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The origin of the synergistic effect of muramyldipeptide with endotoxin and peptidoglycan
Margreet A. Wolfert1, Thomas F. Murray2, Geert-Jan Boons3, and James N. Moore1
Department of Large Animal Medicine1,and Department of Physiology & Pharmacology2, College of Veterinary
Medicine, The University of Georgia, Athens, GA 30602, and
Complex Carbohydrate Research Center3, The University of Georgia, 220 Riverbend Road, Athens, GA 30602
To whom correspondence should be addressed: Margreet A. Wolfert, Ph.D., Department of
Large Animal Medicine, College of Veterinary Medicine, University of Georgia, Athens, GA
30602. Tel.: 706-542-6326; Fax: 706-542-8833; E-mail: [email protected].
Running Title: Synergistic effect of MDP with LPS and PGN.
Copyright 2002 by The American Society for Biochemistry and Molecular Biology, Inc.
JBC Papers in Press. Published on July 31, 2002 as Manuscript M204885200 by guest on January 31, 2018
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SUMMARY
Although the basis for the high mortality rate for patients with mixed bacterial infections is
likely to be multifactorial, there is evidence for a synergistic effect of muramyldipeptide (MDP)
with lipopolysaccharide (LPS) on the synthesis of proinflammatory cytokines by mononuclear
phagocytes. In this study, co-incubation of human Mono Mac 6 cells with MDP and either LPS
or peptidoglycan (PGN) resulted in an apparent synergistic effect on tumor necrosis factor-α
(TNF-α) secretion. Although incubation of cells with MDP alone produced minimal TNF-α, it
caused significant expression of TNF-α mRNA. These findings suggest that the majority of
TNF-α mRNA induced by MDP alone is not translated into protein. Furthermore, simultaneous
incubation of cells with MDP and either LPS or PGN resulted in TNF-α mRNA expression that
approximated the sum of the amounts expressed in response to MDP, LPS, and PGN
individually. These findings indicate that the apparent synergistic effect of MDP on TNF-α
production induced by either LPS or PGN is caused by removal of a block in translation of the
mRNA expressed in response to MDP. In subsequent studies, the effects of MDP alone and its
effect on the production of TNF-α by LPS and PGN were determined to be independent of
CD14, Toll-like receptor 2, and Toll-like receptor 4. These findings indicate that MDP acts
through receptor(s) other than those primarily responsible for transducing the effects of LPS and
PGN. Successful treatment of patients having mixed bacterial infections is likely to require
interventions that address the mechanisms involved in responses induced by a variety of bacterial
cell wall components.
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INTRODUCTION
Bacteremia is a critical problem in intensive care units, accounting for high morbidity and
mortality rates. The mortality rate associated with bacteremia exceeds 30% (1,2). In a recent
12-year clinical study, gram-positive and gram-negative bacteria accounted for 46.9% and 31.5%
of bacteremic episodes in an intensive care unit, respectively, with gram-positive organisms
being cultured from more patients (3). Further, the incidence of combined infections increased
more than 4-fold over the 12-year period and was associated with a mortality rate exceeding
55%. Based on the fact that the majority of the deleterious effects of bacteremia are caused by
inflammatory responses to specific bacterial components, these findings suggest that the patient's
response to a mixture of gram-positive and gram-negative organisms may be heightened to the
detriment of the patient.
The two most commonly studied components of gram-positive and gram-negative bacterial
cell walls are peptidoglycan (PGN)2 and lipopolysaccharide (LPS)2, respectively. Although
gram-negative bacterial cell walls also contain PGN, its concentration is far greater in the walls
of gram-positive bacteria (4). Proinflammatory effects of these bacterial cell wall components
occur both in in vitro after treatment of mononuclear phagocytes and in in vivo after exposure of
whole animals, with cells and animals being more sensitive to LPS than to PGN (5).
The results of recent experimental studies provide evidence for a synergistic effect of LPS
with muramyldipeptide (MDP)2, the minimal structural subunit of PGN accounting for some of
its immunogenicity (6). However, the underlying mechanism of action for MDP has not been
fully elucidated. For example, there are discrepancies regarding the involvement of specific
receptors, with some investigators indicating that MDP exerts its synergistic effect in human
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leukocytes in a CD14-dependent manner (7). Others indicate that the response is CD14- and
Toll-like receptor (TLR)2 4-independent in human monocytic cell lines and that MDP up-
regulates expression of one of the primary components (MyD88 mRNA) in the TLR-mediated
response to LPS (4).
We report here that MDP not only synergizes with LPS but also acts similarly with PGN, to
induce the synthesis of tumor necrosis factor (TNF)-α in the human monocytic cell line Mono
Mac 6. This synergistic effect of MDP with LPS or PGN was investigated in relation to the
expression and stability of TNF-α mRNA. Furthermore, the role of receptors (e.g., CD14 and
TLR2/4) known to be involved in mediating cellular activation in response to bacterial cell wall
components was studied.
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EXPERIMENTAL PROCEDURES
Reagents- PGN from Staphylococcus aureus was obtained from BioChemika, MDP (N-
acetylmuramyl-L-alanyl-D-isoglutamine) from Calbiochem, E. coli 055:B5 LPS and [3H]E. coli
K12 LCD25 LPS from List Biological Laboratories, polymyxin B from Bedford Laboratories,
and actinomycin D from Sigma Chemicals. Affinity purified anti-CD14 antibodies MEM-18
(IgG1) and MY4 (IgG2b) were purchased from SANBIO b.v. and Coulter, respectively.
Functional grade purified anti-human TLR2 (clone TL2.1) and TLR4 (clone HTA125) antibodies
(IgG2a) were from Bioscience. Affinity purified mouse IgG1 (Sigma), IgG2b (Coulter), and
IgG2a (Sigma) were used as control antibodies. There were no effects of preincubation with
these control antibodies for MEM-18, MY4, or TLRs. PGN was assayed for endotoxin using the
Limulus Amoebocyte Lysate assay (BioWhittaker). No significant endotoxin contamination of
this preparation was detected (< 1 ng of endotoxin/mg).
Cell Maintenance- Mono Mac 6 cells, provided by Dr. H.W.L. Ziegler-Heitbrock (University
of Munich, Germany), were cultured in RPMI 1640 medium with L-glutamine (BioWhittaker)
supplemented with 100 u/ml penicillin, 100 µg/ml streptomycin, 1% OPI supplement (Sigma;
containing oxaloacetate, pyruvate and bovine insulin), and 10% fetal calf serum (FCS)2
(HyClone). The cells were maintained in a humid 5% CO2 atmosphere at 37oC. New batches of
frozen cell stock were grown up every 2 months and growth morphology evaluated. Before each
experiment, Mono Mac 6 cells were allowed to differentiate for 2 days in the presence of 10
ng/ml calcitriol (Sigma).
ELISA TNF- Cells were harvested by centrifugation and gently resuspended (106 cells/ml)
in prewarmed (37oC) medium. Cells were then incubated for 6 hours with different
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combinations of stimuli in the presence or absence of polymyxin B or antibodies as described in
the Results section. At the end of the incubation period, cell supernatants were collected and
stored frozen (-80oC) until assayed for TNF-α protein. In the experiments with antibodies, the
cells were incubated with each antibody for 30 min at 4oC, before the stimuli were added.
Concentrations of TNF-α in culture supernatants were determined in duplicate by a solid
phase sandwich ELISA. Briefly, 96-well plates (Nalge Nunc International) were coated with
purified mouse anti-human TNF-α monoclonal antibody (mAb)2 (Pharmingen). TNF-α in
standards and samples was allowed to bind to the immobilized mAb for 2 hours at room
temperature. Biotinylated mouse anti-human TNF-α mAb (Pharmingen) was then added,
producing an antibody-antigen-antibody “sandwich”. After addition of avidin-horseradish
peroxidase conjugate (Pharmingen) and ABTS peroxidase substrate (Kirkegaard & Perry
Laboratories), a green color was produced in direct proportion to the amount of TNF-α present in
the sample. The reaction was stopped by adding peroxidase stop solution (Kirkegaard & Perry
Laboratories), and the absorbance was measured at 405 nm using a microplate reader (Dynatech
Laboratories). All data for TNF-α are presented as the means + SD of duplicate cultures. Each
experiment was repeated at least twice.
Evaluation of materials for contamination by LPS- To ensure that any increase in TNF-α
production was not caused by LPS contamination of the solutions containing the various stimuli,
the experiments were performed in the absence and presence of polymyxin B, an antibiotic that
avidly binds to the lipid A region of LPS, thereby preventing LPS-induced monokine production
(8). TNF-α concentrations in supernatants of cells preincubated with polymyxin B (1 µg/ml) for
30 minutes before incubation with E. coli O55:B5 LPS for 6 hours were reduced from 2,330 +
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111 pg/ml to 10 + 7 pg/ml, whereas preincubation with polymyxin B had no effect on TNF-α
synthesis by cells incubated with PGN (~1,800 pg/ml) or MDP (~80 pg/ml). Therefore, LPS
contamination of the latter preparations was inconsequential.
Preparation of RNA and quantification of TNF- mRNA by real-time polymerase chain
reaction (PCR)2 analysis- Cells were harvested by centrifugation and gently suspended (1.25 x
106 cells/ml) in prewarmed (37oC) medium. In appropriate experiments, cells were incubated
first with antibody or media (control) for 30 min at 4oC. Cells were then incubated with the
indicated concentrations of the stimuli for 1.5 hours after which cells were harvested, and total
RNA was isolated using the StrataPrep Total RNA Miniprep Kit (Stratagene) according to the
manufacturer’s protocol.
TNF-α gene expression was quantified in a two-step reverse transcription-PCR (RT-PCR)2.
In the RT step, cDNA was reverse transcribed from total RNA samples (0.625 µg/50 µl) using
random hexamers from the TaqMan RT reagents (Applied Biosystems). In the PCR step, PCR
products were synthesized from cDNA (22.5 ng/20 µl) using the Taqman universal PCR master
mix and TaqMan PDARs for human TNF-α (Applied Biosystems). Measurements were done
using the ABI Prism 7900 HT sequence detection system (Applied Biosystems), according to the
manufacturer’s protocols. As an endogenous control for these PCR quantification studies, 18S
ribosomal RNA gene expression was measured using the TaqMan ribosomal RNA control
reagents (Applied Biosystems). Results represent means + SD of triplicate measurements. Each
experiment was repeated at least twice.
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LPS binding assay- Kinetic analysis of the [3H]LPS discociation rate (off rate) from Mono
Mac 6 cells was performed by the method of Kitchens and Munford (9). Mono Mac 6 cells were
washed in ice-cold HNE buffer (20 mM HEPES, pH 7.4, 150 mM NaCl, 1 mM EDTA) and
preincubated for 30 min at 37oC in SEBDAF buffer (20 mM HEPES, pH 7.4, 150 mM NaCl, 1
mM EDTA, 300 µg/ml BSA, 10 mM NaN3, 2 mM NaF, 5 mM deoxyglucose) to deplete
intracellular ATP and prevent ligand internalization. The cells were then centrifuged,
resuspended in RPMI containing 300 µg/ml BSA, 10 mM NaN3, 2 mM NaF, and 5 mM
deoxyglucose (1 x 106 cells in 500 µl, final volume). [3H]LPS (final concentration 30 ng/ml),
was first mixed with FCS (final concentration 7.5%) as a source for LPS binding protein. It was
added to the cells and incubated with frequent mixing for 1 hour at 37oC to reach equilibrium.
Next, unlabeled LPS (final concentration 10 µg/ml) or a mixture of unlabeled LPS and MDP
(final concentrations 10 µg/ml and 100 µg/ml, respectively), mixed first with FCS (final
concentration 7.5%), were added to the cells. The cells were harvested at several time points.
To determine non-specific binding, unlabeled LPS alone or in combination with MDP was added
to the cells before addition of [3H]LPS. The cells were harvested by adding ice-cold HNE buffer
(500 µl), and centrifuging the mixtures at 15,000 rpm for 2 min at 4oC. The supernatant was
aspirated, and the cells were washed with 500 µl ice-cold HNE buffer. The cells were lysed in 5
ml liquid scintillation cocktail (Beckman Instruments Inc.), and the cell-associated 3H was
counted. Results represent means + SD of triplicate samples. The experiment was repeated with
similar results.
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Data analysis- LPS and PGN concentration-response data for stimulation of TNF-α
production in Mono Mac 6 cells were analyzed using nonlinear least-squares curve fitting in
Prism (GraphPad Software, Inc.). These data were fit with the following logistic equation:
Y = Emax / (1 + (EC50/X)Hill slope),
where Y is the TNF-α response, X is the LPS or PGN concentration, Emax is the maximum
response2 and EC50 is the concentration of LPS or PGN producing 50% stimulation2.
[3H]LPS dissociation experiments were analyzed by fitting a monoexponential decay equation
to the data
Y = Y0 e –kt,
where Y is the amount of [3H]LPS bound at time t, Y0 is the amount of [3H]LPS bound at time
zero, t is time of dissociation2, and k is the dissociation rate constant2. Half-lives2 for [3H]LPS
dissociation in the presence and absence of MDP were calculated as
t1/2 = 0.693 / k.
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RESULTS
Effect of MDP on the responses of Mono Mac 6 cells to LPS and PGN- The effect of a 30
minute preincubation of Mono Mac 6 cells with MDP on TNF-α secretion induced by a wide
concentration range of LPS and PGN was compared with incubation with either LPS or PGN
alone (Fig. 1). Preincubation of cells with MDP resulted in a LPS concentration-response curve
having a higher maximal level (a two-fold increase), whereas the EC50 and Hill slope values did
not differ significantly from those obtained in the absence of MDP. In the absence of MDP, the
maximum concentration of produced TNF-α in response to LPS was 1,477 pg/ml, with a Hill
slope of 2.7 and an LPS EC50 of 13.2 ng/ml. Pretreatment with MDP increased the maximum
level of LPS-induced TNF-α production to 3,003 pg/ml; the Hill slope and EC50 were 3.1 and 8.6
ng/ml, respectively. Stimulation with MDP alone resulted in a TNF-α concentration of 71 pg/ml.
Similar results were obtained when cells were preincubated with MDP followed by PGN (Fig.
1b). The maximum level of TNF-α after incubation with PGN alone was 3,313 pg/ml with a Hill
slope of 1.1 and a PGN EC50 of 26.1 µg/ml. Preincubation with MDP yielded a maximum level
of 5,469 pg/ml, a Hill slope of 1.2, and an EC50 of 22.4 µg/ml. These results, derived from four-
parameter logistic fits to the data, demonstrate that the singular observable effect of MDP is to
increase the maximum TNF-α responses to LPS or PGN in the absence of an influence on their
respective potencies.
In addition to the experiments presented above, in which Mono Mac 6 cells were incubated
with MDP before addition of LPS or PGN, experiments also were performed in which MDP was
added simultaneously with LPS or PGN. Simultaneous treatment of Mono Mac 6 cells with
MDP and LPS or PGN produced the same increase in TNF-α secretion (data not shown),
suggesting that pretreatment was not necessary for the effect of MDP. Further, we performed
several experiments in which the cells were first exposed to MDP for 10 min or 20 min, washed
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and then exposed to either LPS or PGN. The effect of exposure to MDP for only 10 min on
TNF-α concentration was more than additive, although not as dramatic as that occurring with
simultaneous and continuous treatment of the cells with MDP and LPS or PGN (data not shown).
We also performed experiments to determine if preincubation with MDP had a synergistic effect
with subsequent exposure to a second dose of MDP. In these experiments, the response was
simply additive (data not shown).
To rule out the possibility that Mono Mac 6 cells incubated with MDP produce TNF-α that
remains associated with the cells and was not secreted, TNF-α concentrations in the supernatant
were compared with total (cell-associated and secreted) TNF-α concentrations. Incubation of
cells with LPS, PGN, MDP, MDP plus LPS, and MDP plus PGN, produced only small amounts
of TNF-α that remained cell-associated; total TNF-α concentrations were indistinguishable from
those in the supernatants (data not shown).
Induction of TNF- mRNA by LPS, PGN, and MDP- TNF-α production in response to LPS is
controlled both at the transcriptional and post-transcriptional levels (10-12). To study whether
the synergistic induction of Mono Mac 6 cells by MDP is controlled at the level of gene
transcription, TNF-α mRNA expression was measured after treatment with medium, LPS or
PGN alone, or in combination with MDP. The effects of LPS and PGN each were determined at
their lowest concentrations causing maximal TNF-α protein production (i.e., LPS 30 ng/ml; PGN
100 µg/ml). Data presented in Fig. 2a indicate that incubation with LPS or PGN results in
similar levels of TNF-α mRNA. Incubation with MDP (100 µg/ml) resulted in a 10-fold increase
in TNF-α gene expression compared with control cells, although this was less than that caused
by LPS or PGN. The same concentration of MDP caused very low levels of TNF-α protein
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secretion compared with LPS or PGN. Simultaneous treatment of cells with MDP and either
LPS or PGN resulted in further enhancement of TNF-α mRNA expression, which appears to
represent the additive effect of additing MDP and LPS or PGN individually. In contrast,
concentrations of TNF-α protein in the supernatants from the same samples, even after only 90
min exposure, were suggestive of a pronounced synergistic effect of MDP with either LPS or
PGN (Fig. 2b).
In addition to the experiment presented above, in which Mono Mac 6 cells were stimulated
for 90 min before RNA extraction, the same experiments were also performed in which RNA
was extracted after 30 min, 60 min, and 120 min of stimulation (Table 1). In all cases, only a
small amount of mRNA was expressed after 30 min. Stimulation with LPS or PGN produced
maximal mRNA expression at 60 min, whereas stimulation with MDP or MDP in combination
with LPS or PGN reached their maximal values at 90 min. TNF-α mRNA expression was
reduced markedly by 120 min. Varying the incubation period did not alter the finding that
simultaneous treatment of cells with MDP and either LPS or PGN resulted in an additive effect
on TNF-α mRNA expression.
Comparison of MDP with LPS on TNF- mRNA expression and TNF- protein concentration-
After determining that incubation of cells with MDP at 100 µg/ml resulted in TNF-α gene
expression but minimal protein translation, we wondered if this effect might be dependent on
MDP concentration and if the degree of mRNA expression in response to MDP might be
insufficient to result in protein translation. To address this question, Mono Mac 6 cells were
incubated for 90 min with medium alone as control, LPS ranging from 0.01 to 100 ng/ml, or
MDP ranging from 0.01 to 300 µg/ml, in an effort to determine if there were concentrations of
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LPS causing TNF-α mRNA expression but not translation, and to determine the dose
dependency for the effects of MDP. Even after only 90 min, there was a clear dose response for
LPS, both for TNF-α protein production and the TNF-α mRNA expression (Fig. 3). A plateau in
TNF-α protein production was evident at 30 ng/ml LPS, a finding that was in agreement with
data presented in Fig. 1 where cells were stimulated for 6 hours. However, a plateau was not
observed for TNF-α RNA expression. Although incubation with MDP at 100 µg/ml resulted in
TNF-α protein production comparable with that caused by 0.01 – 0.1 ng/ml LPS, TNF-α mRNA
expression induced by MDP was comparable with that caused by 1 ng/ml LPS. These results
demonstrate that the amount of TNF-α mRNA expressed when cells are incubated with MDP
was not a limiting factor for subsequent protein translation.
Incubation of the cells with MDP yielded dose dependent increases in both TNF-α protein
production and TNF-α mRNA expression. Although TNF-α protein concentrations barely
exceeded background values even at a MDP concentration of 300 µg/ml (Fig. 3a), TNF-α mRNA
expression was increased by MDP at 10 µg/ml and reached a maximum at 100 µg/ml (Fig. 3b).
Consequently, we used MDP at 100 µg/ml in the remainder of the study.
Determination of half-life of TNF- mRNA- The above results suggest that the apparent
synergistic effects of MDP and either LPS or PGN were caused by different patterns of
regulation of mRNA translation. It was reported recently that synergistic production of TNF-α
by macrophages exposed to a combination of bacterial DNA and LPS was at least in part caused
by prolonged half-life of TNF-α mRNA (13). To investigate if this was the case for Mono Mac 6
cells exposed to a combination of MDP and either LPS or PGN, the half-life of TNF-α mRNA
was determined in Mono Mac 6 cells exposed to medium, LPS or PGN alone, or in combination
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with MDP. After a 90 min exposure, further transcription was inhibited by treating the cells with
actinomycin D (10 µg/ml). Total RNA was isolated at various time points after the addition of
actinomycin D, and TNF-α mRNA expression was measured by RT-PCR. The half-lives of
TNF-α mRNA in Mono Mac 6 cells stimulated with LPS, PGN, MDP, MDP plus LPS, and MDP
plus PGN were 14.3, 14.6, 11.5, 14.1, and 13.4 min, respectively (Fig. 4). The half-life of TNF-
α after stimulation with MDP alone was slightly shorter compared with the other stimuli, but the
half-lives of the combinations of MDP and LPS or PGN did not differ significantly from those of
LPS or PGN alone. Therefore, the effect of MDP with either LPS or PGN on release of TNF-α
cannot be explained by altered stability of TNF-α mRNA. Consequently, other post-
transcriptional factors must be responsible for the effect.
Effect of anti-CD14 mAbs on the synergistic effect of MDP- The fact that maximal TNF-α
mRNA expression for cells incubated with LPS or PGN occurred at 60 min, whereas maximal
mRNA expression for cells incubated with MDP alone or MDP with either LPS or PGN
occurred at 90 min suggests that activation of gene expression by MDP might occur through a
different route than LPS and PGN. To address this question, we performed experiments to
explore the possibility that different receptors are involved in the response of the cells to MDP
and either LPS or PGN. Two anti-CD14 mAbs MY4 and MEM-18 were used to assess the
involvement of CD14. It has been reported previously that the binding of LPS and PGN to
CD14 involves amino acids 51-64 or 57-64 in the N-terminal region of the receptor (14,15).
MEM-18, with its epitope at residues 57-64, is an anti-CD14 mAb specific for this region. The
epitope of MY4 is located closer to the N-terminal end of CD14 at amino acids 34-44. MY4 is
quite effective in inhibiting binding of both LPS and sPGN to soluble CD14 (14). In
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experiments performed in our laboratory, both MEM-18 and MY4 completely neutralized the
effect of LPS. Whereas the effect of PGN was reduced by 86% and 54% by MEM-18 and MY4,
respectively (Fig. 5). We have previously reported that increasing the concentration of these
mAbs did not further increase their blocking effect on PGN (16). Even when LPS appeared to be
completely blocked by the anti-CD14 mAbs, the presence of MDP resulted in significant
amounts of TNF-α secretion (Fig. 5a). A similar effect of MDP on the response to PGN was
observed, even when the effects of PGN alone were partially blocked with the anti-CD14 mAbs
(Fig. 5b). These results suggest that the effect of MDP on the subsequent response to LPS and
PGN was CD14-independent.
Expression of TNF-α mRNA was determined for cells incubated for 90 min with medium
(control), LPS or PGN alone, or in combination with MDP in the absence or presence of MY4.
Whereas LPS-induced expression of TNF-α mRNA was almost completely abolished by MY4,
this antibody had minimal effect on the responses to PGN or MDP alone, and the additive effects
of MDP on the response to either LPS or PGN persisted in the presence of MY4 (Table 2).
These results provide further evidence that this effect of MDP was CD14-independent.
Influence of TLR4 and TLR2 on synergism of MDP with LPS and PGN- TLR 2 and 4 have
been implicated as critical receptors responsible for initiation of signaling events and cellular
activation in response to bacterial cell wall components (17). For instance, there is compelling
information that TLR4 plays a critical role in LPS-induced cell signaling and serves as a cell-
surface co-receptor for CD14. These two receptors are necessary for LPS-mediated NF-κB
activation and subsequent cellular events (18). Although incubation with the mAb directed
against TLR4 reduced the effect of LPS by 80% (Fig. 6), it did not affect the synergism between
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MDP and LPS. MDP increased LPS-induced synthesis of TNF-α by 3.3-fold in the absence of
the anti-TLR4 mAb, and by 3.7-fold in the presence of the antibody. These findings provide
evidence that the synergism of MDP with LPS was TLR4-independent.
The results of recent studies suggest that TLR2 is involved in cellular responses to a wide
variety of infectious pathogens and their products, including PGN (17). Incubation of cells with
the mAb against TLR2 reduced the effect of PGN by 55% (Fig. 7). In the absence of the anti-
TLR2 mAb, MDP increased PGN-induced synthesis of TNF-α protein by 2.4-fold. Similarly,
PGN-induced TNF-α protein production was increased 2.0-fold in cells co-incubated with MDP
and the anti-TLR2 mAb. These findings suggest that the synergistic effect of MDP on the
cellular response to PGN was TLR2-independent.
Further, TNF-α mRNA expression was determined for cells incubated for 90 min with
medium (control), LPS or PGN alone, or in combination with MDP in the absence or presence of
anti-TLR2 or anti-TLR4 mAb (Table 3). Neither the anti-TLR2 nor the anti-TLR4 mAbs
reduced TNF-α mRNA expression in response to MDP. Furthermore, the additive effects of
MDP on expression of TNF-α mRNA induced by either LPS or PGN persisted in the presence of
the anti-TLR mAbs, providing additional evidence that MDP exerts its effect independent of
TLR2 and TLR4.
Effect of MDP on the [3H]LPS off rate from Mono Mac 6 cells- Based on the above results
suggesting that MDP exerts its effects independent of CD14, TLR2, and TLR4, we considered
the possibility that MDP might alter the interaction of LPS or PGN with these cell surface
receptors. To investigate the possibility that MDP might exert an allosteric action to increase the
affinity of LPS binding to the cell surface, the dissociation rate of LPS from the Mono Mac 6
cells was compared in the absence and presence of MDP (Fig. 8). The k of LPS was 0.0056 +
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0.0025 in the absence of MDP and 0.0061 + 0.0023 in the presence of MDP. The t1/2 of LPS
alone (125 min) was not appreciably different from that of LPS in the presence of MDP (115
min).
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DISCUSSION
In this study, we investigated the underlying mechanism for the apparent synergistic effect of
MDP on TNF-α production induced by either LPS or PGN. Co-incubation of Mono Mac 6 cells
with MDP and LPS yielded supernatant concentrations of TNF-α that were consistent with a
synergism between the two bacterial components. We observed the same effect with co-
incubation of the cells with MDP and PGN, a finding that has not been reported previously.
Incubation of Mono Mac 6 cells with LPS or PGN alone, showed a clear dose response for LPS
and PGN starting at 2 ng/ml and 2 µg/ml, respectively. Concentrations of LPS (30 ng/ml) and
PGN (100 µg/ml) resulted in peak supernatant concentrations of TNF-α, which exceeded 1,400
pg/ml and 3,300 pg/ml, respectively. In contrast, incubation of the cells with MDP alone at
concentrations up to 200 µg/ml resulted only in slight increases in supernatant concentrations of
TNF-α. The addition of MDP to either LPS or PGN resulted in substantial increases in TNF-α
compared with LPS or PGN alone. This apparent synergistic effect was obvious with different
concentrations of LPS and PGN, was evident after as little as 90 min of incubation, and was
maintained throughout the 6-hour incubation period. The presence of MDP significantly
increased the maximum value of the dose response curve for TNF-α secretion in response to LPS
or PGN, without changing either the Hill slope or the EC50. The primary effect of MDP was
therefore to increase the maximum TNF-α response to LPS or PGN, which was not consistent
with an allosteric action on either the LPS or PGN recognition site.
Others have reported a synergistic effect of muramyl peptides with LPS. Flak et al. reported
synergistic interactions between a naturally occurring PGN fragment (muramyl peptide) from
Bordetella pertussis and LPS in the induction of inflammatory processes (induction of
interleukin (IL)2-1a, type II (inducible) nitric oxide synthase, nitric oxide production, and
inhibition of DNA synthesis) within hamster trachea epithelial cells (19). Yang et al. reported
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the synergistic effect of MDP with LPS or lipoteichoic acid to induce inflammatory cytokine IL-
8 in human monocytic cells in culture (4). Wang et al. reported that co-administration of PGN or
MDP with LPS caused significantly increased concentrations of TNF-α and IL-6 in cultures of
whole human blood, whereas the release of IL-10 was not influenced (7). In contrast, we
observed that TNF-α concentration in the supernatant of Mono Mac 6 cells co-incubated with the
combination of LPS and PGN were only additive (data not shown). A possible explanation for
this discrepancy is that we used insoluble PGN to eliminate potential effects of smaller PGN
fragments on the responses being measured. Because Wang et al. used a sonicated preparation
of PGN, their PGN preparation may have included some smaller fragments that might act like
MDP. Another difference between the two studies is the type of cell used; this may account for
the differences noted. We report here for the first time that MDP, which is the minimum active
fragment of PGN adjuvants (6), also synergizes with PGN in TNF-α production.
A unique finding of this study was that TNF-α mRNA expression showed a completely
different profile than TNF-α protein production. Incubation of the Mono Mac 6 cells with MDP
alone increased TNF-α mRNA, and co-incubation of the cells with MDP and either LPS or PGN
resulted in TNF-α mRNA expression that approximated the sum of the message generated in
response to MDP and either LPS or PGN alone. Although the additive effect on TNF-α mRNA
expression was maximal at 90 min, the same trend was apparent regardless of the incubation
period. With these data regarding the effects of MDP on TNF-α mRNA expression, the
concentration of TNF-α protein simply reflects the additive effects of MDP and either LPS or
PGN and not a synergistic interaction between these toxins. In short, co-incubation of MDP with
either LPS or PGN increases TNF-α gene expression and TNF-α protein production to the same
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extent. Therefore, we conclude that TNF-α mRNA induced by MDP alone is not translated and
that the impediment in translation is circumvented by the presence of either LPS or PGN.
Having identified the lack of correlation between expression of TNF-α mRNA and TNF-α
protein induced by MDP and the subsequent responses to either LPS or PGN, we explored
whether MDP mediates its effect through different cell surface receptors than LPS and PGN. It
is well accepted that LPS and PGN initiate the production of proinflammatory mediators by
interacting with the cluster differentiation antigen CD14 (20-23). CD14 is a
glycosylphosphatidylinositol-anchored protein lacking transmembrane and cytoplasmic domains.
There is compelling evidence that LPS and PGN transmit their signals via individual members of
the TLR family, TLR4 (24,25) and TLR2 (26,27), respectively. An intermediary protein (MD-2)
is required for interactions involving LPS, CD14, and TLR4 (28).
In our studies we utilized two different anti-CD14 mAbs, MY4 and MEM-18, each of which
completely blocked the response to LPS. In contrast, these antibodies only partially inhibited
PGN-induced TNF-α production. However, neither antibody affected the apparent synergistic
effect of MDP with either LPS or PGN, suggesting that LPS and PGN in the presence of
antibodies that block CD14 were still able to initiate translation of the TNF-α mRNA induced by
MDP. The latter finding indicates that the effect of MDP is CD14 independent. This CD14-
independence was further confirmed by enhanced expression of TNF-α mRNA expression in
response to MDP in the presence of MY4. The finding that PGN-induced synthesis of TNF-α
was only partially blocked by MY4 and MEM-18 in this study was not completely unexpected.
Dziarski et al. have reported that MEM-18 almost completely inhibited binding of soluble (s)2
CD14 to sPGN, when sPGN was immobilized on agarose (14). However, we determined in a
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previous study that PGN-induced TNF-α protein production in Mono Mac 6 cells could only be
partially blocked by anti-CD14 mAbs, suggesting that PGN exerts its effect through CD14 and
another unidentified receptor (16). This latter finding corroborates the inability of MY4 and
MEM-18 to completely block the effects of PGN in the present study. The unidentified receptor
could be TLR2, which may be activated by PGN independent of CD14 (29).
Unfortunately, commercially available anti-TLR mAbs do not completely block the effects of
either LPS or PGN, probably because of partial neutralization by these antibodies. Nevertheless,
the finding that these mAbs against TLR2 and TLR4 do not affect the apparent synergistic effect
of MDP with either LPS or PGN, strongly suggests that the effect of MDP is TLR2- and TLR4-
independent. The strength of this conclusion was increased by the lack of effect of either the
anti-TLR2 or the anti-TLR4 mAb on TNF-α mRNA expression by cells incubated with MDP.
Additional findings in the present study supporting our contention that MDP acts through
receptor(s) other than those utilized by LPS and PGN are the following: (1) the fact that MDP
increased the production of TNF-α protein induced by concentrations of LPS or PGN that by
themselves already caused maximal TNF-α production, (2) the observation that maximal TNF-α
mRNA expression occurs later when cells are incubated either with MDP alone or with MDP
combined with either LPS or PGN than after stimulation with either LPS or PGN alone, and (3)
the fact that the dissociation rate of LPS did not change in the presence of MDP suggests that
MDP does not alter the interaction of LPS with its cell surface receptor, inasmuch as such an
allosteric effect would have influenced [3H]LPS dissociation kinetics.
Our findings suggesting that MDP acts through receptors other than those utilized by LPS
and PGN are in agreement with results reported for hamster tracheal epithelial cells and another
monocytic cell line (4,19). Although our finding that MDP acts independently of CD14 appears
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to contradict an earlier report that MDP binds to CD14 and prevents the binding of FITC-labeled
sPGN to human monocytes (30), the amount of MDP required to inhibit sPGN binding in that
study was very high, indicating that MDP bound to those cells with low-affinity. More recently,
Dziarski et al. reported that sCD14 can bind to MDP when coupled to agarose and that this
binding could not be inhibited by monomeric sMDP (14). Those investigators concluded that
polymeric, aggregated or solid-bound PGN or MDP is needed for CD14 binding, which is in
agreement with our findings that the effect of MDP is CD14-independent.
What remains to be determined are the mechanism(s) responsible for inhibiting translation of
the TNF-α mRNA expressed in response to MDP alone and the pathway of cellular activation
induced by MDP. It is well known that the synthesis of TNF-α is tightly controlled at many
different levels and is subject to several negative feedback mechanisms (11,31). For example,
post-transcriptional regulation of TNF-α production is mediated by the AU-rich element located
in the TNF-α mRNA 3’ untranslated region, which controls its translation and stability (32).
Metabolism of the 3’ poly(A) tail region of TNF-α mRNA plays a critical regulatory function in
TNF-α translation (33). In unstimulated, but adherent cells, shortening of the length of the
poly(A) tail prevents the initiation of TNF-α translation. This process is reversed by LPS,
allowing synthesis of translatable polyadenylated TNF-α mRNA. It is possible that MDP lacks
the ability to exert the same effect as LPS, rendering mRNA transcribed in response to MDP in a
form that requires an additional stimulus (e.g., LPS) in order for protein translation to occur. The
end result of cellular activation by LPS or PGN is up-regulation of more than 120 genes,
including differential activation of MAP kinases and slightly different patterns of gene activation
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(34). It is possible that activation of some of these genes are important for translational control
of TNF-α production and are not activated by MDP alone.
In an effort to explain the apparent synergistic effect of MDP and LPS on TNF-α protein
production, some possibilities have been addressed by other investiators. Wang et al. reported a
twofold increase in surface expression of CD14 on monocytes and suggested that both PGN and
MDP prime leukocytes for LPS-induced release of pro-inflammatory cytokines (7). However,
this effect could not explain the observed synergism between PGN or MDP and LPS, as up-
regulation of CD14 up-regulation occurred after the synergistic effect was evident. Other
investigators have suggested involvement of MyD88, an adapter molecule essential for cell
signaling events initiated via the TLR family (35). In those studies, MDP and LPS individually
up-regulated MyD88 mRNA expression in THP-1 cells, but there was no synergistic up-
regulation of MyD88 mRNA in cells incubated with the combination of MDP and LPS (4).
Furthermore, the up-regulation of MyD88 that was detected, occurred later than the synergistic
effect and so could not account for the synergism. In this study, we have shown that the inability
to translate mRNA induced by MDP is not due to alterations in mRNA stability. We also
determined that there were no differences in transport between TNF-α induced by MDP and LPS
or PGN, as secreted TNF-α concentrations were indistinguishable from total TNF-α
concentrations.
In summary, we have demonstrated for the first time that MDP induces TNF-α gene
expression, without significant TNF-α translation. Furthermore, the block in translation is
removed in the presence of LPS and PGN, thereby accounting for the apparent synergistic effect
of MDP on TNF-α protein production. The amount of TNF-α protein produced in response to
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the combination of either MDP and LPS or MDP and PGN is as expected in light of the amount
of TNF-α mRNA expressed individually in response to MDP, LPS, or PGN. Furthermore, we
have demonstrated that the effect of MDP alone and its apparent synergism with either LPS or
PGN is CD14-, TLR2- and TLR4-independent. Thus, we conclude that MDP exerts its effects
via different receptors than those responsible for the effects of LPS and PGN. Taken in the
context of reports documenting the deleterious effects of MDP in animals subjected to
experimentally induced septicemia (36) and the fact that antimicrobial drugs exert their effects
by causing the breakdown of bacterial cell wall components such as PGN (37), the results of the
current study underscore the importance of considering both serum concentrations of
inflammatory mediators as well as gene expression profiles when interpreting findings associated
with septicemia.
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REFERENCES
1. Roberts, F. J., Geere, I. W., and Coldman, A. (1991) Rev. Infect. Dis. 13, 34-46
2. Pittet, D., Tarara, D., and Wenzel, R. P. (1994) J.A.M.A. 271, 1598-1601
3. Crowe, M., Ispahani, P., Humphreys, H., Kelley, T., and Winter, R. (1998) Eur. J. Clin.
Microbiol. Infect. Dis. 17, 377-384
4. Yang, S., Tamai, R., Akashi, S., Takeuchi, O., Akira, S., Sugawara, S., and Takada, H.
(2001) Infect. Immun. 69, 2045-2053
5. Lichtman, S. N., Wang, J., Schwab, J. H., and Lemasters, J. J. (1994) Hepatology 19,
1013-1022
6. Baschang, G. (1989) Tetrahedron 45, 6331-6360
7. Wang, J. E., Jorgenson, P. F., Ellingsen, E. A., Almlof, M., Thiemermann, C., Foster, S.
J., Aasen, A. O., and Solberg, R. (2001) Shock 16, 178-182
8. Tsubery, H., Ofek, I., Cohen, S., and Fridkin, M. (2000) Biochemistry 39, 11837-11844
9. Kitchens, R. L., and Munford, R. S. (1995) J. Biol. Chem. 270, 9904-9910
10. Biragyn, A., and Nedospasov, S. A. (1995) J. Immunol. 155, 674-683
11. Beutler, B. (1992) Am. J. Med. Sci. 303, 129-133
12. Kontoyiannis, D., Pasparakis, M., Pizarro, T. T., Cominelli, F., and Kollias, G. (1999)
Immunity 10, 387-398
13. Gao, J. J., Xue, Q., Papasian, C. J., and Morrison, D. C. (2001) J. Immunol. 166, 6855-
6860
14. Dziarski, R., Tapping, R. I., and Tobias, P. S. (1998) J. Biol. Chem. 273, 8680-8690
15. Juan, T. S.-C., Hailman, E., Kelley, M. J., Busse, L. A., Davy, E., Empig, C. J., Narhi, L.
O., Wright, S. D., and Lichenstein, H. S. (1995) J. Biol. Chem. 270, 17237-17242
by guest on January 31, 2018http://w
ww
.jbc.org/D
ownloaded from
26
16. Siriwardena, A., Jorgenson, M. R., Wolfert, M. A., Vandenplas, M. L., Moore, J. N., and
Boons, G.-J. (2001) J. Am. Chem. Soc. 123, 8145-8146.
17. Tapping, R. I., Akashi, S., Miyake, K., Godowski, P. J., and Tobias, P. S. (2000) J.
Immunol. 165, 5780-5787
18. Chow, J. C., Young, D. W., Golenbock, D. T., Christ, W. J., and Gusovsky, F. (1999) J.
Biol. Chem. 274, 10689-10692
19. Flak, T. A., Heiss, L. N., Engle, J. T., and Goldman, W. E. (2000) Infect. Immun. 68,
1235-1242
20. Gupta, D., Kirkland, T. N., Viriyakosol, S., and Dziarski, R. (1996) J. Biol. Chem. 271,
23310-23316
21. Lee, J.-D., Kravchenko, V., Kirkland, T. N., Han, J., Mackman, N., Moriarty, A.,
Leturcq, D., Tobias, P. S., and Ulevitch, R. J. (1993) Proc. Natl. Acad. Sci. U. S. A. 90,
9930-9934
22. Wright, S. D., Ramos, R. A., Tobias, P. S., Ulevitch, R. J., and Mathison, J. C. (1990)
Science 249, 1431-1433
23. Dziarski, R., Ulmer, A. J., and Gupta, D. (2000) Chem. Immunol. 74, 83-107
24. Beutler, B. (2000) Curr. Opin. Immunol. 12, 20-26
25. Jiang, Q., Akashi, S., Miyake, K., and Petty, H. R. (2000) .J. Immunol. 165, 3541-3544
26. Schwandner, R., Dziarski, R., Wesche, H., Rothe, M., and Kirschning, C. J. (1999) J.
Biol. Chem. 274, 17406-17409
27. Yoshimura, a., Lien, E., Ingalls, R. R., Tuomanen, E., Dziarski, R., and Golenbock, D.
(1999) J. Immunol. 163, 1-5
by guest on January 31, 2018http://w
ww
.jbc.org/D
ownloaded from
27
28. Yang, H., Young, D. W., Gusovsky, F., and Chow, J. C. (2000) J. Biol. Chem. 275,
20861-20866
29. Triantafilou, K., Triantafilou, M., and Dedrick, R. L. (2001) Nature Immunol. 2, 338-345
30. Weidemann, B., Schletter, J., Dziarski, R., Kusumoto, S., Stelter, F., Rietschel, E. T.,
Flad, H.-D., and Ulmer, A. J. (1997) Infect. Immun. 65, 558-564
31. Jongeneel, C. V. (1995) Immunobiology 193, 210-216
32. Mijatovic, T., Houzet, L., Defrance, P., Droogmans, L., Huez, G., and Kruys, V. (2000)
Eur. J. Biochem. 267, 6004-6011
33. Crawford, E. K., Ensor, J. E., Kalvakolanu, I., and Hasday, J. D. (1997) J. Biol. Chem.
272, 21120-21127
34. Wang, Z.-M., Liu, C., and Dziarski, R. (2000) J. Biol. Chem. 275, 20260-20267
35. Takeuchi, O., Takeda, K., Hoshino, K., Adachi, O., Ogawa, T., and Akira, S. (2000)
Internat. Immunol. 12, 113-117
36. Ribi, E. E., Cantrell, J. L., Von Eschen, K. B., and Schwartzman, S. M. (1979) Cancer
Res. 39, 4756-4759
37. van Langevelde, P., Ravensbergen, E., Grashoff, P., Beekhuizen, H., Groeneveld, P. H.,
and van Dissel, J. T. (1999) Antimicrob. Agents Chemother. 43, 2984-2989
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FOOTNOTES
1. This work was supported in part by grants from the Georgia Heart Association
(0051229B) and National Institutes of Health (GM61761-02).
2. The abbreviations used are: PGN, peptidoglycan; LPS, lipopolysaccharide; MDP,
muramyldipeptide (N-Acetylmuramyl-L-alanyl-D-isoglutamine); TLR, Toll-like receptor;
TNF, tumor necrosis factor; FCS, fetal calf serum; RT-PCR, reverse transcription-
polymerase chain reaction; Emax, maximum response; EC50,concentration producing 50%
activity; t, time of dissociation; k, dissociation rate constant; t1/2, half-life; mAb(s),
monoclonal antibody(ies); IL, interleukin; s, soluble.
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FIGURE LEGENDS
Fig. 1. Effect of MDP on LPS and PGN concentration-response curves. Mono Mac 6
cells were preincubated with 100 µg/ml MDP or medium as control for 30 min at 37oC. After 6
hours of stimulation with increasing concentrations of LPS (Fig. 1a) or PGN (Fig. 1b) as
indicated TNF-α was determined. Stimulation with MDP alone (100 µg/ml) resulted in a TNF-α
concentration of 71 + 21 pg/ml.
Fig. 2. Induction of TNF- mRNA and TNF- production by Mono Mac 6 cells treated
with LPS, PGN and MDP. Mono Mac 6 cells were stimulated with medium alone, medium
containing 30 ng/ml LPS, or 100 µg/ml PGN in the absence or the presence of 100 µg/ml MDP
for 90 min before RNA was isolated for RT-PCR analysis of TNF-α message (40 PCR cycles)
(Fig. 2a). mRNA of the 18S ribosomal gene amplified under the same conditions was used as
the internal controls. Supernatants of the same samples were assayed for TNF-α protein (Fig.
2b).
Fig. 3. Comparison of MDP with LPS on TNF- mRNA and TNF- protein levels.
Mono Mac 6 cells were stimulated with medium alone, increasing concentrations of LPS (0.01 -
100 ng/ml), or increasing concentrations of MDP (0.01 - 300 µg/ml) as indicated. After 90 min,
TNF-α production was measured (Fig. 3a), and total RNA was extracted and subjected to RT-
PCR analysis for detection of TNF-α mRNA (Fig. 3b).
Fig. 4. TNF- mRNA stability in Mono Mac 6 cells treated with different combinations
of stimuli. Mono Mac 6 cells were stimulated with medium alone, medium containing 30 ng/ml
LPS, or 100 µg/ml PGN in the absence or the presence of 100 µg/ml MDP for 90 min. At this
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time, new transcription was stopped with actinomycin D (10 µg/ml). Over a period of 2 hours,
total RNA was isolated at different time points and TNF-α mRNA was measured by RT-PCR.
Treatment with 10 µg/ml actinomycin D did not affect cell viability as judged by cellular
exclusion of trypan blue.
Fig. 5. Effect of anti-CD14 mAbs on MDP synergism with LPS and PGN. Mono Mac 6
cells were preincubated with anti-CD14 mAb MEM-18 (16 µg/ml), anti-CD14 mAb MY4 (20
µg/ml), or medium as control for 30 min at 4oC. TNF-α was measured after 6 hours of
stimulation with 10 ng/ml LPS (Fig. 5a) or 10 µg/ml PGN (Fig. 5b) either in the absence (LPS or
PGN alone) or presence (MDP & LPS or PGN) of 100 µg/ml MDP. No effects were observed
for the control antibodies IgG1 (MEM-18) and IgG2b (MY4).
Fig. 6. Effect of anti-TLR4 on synergistic effect between MDP and LPS. Mono Mac 6
cells were preincubated with anti-TLR4 mAb (40 µg/ml) or medium as control (alone) for 30
min at 4oC. After 6 hours of stimulation with MDP (100 µg/ml), LPS (10 ng/ml), or MDP
together with LPS at the same concentrations, TNF-α production was measured. (NA = not
analyzed). Preincubation with the control antibody IgG2a had no effect.
Fig. 7. Effect of anti-TLR2 on synergistic effect between MDP and PGN. Mono Mac 6
cells were preincubated with anti-TLR2 mAb (40 µg/ml) or medium as control (alone) for 30
min at 4oC. After 6 hours of stimulation with MDP (100 µg/ml), PGN (10 µg/ml), or MDP
together with PGN at the same concentrations, TNF-α production was measured. (NA = not
analyzed). Preincubation with the control antibody IgG2a had no effect.
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Fig. 8. Effect of MDP on the [3H]LPS off rate from Mono Mac 6 cells. After Mono Mac 6
cells were preincubated with [3H]LPS (30 ng/ml) for 1 hour at 37oC, unlabeled (cold) LPS (10
µg/ml) or a mixture of MDP (100 µg/ml) and unlabeled LPS (10 µg/ml) were added to the cells.
Cells were harvested at various time points and cell-associated 3H was counted.
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TABLES
Table 1 Relative TNF-α gene expression by Mono Mac 6 cells after stimulation with
different stimuli followed in time.
Relative Quantity of TNF- mRNA(95% confidence intervals)
30min
60min
90min
120min
Untreated 1.0 - 1.0 0.8 - 1.2 1.0 - 1.0 0.9 - 1.1
LPS (30 ng/ml) 1.1 - 1.6 17.3 - 18.1 15.8 - 19.4 11.5 - 12.5
PGN (100 µg/ml) 1.9 - 2.2 29.1 - 32.1 17.5 - 22.5 13.0 - 15.4
MDP (100 µg/ml) 0.9 - 1.5 3.2 - 4.2 7.2 - 8.5 2.4 - 2.6
MDP (100 µg/ml) & LPS (30 ng/ml) 1.2 - 1.4 21.2 - 22.5 26.8 - 33.6 8.9 - 11.7
MDP (100 µg/ml) & PGN (100 µg/ml) 2.0 - 2.3 22.4 - 30.4 27.1 - 31.2 14.2 - 18.6
Table 2 Effect of anti-CD14 mAb MY4 on the induction of TNF-α mRNA by Mono Mac 6
cells incubated with different stimuli for 90 min.
Relative Quantity of TNF- mRNA(95% confidence intervals)
Control MY4(20 µg/ml)
Untreated 0.9 - 1.2 1.0 - 1.1
LPS (30 ng/ml) 7.7 - 9.7 1.0 - 1.9
PGN (100 µg/ml) 7.1 - 8.9 6.2 - 8.2
MDP (100 µg/ml) 4.5 - 5.5 4.9 - 5.7
MDP (100 µg/ml) & LPS (30 ng/ml) 12.3 - 15.7 5.0 - 6.3
MDP (100 µg/ml) & PGN (100 µg/ml) 9.2 - 11.2 8.1 - 10.9
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Table 3 Effect of anti-TLR mAbs on the induction of TNF-α mRNA by Mono Mac 6 cells
incubated with different stimuli for 90 min.
Relative Quantity of TNF- mRNA(95% confidence intervals)
Control Anti-TLR2(40 µg/ml)
Anti-TLR4(40 µg/ml)
Untreated 1.0 – 1.0 0.8 – 1.0 0.8 – 0.9
LPS (30 ng/ml) 13.0 – 14.3 NA 10.3 – 12.4
PGN (100 µg/ml) 10.9 – 11.6 7.8 – 8.8 NA
MDP (100 µg/ml) 6.1 – 8.6 6.5 – 8.3 6.8 – 7.9
MDP (100 µg/ml) & LPS (30 ng/ml) 22.5 – 23.0 NA 16.7 – 18.1
MDP (100 µg/ml) & PGN (100 µg/ml) 17.4 – 19.5 15.0 – 16.3 NA
Note: NA = not analyzed
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Margreet A. Wolfert, Thomas F. Murray, Geert-Jan Boons and James N. Moorepeptidoglycan
The origin of the synergistic effect of muramyldipeptide with endotoxin and
published online July 31, 2002J. Biol. Chem.
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