchased from Coulter Diagnostics (Hialeah, FL).
Mycoplasma culture, inactivation, and membrane lipoproteins preparation
Mycoplasma fermentans (PG18 strain) and Mycoplasma pneumoniae (FH LIV strain) were cultivated in medium containing 20% horse serum (Life Technologies, Cergy Pontoise, France), 10% freshly prepared yeast extract, 1% glucose and 1000 U/ml penicillin G. Mycoplasma cultures were incubated at 37°C and 5% CO2, then quantified as described by Rodwell and Whitcomb (9) and expressed as CCU (color changing unit) per milliliter. For heat inactivation, mycoplasmas were isolated by centrifugation (15,000 x g at 4°C for 30 min), washed, and resuspended in Hayflick medium at 107 CCU/ml, followed by incubation at 60°C for 30 min. Heat-inactivated mycoplasmas (HIM) were stored at -70°C until needed. Complete heat inactivation was verified by inoculation of 106 CCU into 10 ml of culture medium to monitor the growth of mycoplasmas; no growth could be observed over a 2-wk period. Mycoplasma membrane lipoproteins were prepared by hydrophilic/hydrophobic fractionation using the TX-114 partitioning method as described previously (10). Protein concentrations were determined by microBCA assay (Pierce, Rockford, IL). The endotoxin level of both HIM and LAMP preparations was <60 pg/ml, as determined by Limilus amebocyte lysate assay (Haemachem, St. Louis, MO).
Cell cultures, stimulation, and lysate preparation
The murine macrophage cell line RAW 264.7, HeLa cell line, and 3T6 cell line (from American Type Culture Collection, Rockville, MD) were cultured (37°C, 5% CO2) in DMEM culture medium (Life Technologies) containing 10% FCS (Life Technologies), 2 mM L-glutamine, and antibiotics. The human monocytic cell line THP-1 was cultured (37°C, 5% CO2) in RPMI culture medium (Life Technologies) containing 10% FCS, 2 mM L-glutamine, and antibiotics. Cell lines were tested every 2 wk by a PCR-based detection assay for mycoplasma contamination (11). For stimulation experiments, cells were seeded at 106 cells/ml density and then cultivated overnight. Cells were stimulated with either HIM (106 CCU/ml) or LAMP (1 µg/ml) 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, cell lysates were prepared as described elsewhere (8). Briefly, cells were washed twice with ice-cold PBS containing 1 mM Na3VO4. For each 106 cells that were 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 DTT, 75 mM ß-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 x g for 10 min at 4°C), and supernatant was aliquoted and stored at -80°C. Protein concentration in cell lysates was determined by microBCA assay (Pierce).
In experiments in which chemical inhibitors or anti-CD14 Ab were used, cells were pretreated for 1 h with various concentrations of each item before stimulation.
Human monocyte culture and stimulation
Monocytes were prepared from PBMC by Ficoll/Hypaque density gradient centrifugation (Pharmacia LKB, Uppsala, Sweden). 12 x 106 PBMC/well were allowed to adhere to six-well tissue culture plates (Costar, Cambridge, MA) for 1 h at 37°C, 10% CO2, in DMEM containing 1% human serum (CNTS, Saint Antoine, France), 2 mM L-glutamine, and antibiotics. To remove nonadherent cells, wells were washed twice with prewarmed culture medium. Adherent cells (approximately 1.2 x 106 cells/well) were cultured (37°C, 10% CO2) in 2 ml of culture medium overnight. Human monocytes were stimulated with LAMPf (1 µg/ml) for appropriate time intervals, and cell lysates were prepared for phosphotransferase assays as described above.
Assay for cytokines
RAW 267.4 cells and THP-1 cells were cultivated and stimulated
in 24-well tissue culture plates (Costar) as described above. After
24 h of stimulation, cells were lysed by two consecutive cycles of
freezing/thawing. Thus, the samples represented the total amount of
cytokines produced (both intracellular and those that have been
released into the supernatant). The cytokine concentration from murine
macrophages was measured using murine IL-1ß, IL-6, and TNF- ELISA
from Genzyme (Boston, MA). For cytokine measurement from the human
monocytic cell line THP-1, cytokine ELISA kits from R&D Systems
(Abingdon, U.K.) were used. The assays were performed according to the
manufacturer’s instructions.
Immunoprecipitation of PTK, ERK, JNK, and p38 from stimulated cell lysates
For total tyrosine-phosphorylated protein immunoprecipitation, 500 µg of proteins from cell lysates were incubated with 10 µg of agarose-coupled anti-PY 4G10 Ab overnight at 4°C with continuous rotation. The mixtures were then centrifuged (7000 x g for 2 min at room temperature), and agarose beads were washed three times with PBS buffer and resuspended in 20 µl of SDS sample buffer. Following electrophoresis, immunoblot was performed using a second peroxidase-coupled ant-PY RC20 Ab.
ERK1/2 and JNK were immunoprecipitated by incubating 500 µg of cell lysates with 2 µg of anti-JNK1 Ab or 5 µg of anti-ERK1-CT Ab, 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 2 more hours. The mixtures were then centrifuged (7000 x 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 ß-glycerol phosphate, 7.5 mM MgCl2, 1 mM DTT, 1% Nonidet P-40) containing 250 mM NaCl. The beads were resuspended either in 50 µl of buffer B containing 10 mM MgCl2 and 1 mM MnCl2 for phosphotransferase assays or in 20 µl of SDS sample buffer for electrophoresis and immunoblotting using anti-PY 4G10 Ab. For p38 immunoprecipitation, the protocol was slightly modified. Anti-p38 Ab (10 µg) was first coupled to A/G-Sepharose beads (Santa Cruz Biotechnology) for 2 h at 4°C, then washed with buffer B, and then added to 500 µg of cell lysates. Immunoprecipitation was allowed overnight at 4°C with continuous rotation and immunoprecipitates were analyzed as described above.
Electrophoresis and immunoblotting
SDS-PAGE was performed on 12% separating gels. Samples were boiled for 10 min in the presence of SDS sample buffer. After electrophoresis, proteins were transferred onto a polyvinylidene difluoride membrane (Immobilon-P; Millipore, Molshein, France). Membranes were blocked overnight at 4°C with PBS containing 3% BSA, 1% goat serum, and 0.01% Tween-20. The membranes were washed twice with PBS containing 150 mM NaCl and 0.1% Tween-20, then incubated for 1 h at room temperature with the anti-PY 4G10 Ab or peroxidase-coupled anti-PY RC20 Ab at 1/1500 dilution in PBS containing 0.3% BSA and 0.1% Tween-20. Membranes were washed five times and directly subjected to ECL detection when peroxidase-coupled anti-PY RC20 Ab was used. Otherwise, membranes were incubated with a second Ab, a goat anti-rabbit coupled to peroxidase, washed five times, and then processed for ECL detection as described in the manufacturer’s instructions.
Measurement of phosphotransferase activity
10 µg of MBP, 2 µg of GST-c-Jun, or 200 ng of GST-MAPKAP K2
in the presence 50 µM [-32P]ATP were added to
ERK1/2, p38, or SAPK/JNK immunoprecipitates, respectively. The
reactions were conducted at 30°C for 30 min duration, then terminated
by adding SDS sample buffer to 1x final concentration. Samples were
analyzed by SDS-PAGE using 12% gels for GST-MAPKAP K2 and GST-c-Jun
and 16% gels for MBP. Gels were fixed in 10% acetic acid and 50%
methanol, then embedded in cellophane sheets and dried. Dried gels were
first autoradiographed for 3 to 16 h to qualitatively determine
ERK1/2, JNK, and p38/Mpk2 activity by visualization of phosphorylated
MBP, GST-c-Jun, or GST-MAPKAPK2, respectively, and quantitatively
evaluated using a PhosphorImager and ImageQuant software (Molecular
Dynamics, Sunnyvale, CA).
ERK and p38 activation was additionally determined by measuring radioactively their respective phosphotransferase activities toward peptide substrate using p42/44MAPK or p38/MpK detection kits. Assays were performed according to the manufacturer’s instructions. Activity was expressed as [32P]ATP cpm.
Plasmid and cell transfection
Constructs expressing JNK1 dominant negative mutant (dnJNK-APF) and JNK1 wild-type (JNK-WT) were kindly provided by Dr. B. Dérijard (CNRS, Nice, France) and Dr. R. J. Davis (University of Massachusetts, Boston, MA). RAW 264.7 cells were grown up to 80% confluence, then transfected with the indicated plasmids by the electroporation method described by Stacey et al. (12). After transfection, cells were cultivated for 36 h before subsequent treatment. Cells were stimulated with LAMPf, and cytokine production was measured as described above.
|
Results |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Mycoplasmas, and more precisely their lipid-associated membrane
proteins (or LAMPs), have been demonstrated to interact with monocytes
and induce proinflammatory cytokine synthesis by these cells. We
selected the murine macrophage cell line RAW 264.7 to analyze the
biochemical events triggered by mycoplasmas in macrophages, because
this cell line have been extensively used to characterize signaling
pathways in response to extracellular stimuli such as bacterial LPS
(8). First, we assessed the capability of heat-inactivated M.
fermentans (HIMf) as well as LAMP extracted from this species
(LAMPf) to induce cytokine production by RAW 264.7 cells. Cells treated
with either HIMf (106 CCU/ml) or LAMPf (1 µg/ml) for an
18-h period produced a significant amount of IL-1ß, IL-6, and TNF-
(Fig. 1
A). As with
other monocytic cell lines, IL-1ß was found to be almost exclusively
intracellular in RAW 264.7 (4, 13). M. pneumoniae-derived
LAMP preparations (LAMPp) (Fig. 1A) as well as
heat-inactivated M. pneumoniae (data not shown) failed to
induce cytokine production by murine macrophages, thus indicating that
this biologic activity is not a general rule for any mycoplasma
species. Cytokine production by LAMPf-treated murine macrophages was
found to be dose dependent in a concentration range from 10 ng/ml to 1
µg/ml of lipoproteins (Fig. 1B). At concentrations
higher than 1 µg/ml, the stimulation effect declined. Similar results
were obtained with the human monocytic cell line THP-1 (data not shown;
(14)).
|
A for TNF-, and data not
shown). Additionally, the anti-CD14 mAb MY4, which inhibited
LPS-induced cytokine production by human monocytes, had no effect on
cytokine production by LAMPf-stimulated human monocytic THP-1 cells
(Fig. 2B).
|
PTK activation is one of the earliest responses of monocytes to
LAMPf (4); we therefore examined phosphotyrosine-containing proteins in
RAW 264.7-LAMPf-stimulated cells. Cell lysates were immunoprecipitated
with an agarose-coupled anti-PY 4G10 mAb, electrophoresed on
SDS-PAGE, and immunoblotted using a second anti-PY RC20 mAb. As
shown in Figure 3A, LAMPf
enhanced tyrosine phosphorylation of several proteins, with the maximal
effect occurring at 30 min after challenging the cells. PTK activation
plays a crucial role in cytokine induction by mycoplasmas because
genistein, a well-known inhibitor of PTKs, completely blocked cytokine
synthesis induced in RAW 264.7 cells by LAMPf (Fig. 3B).
|
It has recently been demonstrated that LPS stimulation of murine
macrophages leads to increased tyrosine phosphorylation and activation
of the three subgroups of MAPK identified in mammals: ERK, JNK, and p38
(8). Since both LAMPf and LPS have been demonstrated to induce the
synthesis of proinflammatory cytokines by monocytic cells, we examined
whether LAMPf was capable of activating the MAPK pathways. RAW 264.7
cells were treated with LAMPf for different time intervals and cell
lysates tested for ERK1/2, SAPK/JNK, and p38 activation by measuring
their respective phosphotransferase activities toward different
substrates. Activation of ERK1/2 was first determined by measuring the
phosphorylation of MBP after the immunoprecipitation of lysates with an
anti-ERK Ab. This assay allowed the detection of a significant
phosphotransferase activity in lysates from LAMPf (1 µg/ml)-treated
macrophages (Fig. 4A).
Similar results were obtained when an ERK1/2-specific peptide was used
as a substrate to determine the phosphotransferase activity in total
cell lysates (Fig. 4B). In both assays maximal
activity was detected at 30 min of stimulation. At 60 min, this
activity declined to 60% of the maximal value. Substrate
phosphorylation paralleled an increase in both ERK1 and ERK2 tyrosine
phosphorylation. As shown in Figure 4C, immunoprecipitation
with an ERK1/2 polyclonal Ab and Western blotting with an anti-PY
4G10 mAb allowed the detection of two bands at a molecular mass of 44
and 42 kDa.
|
A) or a
p38-specific peptide as substrate (Fig. 5B). In both
assays, significant p38 phosphotransferase activity was found in cell
lysates from LAMPf-treated RAW 264.7 macrophages compared with
untreated cells. p38 activation in response to LAMPf peaked at 30 min,
but the kinetics differed from ERK1/2 activation: 1 h after LAMPf
challenge, the p38 phosphotransferase activity dramatically dropped to
10% of maximal activity. p38 was demonstrated to undergo tyrosine
phosphorylation by immunoprecipitation with a specific p38 Ab followed
by immunoblotting with the anti-PY 4G10 mAb (Fig. 5C).
|
,
stimulation of RAW 264.7 cells with LAMPf strongly induced JNK
activity. Once again, the maximal activation occurred at 30 min after
stimulation.
|
show that M.
fermentans lipoproteins induce the activation of ERK (Fig. 7A), p38 (Fig. 7B), and JNK (Fig. 7C) in these cells. The time course activation of
MAPK pathways observed in human monocytes was comparable with that
observed using the murine macrophage cell line RAW 264.7. The same
results were obtained using elutriated human monocytes from healthy
donors (data not shown).
|
A). Interestingly, LAMPp
also did not induce any MAPK activation in these cells (data not
shown). We did not observe any differences in MAPK activation when
LAMPf preparations were preincubated with polymyxin B (1000 U/ml) for
1 h before cell stimulation (data not shown), thus indicating that
the observed effect does not account for endotoxin contamination.
A serum protein named LBP (i.e., LPS-binding protein) has been
identified as a major mediator of LPS effects (15, 16). We therefore
investigated the requirement of serum to mediate the effect of LAMPf on
murine macrophages. While the absence of serum completely abolished the
activation of MAPK pathways in LPS-treated RAW 264.7 cells, it did not
significantly affect the MAPK activation induced by LAMPf treatment
(Fig. 8A).
Interestingly, the presence of serum is not absolutely required for the
induction of cytokines by mycoplasmas. As depicted in Figure 8B, 18-h serum starvation slightly reduced the cytokine
production by mycoplasma-stimulated macrophages, whereas it prevented
cytokine induction by LPS.
|
).
Control treatment with PMA (50 ng/ml) showed an increased MAPK and p38
phosphotransferase activity in a time interval as short as 10 min after
challenge. The same results were obtained with the murine fibroblast
cell line 3T6 (data not shown). This suggests that M.
fermentans triggers MAPK activation in a cell-specific manner.
|
To examine the involvement of MAPKs in mycoplasma-mediated cytokine production by murine macrophages, we specifically blocked each of the three MAPK pathways and monitored the cytokine production when RAW 264.7 cells were challenged with LAMPf.
PD-98059 is a synthetic compound that specifically inhibits the
ERK-activating MAPK kinase MEK-1 (17, 18). This compound selectively
inhibited the LAMPf-mediated activation of ERK1/2 in murine macrophages
without significantly affecting the stimulation of p38 or JNK (data not
shown). As shown in Figure 10A, PD-98059 decreased
TNF- and IL-1ß production by LAMPf-stimulated RAW 264.7 cells in a
dose-dependent manner. TNF- production was completely blocked at 10
µM of PD-98059, and IL-1ß production was inhibited up to 80% at 30
µM. On the contrary, only 20% IL-6 inhibition could be observed at
the highest dose used (30 µM).
|
B, pretreatment of cells with 1 µM of SB203580
completely abrogates mycoplasma-mediated IL-1ß and TNF-
production. A higher dose of this compound was necessary to completely
block the IL-6 production (Fig. 10B).
To investigate the involvement of the JNK pathway, RAW 264.7 cells were
transiently transfected with a dominant negative mutant of JNK1,
dnJNK-APF, in which the phosphorylation residues Thr183 and
Tyr185 were changed to Ala and Phe, respectively (20). The
expression of dnJNK-APF has been shown to efficiently block JNK1
phosphorylation in response to extracellular stimuli such as UV
irradiation (20, 21). In our hands, dnJNK-APF selectively affected the
LAMPf-mediated activation of JNK without changing ERK1/2 or p38
activities (data not shown). The transfection of RAW 264.7 cells with
JNK1-APF significantly reduced LAMPf-induced IL-6 secretion as compared
with cells transfected with the wild-type JNK1 DNA (JNK-WT), whereas no
changes were detected in the IL-1ß or TNF- levels (Fig. 11).
|
production induced by LAMPf, while
the JNK pathway is most likely implicated in IL-6 production
only. |
Discussion |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
This capacity of M. fermentans to stimulate MAPK pathways is
not shared by any species of mycoplasma because our findings indicate
that M. pneumoniae-derived LAMP failed to induce the
activation of MAPKs. Interestingly, this species is not capable of
inducing the production of cytokines by monocytic cells (Fig. 1A; (4)). However, M. pneumoniae has been shown
to stimulate cytokines production by total blood cells (22), suggesting
that this pathogen might induce the cytokine production by stimulating
a cell population other than one of monocyte/macrophage lineage. Recent
findings indicate that M. pneumoniae interacts with CD4 T
cells to induce proinflammatory cytokines (23), supporting the
foregoing hypothesis. It could be speculated that the failure of
M. pneumoniae to stimulate macrophages is part of a survival
strategy that this pathogen employs.
LPS is a potent stimulator of the MAPK pathways in monocytes and macrophages and a very efficient inducer of proinflammatory cytokines in these cells (8). Classical Gram negative bacterial LPS is absent from mycoplasmas, but membranes of these microorganisms are rich in lipoproteins; therefore, it is interesting to compare the effects of LPS and LAMPs on monocytic cell lines. The first clear difference between LAMPf and LPS is the quite dissimilar kinetics of MAPK activation. MAPKs peak activity induced in RAW 264.7 cells in response to LPS have been observed between 10 and 15 min after stimulation (data not shown; (8)), while LAMPf induced MAPK’s maximal activity 30 min after challenge.
A second major difference consists in the lack of serum requirement for
LAMPf-mediated MAPK activation. Although the absence of serum did not
affect ERK1/2, p38, or JNK activation, it slightly reduced
LAMPf-induced cytokine production, suggesting that pathways other than
MAPKs could contribute to the signaling. The possible involvement of a
Ca2+-dependent biochemical pathway, but not PKC, in
mycoplasma-induced TNF- by THP-1 cells has been pointed out (24),
suggesting that other secondary messengers, such as phospholipase C,
might also contribute to this effect. This issue should be addressed in
further studies. LPS-induced cell activation involves the membrane
receptor CD14, which recognizes LPS complexed to the serum protein LBP
(LPS-binding protein). This could explain the inability of LPS to
activate MAPK pathways and stimulate cytokine synthesis after a 12-h
serum starvation. Interestingly, an anti-CD14 human mAb, MY4, able
to block LPS activation, did not effectively reduce cytokine levels
produced by LAMPf-stimulated human monocytic cell line THP-1. From our
data, it can be hypothesized that M. fermentans membrane
lipoproteins may recognize specific membrane receptor(s) present in the
monocyte/macrophage lineage and most probably absent in epithelial
cells and fibroblasts, since this type of cell does not respond to
mycoplasma challenge by activating the MAPK pathways. The putative
receptor(s) might be coupled, directly or indirectly, to PTKs that
subsequently trigger the MAPKs activation.
Presently, it is not clearly demonstrated whether lipid fraction of LAMPf or protein is involved in monocytes/macrophages activation. Based on proteinase K digestions, several reports have suggested that lipid fractions is the active component (4, 14), but the possibility that activity resides in small lipopeptides or small peptides resulting from this treatment cannot be ruled out. Hall et al. (25) have recently characterized a new lipoprotein gene (p48) from M. fermentans. When p48 gene was expressed as a MBP-P48 fusion protein in E. coli, the fusion protein was shown able to activate the HL-60 cell line, although it is not anchored to lipids. Furthermore, Muhlradt et al. (26) have also reported that the N-terminal peptide (the first 14 amino acids) of P48 coupled to fatty acid is also capable of stimulating monocytes. Lipid-uncoupled N-terminal peptide from P48 has not been tested for its ability to interact with monocytes/macrophages. We are currently investigating this issue.
The importance of the different intracellular signaling routes for
cytokine production has not been addressed extensively. In response to
extracellular stimuli, activated MAPKs can activate a number of
substrates, including transcription factors, and control the synthesis
of cytokines. In this way, the LPS-mediated induction of IL-1ß and
TNF- has been demonstrated to be controlled by p38 (19). Recently,
it has been shown that the activation of ERK1 is necessary for FcR
cross-linking-induced TNF- synthesis (27, 28). Our study points out
the importance of MAPK pathways in mycoplasma-mediated cytokine
production by monocytic cells. By using specific inhibitors of MEK-1
(the kinase upstream ERK1/2) and p38 or a JNK dominant negative
construct, we could delineate the contribution of each of these
signaling pathways in the cytokine induction by LAMPf. Results obtained
with the specific inhibitor of p38 SB203580 clearly show that the p38
pathway is crucial for IL-1ß, TNF-, and IL-6 production by
LAMPf-stimulated RAW 264.7 cells. MEK-1 inhibitor PD-98059 blocked
LAMPf-induced IL-1ß and TNF- but not IL-6, while the JNK dominant
negative mutant inhibited only IL-6. Our results suggest that at least
two MAPK-derived signals are required for the synthesis of a given
cytokine in response to mycoplasmas. Whereas p38 appears to trigger a
signal common to the three studied proinflammatory cytokines, ERK and
JNK differentially contribute to the synthesis of IL-1ß, TNF-,
and IL-6.
In summary, we have demonstrated that M. fermentans membrane lipoproteins activate in macrophages the three MAPK identified in mammals, and we shown the importance of these pathways in the induction of proinflammatory cytokines by these microorganisms. Further studies to characterize molecules involved in the interaction between mycoplasmas and monocytic cells should improve the understanding of the immunomodulatory activity and pathogenicity of these infectious agents.
|
Acknowledgments |
|---|
|
Footnotes |
|---|
2 Abbreviations used in this paper: LAMP, lipid-associated membrane protein; LAMPf, M. fermentans-derived LAMP; LAMPp, M. pneumoniae-derived LAMP; ERK1/2, extracellular signal-regulated kinases 1 and 2; GST, glutathione S-transferase; HIM, heat-inactivated mycoplasma; HIMf, heat-inactivated M. fermentans; JNK, c-Jun N-terminal kinase; SAPK, stress-activated protein kinase; MAPK, mitogen-activated protein kinase; MBP, myelin basic protein; MAPKAPK2, MAPK-activated protein kinase 2; MEK-1, MAPK/extracellular signal-regulated kinase 1; PTK, protein tyrosine kinase; PY, phosphotyrosine; MOPS, 3-(N-morpholino)propanesulfonic acid.
Received for publication July 17, 1997. Accepted for publication October 21, 1997.
|
References |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
receptor-stimulated TNF-alpha synthesis. J. Immunol. 158:3433.[Abstract]
This article has been cited by other articles:
|
|
 |
|
  L. A. Tephly and A. B. Carter Asbestos-Induced MKP-3 Expression Augments TNF-{alpha} Gene Expression in Human Monocytes Am. J. Respir. Cell Mol. Biol., July 1, 2008; 39(1): 113 - 123. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. Miksa, R. Wu, X. Cui, W. Dong, P. Das, H. H. Simms, T. S. Ravikumar, and P. Wang Vasoactive Hormone Adrenomedullin and Its Binding Protein: Anti-Inflammatory Effects by Up-Regulating Peroxisome Proliferator-Activated Receptor-{gamma} J. Immunol., November 1, 2007; 179(9): 6263 - 6272. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 R. L. Chelvarajan, Y. Liu, D. Popa, M. L. Getchell, T. V. Getchell, A. J. Stromberg, and S. Bondada Molecular basis of age-associated cytokine dysregulation in LPS-stimulated macrophages J. Leukoc. Biol., June 1, 2006; 79(6): 1314 - 1327. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 N. Tanaka, T. Morita, A. Nezu, A. Tanimura, I. Mizoguchi, and Y. Tojyo Signaling Mechanisms Involved in Protease-Activated Receptor-1-Mediated Interleukin-6 Production by Human Gingival Fibroblasts J. Pharmacol. Exp. Ther., November 1, 2004; 311(2): 778 - 786. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 C. L. Galindo, A. A. Fadl, J. Sha, C. Gutierrez Jr., V. L. Popov, I. Boldogh, B. B. Aggarwal, and A. K. Chopra Aeromonas hydrophila Cytotoxic Enterotoxin Activates Mitogen-activated Protein Kinases and Induces Apoptosis in Murine Macrophages and Human Intestinal Epithelial Cells J. Biol. Chem., September 3, 2004; 279(36): 37597 - 37612. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 C. Stambe, D. J. Nikolic-Paterson, P. A. Hill, J. Dowling, and R. C. Atkins p38 Mitogen-Activated Protein Kinase Activation and Cell Localization in Human Glomerulonephritis: Correlation with Renal Injury J. Am. Soc. Nephrol., February 1, 2004; 15(2): 326 - 336. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C. Stambe, R. C. Atkins, G. H. Tesch, T. Masaki, G. F. Schreiner, and D. J. Nikolic-Paterson The Role of p38{alpha} Mitogen-Activated Protein Kinase Activation in Renal Fibrosis J. Am. Soc. Nephrol., February 1, 2004; 15(2): 370 - 379. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 X. Li, N. Udagawa, M. Takami, N. Sato, Y. Kobayashi, and N. Takahashi p38 Mitogen-Activated Protein Kinase Is Crucially Involved in Osteoclast Differentiation But Not in Cytokine Production, Phagocytosis, or Dendritic Cell Differentiation of Bone Marrow Macrophages Endocrinology, November 1, 2003; 144(11): 4999 - 5005. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 B. Wang, P. P. Cleary, H. Xu, and J.-D. Li Up-Regulation of Interleukin-8 by Novel Small Cytoplasmic Molecules of Nontypeable Haemophilus influenzae via p38 and Extracellular Signal-Regulated Kinase Pathways Infect. Immun., October 1, 2003; 71(10): 5523 - 5530. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
L. Neff, M. Zeisel, V. Druet, K. Takeda, J.-P. Klein, J. Sibilia, and D. Wachsmann ERK 1/2- and JNKs-dependent Synthesis of Interleukins 6 and 8 by Fibroblast-like Synoviocytes Stimulated with Protein I/II, a Modulin from Oral Streptococci, Requires Focal Adhesion Kinase J. Biol. Chem., July 18, 2003; 278(30): 27721 - 27728. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C. Stambe, R. C. Atkins, G. H. Tesch, A. M. Kapoun, P. A. Hill, G. F. Schreiner, and D. J. Nikolic-Paterson Blockade of p38{alpha} MAPK Ameliorates Acute Inflammatory Renal Injury in Rat Anti-GBM Glomerulonephritis J. Am. Soc. Nephrol., February 1, 2003; 14(2): 338 - 351. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
G. Mancuso, A. Midiri, C. Beninati, G. Piraino, A. Valenti, G. Nicocia, D. Teti, J. Cook, and G. Teti Mitogen-Activated Protein Kinases and NF-{kappa}B Are Involved in TNF-{alpha} Responses to Group B Streptococci J. Immunol., August 1, 2002; 169(3): 1401 - 1409. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
< |
) from healthy
donors were stimulated with LAMPf (1 µg/ml) for different time
intervals (15, 30, and 60 min); untreated cells were used as control (0
min). A, ERK1/2 were immunoprecipitated from cell lysates
using anti-ERK1-CT Ab as indicated in Materials and
Methods. ERK1/2 activation was quantified using the
p42/44MAPK detection kit according to the manufacturer’s
instruction. Results are the mean of three independent assays.
B, p38 was immunoprecipitated from cell lysates using
anti-p38 Ab as indicated in Materials and Methods. p38
activation was quantified using the p38/RK/Mpk2 detection kit according
to the manufacturer’s instructions. Results are the mean of three
independent assays. C, JNK was immunoprecipitated (IP) from
cell lysates using JNK1 Ab (anti-JNK1 Ab). Immunoprecipitates were
incubated with GST-c-Jun under phosphorylating conditions. The
GST-c-Jun phosphotransferase activity (PA) was analyzed by SDS-PAGE as
indicated in Materials and Methods. Arrow indicates
phosphorylated GST-c-Jun.
