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Infection and Immunity, January 2001, p. 378-385, Vol. 69, No. 1
0019-9567/01/$04.00+0 DOI: 10.1128/IAI.69.1.378-385.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
Role of Plasma, Lipopolysaccharide-Binding Protein,
and CD14 in Response of Mouse Peritoneal Exudate Macrophages to
Endotoxin
Didier
Heumann,1,*
Yoshiyuki
Adachi,2
Didier
Le
Roy,1
Naohito
Ohno,2
Toshiro
Yadomae,2
Michel Pierre
Glauser,1 and
Thierry
Calandra1
Division of Infectious Diseases, CHUV,
Lausanne, Switzerland,1 and Laboratory
of Immunopharmacology of Microbial Products, Tokyo University of
Pharmacy and Life Science, Tokyo, Japan2
Received 20 July 2000/Returned for modification 21 September
2000/Accepted 15 October 2000
 |
ABSTRACT |
Plasma lipopolysaccharide (LPS)-binding protein (LBP) and membrane
CD14 function to enhance the responses of monocytes to low
concentrations of endotoxin. Surprisingly, recent reports have
suggested that LBP or CD14 may be dispensable for macrophage responses
to low concentrations of LPS or may even exert an inhibitory effect in
the case of LBP. We therefore investigated whether LBP and CD14
participated in the response of mouse peritoneal exudate macrophages
(PEM) to LPS stimulation. In the presence of a low amount of plasma
(<1%) or of recombinant mouse or human LBP, PEM were found to respond
to low concentrations of LPS (<5 to 10 ng/ml) in an LBP- and
CD14-dependent manner. However, tumor necrosis factor production (not
interleukin-6 production) by LPS-stimulated PEM was reduced when cells
were stimulated in the presence of higher concentrations of plasma or
serum (5 or 10%). Yet, the inhibitory effect of plasma or serum was
not mediated by LBP. Taken together with previous results obtained with
LBP and CD14 knockout mice in models of
experimental endotoxemia, the present data confirm a critical part for
LBP and CD14 in innate immune responses of both blood monocytes and
tissue macrophages to endotoxins.
| |
INTRODUCTION |
Lipopolysaccharide (LPS) has been
shown to play a central role in the pathogenesis of severe sepsis and
septic shock caused by gram-negative bacteria. LPS stimulates monocytes
and macrophages to release proinflammatory mediators, such as
cytokines. In circulating monocytes, this pathway is under the control
of two proteins, plasma LPS-binding protein (LBP) and membrane CD14.
Indeed, neutralizing anti-CD14 and anti-LBP antibodies have been shown
to inhibit LPS-induced proinflammatory responses in vitro and in vivo
(8, 9, 20, 21). Similar results have been obtained for
mice with deletion of the CD14 or LBP genes
(10, 14). When LPS is bound to LBP, manyfold smaller
concentrations of LPS can activate monocytes through CD14 (7, 11,
30). Recent observations help in understanding the mechanism by
which LPS-LBP engagement of CD14 leads to monocyte activation, since
CD14 lacks a transmembrane domain. The recently identified Toll-like
receptors (TLRs), and in particular TLR4, are likely candidates to
transmit the LPS signal from CD14 to the cell (4, 12, 27,
29). At high concentrations of LPS, neither LBP nor CD14 is
required for activation of circulating monocytes (7, 11,
30). It is not yet clear whether under these circumstances LPS
stimulation occurs through TLRs or by another as yet unidentified pathway.
Whereas most studies indicating an important contribution of plasma LBP
and membrane-bound CD14 in LPS-induced activation have been performed
with circulating monocytes, other reports have suggested that
macrophages may be activated by LPS without the participation of serum
LBP or CD14 (13, 15, 16, 22, 26, 32). Furthermore, in
contrast with what was shown to occur in human monocytes, purified or
recombinant human LBP (rhLBP) was found to inhibit rather than to
enhance tumor necrosis factor (TNF) production by mouse peritoneal
exudate macrophages (PEM) stimulated with LPS (2, 3, 25).
These unexpected observations raised some doubts about the
well-documented contribution of LBP in amplifying the response of
monocytes to LPS.
We thus investigated further the role played by LBP and CD14 in the
activation of PEM by LPS. Experiments were performed with (i)
recombinant mouse LBP (rmLBP), (ii) plasma from LBP knockout mice, (iii) plasma or serum of various sources (human, mouse, calf),
and (iv) a neutralizing anti-CD14 monoclonal antibody.
| |
MATERIALS AND METHODS |
Sources of plasma and serum.
Heparinized human plasma and
human serum derived from clotted whole blood were obtained from healthy
human volunteers and were aliquoted and frozen at 
80°C. Eight- to
ten-week-old female OF1, NMRI, BALB/c, and C57BL/6J mice (Iffa Credo,
Lyon, France) were bled to obtain heparinized plasma or serum, which
was aliquoted and kept frozen at 80°C. Plasma or serum was also
obtained from LBP+/
heterozygous and
LBP/ mice generated on a BALB/c background
(14) (a kind gift of C. Schütt, Greifswald, Germany).
Plasma and serum were equivalent in their capacity to enhance
LPS-induced responses, as shown by the fact that similar dilutions of
plasma or serum induced similar TNF responses of monocytes in the
presence of LPS (data not shown). Heat-inactivated (56°C for 45 min)
plasma and serum were used in some experiments. LBP is stable under
these conditions (7). Normal plasma (not heat-inactivated) was used in all experiments reported in the tables and figures.
rmLBP was cloned in baculovirus using the cotransfection method with
Baculo-Gold (Pharmingen, San Diego, Calif.) and was expressed
in SF9
insect cells and cultivated in Excell medium, as described
previously
(
19,
20). Experiments were performed with cell
culture
supernatant or with purified rmLBP. In selected experiments,
supernatants of insect SF9 cells transfected with the empty plasmid
were used as controls for the LBP-containing cell culture supernatant.
CHO cells transfected with the human
LBP gene and secreting
rhLBP
were a kind gift of P. S. Tobias (Scripps, La Jolla,
Calif.).
The concentration of LBP in the supernatants of SF9 and CHO
cells
was measured by enzyme-linked immunosorbent assay, as previously
described (
19).
Antibodies.
The neutralizing anti-mouse LBP monoclonal
antibody (MAb) (clone M330-19) and the nonneutralizing anti-mouse LBP
MAb (clone M306-5) have been previously described (20).
These two rat MAbs (both immunoglobulin G2a isotypes) were purified by
protein G chromatography, dialyzed into phosphate-buffered saline, and
stored at 80°C. The LPS content of the anti-LBP MAbs was <5
pg/µg of protein.
4C1 is a newly developed rat antibody that neutralizes mouse CD14. 4C1
was found to block the binding of LPS to mouse macrophage
RAW264.7 and
to reduce LPS-mediated production of cytokines of
these cells
(
1).
Macrophage isolation and culture conditions.
OF1 mice were
injected intraperitoneally with 3 ml of 4% autoclaved Brewer
thioglycolate (Difco Laboratories, Detroit, Mich.). PEM were collected
3 or 5 days after injection and were washed and used without further
purification. More than 90% of the cells were macrophages by
morphological examination. All experiments were carried out with both
3- and 5-day PEM, which gave similar results. For the sake of
simplicity, only data concerning 3-day PEM are reported.
Cells (50,000/well) were plated into 96-well culture plates (Costar,
Cambridge, Mass.) and were stimulated with the indicated
concentrations
of LPS (from
Escherichia coli O111; Sigma, St.
Louis, Mo.)
in the presence of plasma or serum diluted in RPMI
1640 medium
supplemented with
L-glutamine, penicillin (50 U/ml),
and
streptomycin (50 µg/ml). Cells were incubated for 6 h at 37°C
in 5% CO
2 in a final volume of 200 µl. In some
experiments, anti-LBP
MAbs or anti-CD14 MAbs were added at a
concentration of 10 µg/ml
10 min before adding LPS. In other
experiments purified rmLBP,
SF9 insect cells supernatant containing
LBP, or control SF9 supernatant
with the empty plasmid was added to the
cell culture medium. Similarly,
CHO cell supernatants secreting rhLBP
or control supernatants
were added to PEM. Supernatants of stimulated
PEM were collected,
and concentrations of TNF and of interleukin-6
(IL-6) were measured
as described
below.
TNF and IL-6 determination.
PEM culture supernatants were
assayed for TNF by bioassay using WEHI clone 13 as targets and were
assayed for IL-6 by bioassay using 7TD1 cells as targets, as described
previously (11).
Presence of CD14.
Membrane-bound CD14 was assessed by
incubating PEM with the anti-CD14 MAb 4C1 (1 µg/ml) or with an
irrelevant MAb (1 µg/ml). After washing, cells were further incubated
with goat F(ab)'2 anti-rat immunoglobulin G labeled with
fluorescein isothiocyanate (Sigma), and binding of the antibodies was
measured by flow cytometry.
Data and statistics.
Statistical analyses were done using
the nonparametric analysis of variation (ANOVA) test on ranks for
multiple comparisons.
| |
RESULTS |
Contribution of plasma in the response of PEM to endotoxin.
To
examine the role of plasma in the response of macrophages to LPS, we
stimulated PEM with increasing concentrations of LPS. The cells were
cultured in plasma-free RPMI medium or in RPMI medium containing 0.2 or
1% mouse or human plasma. Cell culture supernatants were assessed for
TNF and IL-6. In the absence of plasma, 10 ng of LPS/ml was necessary
to induce cytokine production. The addition of 0.2% human or mouse
plasma (Fig. 1) or 1% plasma (data not
shown) resulted in increased production of TNF or IL-6 over that
measured in plasma-free medium. The addition of 0.2% plasma markedly
potentiated TNF or IL-6 production, especially at low concentrations of
LPS. Under these conditions, PEM started to produce cytokines at
concentrations of LPS as low as 100 pg/ml. At a concentration of 100 ng
of LPS/ml, the production of TNF was similar for cells incubated in
plasma-free medium or in medium enriched with 0.2% plasma. However,
the addition of 0.2% plasma potentiated the IL-6 response at high
concentrations of LPS.
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FIG. 1.
TNF and IL-6 production by PEM stimulated with LPS in
the presence of low doses of plasma. PEM (50,000 cells/well) were
stimulated for 6 h with LPS in the presence of RPMI medium with no
plasma (squares), RPMI medium containing 0.2% human plasma (circles,
panel A), or 0.2% autologous mouse plasma (triangles, panel B). Data
are the mean ± SD of five different experiments run in
duplicates. *, P < 0.05 by ANOVA.
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|
To determine if other sources of plasma or serum would also augment
cytokine production after LPS challenge, experiments were
repeated comparing native versus heat-inactivated plasma or serum
from calves, humans, or mice. In all conditions, 0.2 or 1% of
plasma
or serum increased the production of TNF or IL-6 (data
not shown),
suggesting that this effect could be mediated by a
protein such as
heat-stable
LBP.
Role of LBP present in plasma in the response of PEM to
endotoxin.
We then investigated whether LBP could account for the
enhancing effect of plasma. A neutralizing rat anti-mouse LBP MAb
(clone M330-19), shown to prevent the binding of LPS to LBP
(20), was used to inhibit LBP activity. A nonneutralizing
anti-LBP MAb (clone M306-5) was used as the control (20).
The addition of clone M306-5 to cells stimulated with LPS in the
presence of 1% mouse plasma did not affect the production of TNF or of
IL-6 (data not shown). The addition of 1% autologous (OF1) mouse
plasma induced an increase of the TNF or IL-6 production over controls
in plasma-free conditions, an effect that was largely inhibited by the
neutralizing anti-LBP MAb (clone M330-19) (Fig.
2). LBP blockade completely suppressed
cytokine production induced by 5 ng of LPS/ml. However, LBP blockade
had no effect when the cells were stimulated with a high concentration
of LPS (50 ng/ml). Similar results were obtained with the plasma or
serum of OF1, NMRI, BALB/c or C57BL/6J mice (data not shown).
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FIG. 2.
Effect of neutralization of mouse plasma LBP on the
cytokine response of PEM to LPS. PEM (50,000 cells/well) were
stimulated for 6 h with LPS in the presence of RPMI medium
containing 1% autologous mouse plasma (A) or RPMI medium with no
plasma (B) and in the presence of nonneutralizing anti-LBP MAb (clone
M306-5) (squares) or neutralizing anti-LBP MAb (clone M 330-19)
(circles). Data are the mean ± SD of five different experiments
run in duplicates. *, P < 0.05 by ANOVA.
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|
To further evaluate the contribution of LBP in the LPS-induced
activation of PEM, similar experiments were performed with
plasma of
mice deficient in LBP (
14). Plasma (1%) of heterozygous
LBP+/ and knockout
LBP/ mice was added to PEM, and cells were
then stimulated with increasing
concentrations of LPS (Fig.
3). As anticipated, plasma of
LBP+/ mice but not of
LBP/ mice increased cytokine production.
This effect was observed
at all LPS concentrations. In the presence of
LBP/ plasma, cytokine production by
LPS-stimulated PEM was similar
to that of cells cultivated in
plasma-free conditions. The difference
between these two plasmas was
indeed due to the presence of LBP,
as demonstrated by the suppression
of enhanced cytokine production
upon blocking LBP activity with the
neutralizing anti-LBP MAb
(clone M330-19) in
LBP+/ plasma (data not shown). To ensure that
the absence of LBP in
LBP/ plasma was the
sole factor responsible for the lack of enhancing
effect in the
response of PEM to LPS, we reconstituted
LBP/ plasma with 10 ng of rmLBP/ml
(corresponding to a 1% plasma concentration,
as the normal mouse
plasma level is 1 µg/ml). The addition of
exogenous LBP to the
LBP/ plasma reconstituted the
cytokine-enhancing effect that was blocked
by neutralizing anti-LBP MAb
(clone M330-19) (Table
1).
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FIG. 3.
Effect of heterozygous LBP+/
mice and LBP/ plasma on the cytokine
response by LPS-stimulated PEM. PEM (50,000 cells/well) were stimulated
for 6 h with LPS in the presence of RPMI medium containing either
1% LBP+/ plasma (squares) or 1%
LBP/ plasma (circles). Data are the
mean ± SD of five different experiments run in duplicates. *,
P < 0.05 by ANOVA.
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|
To determine whether higher doses of LBP in plasma might have an
inhibitory effect on the LPS-induced production of TNF, as
suggested by
previous studies (
2,
3,
18,
25), we compared
the cytokine
production of PEM stimulated with LPS in the presence
of 1% autologous
plasma or 1% autologous plasma spiked with 100
ng of rmLBP/ml (i.e., a
10-fold increase of LBP concentration).
As shown in Table
2, the addition of a 10-fold excess of
LBP
marginally affected the response, whereas blockade of LBP activity
with anti-LBP MAb suppressed the response to LPS.
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TABLE 2.
Effect of anti-LBP MAb or rmLBP on TNF production by
LPS-stimulated PEM cultured with 1%
autologous plasmaa
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Role of recombinant LBP in the response of PEM to endotoxin.
Having shown that LBP controls the cytokine response of PEM under
conditions of low doses of both plasma and LPS, we next investigated
the role of rmLBP in culture conditions carried out in the absence of
plasma. Various doses of purified rmLBP or dilutions of a titrated
supernatant of SF9 cells containing LBP or the empty vector (used as a
control) were added to PEM, which were then stimulated with LPS (Fig.
4). In the presence of plasma-free medium or of control SF9 supernatant, PEM did not produce TNF after
stimulation with 1 ng of LPS/ml. rmLBP (purified or cell culture
supernatant) enhanced the TNF response by LPS-stimulated PEM. No
difference in the level of TNF produced was obtained with
concentrations of rmLBP from 3 pg/ml to 100 ng/ml. A high dose of LBP
did not inhibit TNF production.
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FIG. 4.
Effect of recombinant mouse LBP on TNF production by PEM
stimulated with LPS. PEM (50,000 cells/well) were stimulated for 6 h with 1 ng of LPS/ml in the presence of increasing concentrations of
purified rmLBP (squares) or of SF9 supernatant containing known
concentrations of LBP (circles). TNF was not detected in cultures
containing control SF9 supernatant or RPMI medium (data not shown).
Data are the mean ± SD of four different experiments run in
duplicates.
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|
Recombinant human and mouse LBPs were found to be equipotent. PEM were
stimulated with 1 ng of LPS/ml in the presence of 3
ng/ml of titrated
supernatants containing rmLBP or rhLBP. TNF
production (the mean ± the standard deviation [SD] of three different
experiments) was
2,550 ± 865 using rmLBP and 2,620 ± 1,145 using
rhLBP.
Role of CD14 in the response of PEM to endotoxin.
PEM express
high levels of membrane CD14, as revealed by the binding of the
anti-CD14 MAb 4C1. By flow cytometry, 3-day PEM that have reacted with
an irrelevant MAb express 3.5 fluorescence units, whereas cells that
have reacted with the anti-CD14 MAb 4C1 express 32.5 ± 5.6 fluorescence units (mean of five different determinations).
PEM were pretreated with the neutralizing anti-CD14 MAb 4C1 and were
then stimulated with increasing concentrations of LPS
in plasma-free
medium or in medium containing 1% mouse plasma.
As shown in Fig.
5, anti-CD14 MAb completely suppressed
TNF and
IL-6 responses of cells stimulated with 1 ng of LPS/ml
irrespective
of the culture conditions (with or without plasma). In the
presence
of plasma, the anti-CD14-mediated inhibition of cytokine
production
was less effective at 5 ng of LPS/ml and was ineffective at
100
ng of LPS/ml. However, in plasma-free conditions, CD14 blockade
suppressed cytokine production even at the highest concentrations
of
LPS.
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FIG. 5.
Effect of anti-CD14 MAb on cytokine production by PEM
stimulated with LPS. PEM (50,000 cells/well) were pretreated with 10 µg of the anti-CD14 MAb/ml (circles) or with an irrelevant MAb
(squares) and were stimulated for 6 h with LPS in the presence of
RPMI medium containing 1% OF1 autologous mouse plasma (A) or RPMI
medium with no plasma (B). Data are the mean ± SD of five
different experiments run in duplicates. *, P < 0.05
by ANOVA.
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|
Response of PEM in the presence of high concentrations of
plasma.
Whereas the addition of low doses of plasma (1% or less)
enhanced the LPS-induced cytokine responses of PEM, the addition of 5 or 10% plasma decreased the TNF response as compared to that obtained
with plasma-free medium. This was observed with human plasma and mouse
plasma of various sources (autologous OF1 plasma as in Fig.
6 or heterologous NMRI, BALB/c, or
C57BL/6J plasma [data not shown]). In contrast to TNF, IL-6
production was similar whether the cells were stimulated with LPS in
the presence or in the absence of 5% human or mouse plasma.
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FIG. 6.
TNF and IL-6 production by PEM stimulated with LPS in
the presence of high doses of plasma. PEM (50,000 cells/well) were
stimulated for 6 h with LPS in the presence of RPMI medium with no
plasma (squares), RPMI medium containing 5% human plasma (circles,
panel A) or RPMI medium containing 5% autologous mouse plasma
(triangles, panel B). Data are the mean ± SD of five different
experiments run in duplicates. *, P < 0.05 by
ANOVA.
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Finally, to investigate whether LBP played a role in experiments
carried out with high doses of plasma, we stimulated PEM
in the
presence of 10% mouse plasma, which was shown to suppress
TNF
production of PEM stimulated by LPS. As shown in Table
3,
the addition of 200 ng of LBP/ml
(which represents a fourfold
increase in the concentration of LBP over
that present in 10%
normal mouse plasma) did not restore the
LPS-induced TNF response
of PEM stimulated in the absence of LBP. The
addition of the neutralizing
anti-LBP antibody also did not modify the
response of PEM to LPS,
suggesting that the inhibitory effect of a high
concentration
of plasma was not mediated by LBP.
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TABLE 3.
Effect of anti-LBP MAb or rmLBP on TNF production by
LPS-stimulated PEM cultured with 10%
autologous plasmaa
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| |
DISCUSSION |
Circulating monocytes and tissue macrophages play a central role
in the mediation of the biological effects of LPS, releasing a large
array of mediators and cytokines. The present findings provide further
evidence that LBP and CD14 mediate responses of activated peritoneal
exudate macrophages to low concentrations of LPS, as has been described
for circulating monocytes. In agreement with previous results obtained
with circulating monocytes, we observed that the response of PEM was
LBP- and CD14-dependent at low concentrations of LPS (<5 to 10 ng/ml).
Indeed, recombinant LBP or the presence of low doses of plasma (<1%)
increased the production of TNF and of IL-6 by LPS-stimulated PEM.
Under similar plasma conditions, antibody-mediated blockade of LBP or
CD14 suppressed the LPS response. However, it was also clear that PEM
responded to high concentrations of LPS in the absence of LBP or CD14,
as previously reported for other monocytic cell types (7, 11, 30).
CD14 blockade was more effective in plasma-free conditions than in the
presence of plasma at high LPS concentrations. No explanation is
available for the observation that CD14 blockade still diminished the
TNF response in plasma-free conditions but not in plasma conditions, when the cells were stimulated with 50 ng of LPS/ml. Experiments were
done with 10 µg of anti-CD14 MAb/ml, which is a large excess of MAb
compared to soluble CD14 present in 1% plasma as well to CD14
expressed in macrophages. The fact that the TNF response was abolished
at 1 ng of LPS/ml and was reduced at 5 ng of LPS/ml in both plasma-free
and plasma conditions indicates that the problem was not the
concentration of the anti-CD14 MAb.
It is now clearly established that CD14 and LBP are important partners
in the LPS response of a large variety of monocytes and macrophages,
including circulating human, rabbit, and calf alveolar macrophages
(17, 28, 31), human or rabbit peritoneal or alveolar
macrophages (6, 23, 24), and mouse whole blood (7). Macrophages that are derived from different tissues
or that are at different stages of differentiation might exhibit variable responses to LPS. In fact, several investigators have hypothesized the existence of signaling pathways other than LBP/CD14 in
macrophages. Recent reports have challenged the concept that serum
proteins (LBP) and CD14 are required for the LPS-induced responses of
macrophages: (i) both CD14- and LBP-dependent and CD14- and
LBP-independent responses have been implicated in the response of
bovine macrophages to LPS (5, 16); (ii) an established human monocytic cell line, derived from THP-1 cells, was found to
respond to LPS when grown in the absence of serum proteins for more
than 20 generations (22); (iii) CHO cells not expressing CD14 but transfected with CD11c/CD18 respond to LPS, although at higher
concentrations of LPS than CHO cells transfected with CD14
(13); and (iv) in the absence of serum, mouse
thioglycolate-elicited peritoneal macrophages were found to express
high levels of TNF and IL-1
mRNA upon stimulation with a wide range
of low concentrations of LPS (25). In fact, the addition
of rhLBP to these cells did not enhance but actually decreased TNF and
IL-1 mRNA while increasing that of interferon-inducible protein 10 (IP-10) (25). This observation was confirmed by Amura et
al., who showed that rhLBP suppressed in a dose-dependent manner
LPS-induced TNF production but not NO production by mouse peritoneal
exudate macrophages (2, 3). These findings suggested the
possibility that the modulatory properties of LBP and CD14 vary with
the cellular targets, with the source of LBP or with the mediator under
investigation. In the present study, no difference was observed between
rmLBP and rhLBP, a finding which contrasted with the inhibitory role of
rhLBP (2, 3, 25). Different PEM (from OF1 mice in the
present study versus C3H/OuJ or C3HeB/FeJ mice) or different
preparations of LPS (rough versus smooth) could account for these
differences. All these data illustrate the difficulty of studying
thioglycolate-induced PEM, all the more since various preparations of
commercial thioglycolate may induce various populations of PEM. The
time at which elicited macrophages were harvested (3- or 5-day PEM) did
not play a significant role, as observed in the present study and in
previous reports (2, 3, 25).
Yet, serum or plasma LBP was quite effective in enhancing cytokine
production in response to low concentrations of LPS. Importantly, in
the present study, TNF production was undetectable in serum-free medium
under low LPS conditions and the addition of plasma or purified LBP
allowed TNF production. Mouse plasma contains 1 to 2 µg of LBP/ml
(8). Thus, the present experiments showing a potentiating
effect of 0.2 or 1% mouse plasma were conducted with concentrations of
LBP in the range of 4 to 20 ng/ml. Identical results were obtained with
similar concentrations of rmLBP. rmLBP did not exert an inhibitory
effect at concentrations as high as 100 ng/ml (corresponding to the
concentration of LBP present in 10% plasma). However, in the presence
of 5 and 10% plasma, the TNF response of PEM to LPS was reduced and
was not influenced by the addition of exogenous rmLBP or by the
blockade of endogenous LBP with anti-LBP antibody. Thus, factors other
than LBP are clearly involved in the suppression of LPS-induced TNF
response at high doses of plasma. The mechanisms underlying these
inhibitory effects remain to be identified. Of note, the TNF but not
the IL-6 response of PEM to LPS was reduced when cells were stimulated
in the presence of 5% plasma or serum.
It could be speculated that if circulating monocytes are adapted to
respond to LPS in the presence of 100% plasma, tissue macrophages and,
in particular, PEM may not be capable of responding to LPS in the
presence of high doses of plasma, at least ex vivo. During severe
acute-phase responses, LBP concentrations did not exceed 50 ng/ml in
the peritoneal cavity (our unpublished observations). Similarly,
protein concentrations were approximately 1% of those measured in
plasma. This indicates that in vitro studies with macrophages are
likely more relevant when macrophages are stimulated in the presence of
low amounts of plasma. This also suggests that in vivo PEM are in a
situation in which mechanisms of responses are LBP and CD14 dependent.
In summary, the present study indicates that the response of PEM to low
concentrations of LPS is LBP and CD14 dependent. These observations
confirm and extend previous results obtained in mouse models of
endotoxemia, for which it is not yet clear whether circulating monocytes or tissue macrophages are the major targets of LPS. Mice
treated with anti-LBP antibodies, LBP knockout mice, and CD14 knockout mice were shown to be resistant to LPS-induced
cytokine production and resistant to injections of low concentrations
of LPS (8-10, 14, 21). Yet all these mice succumb to high
concentrations of LPS. These observations appear to rule out an
important contribution of LBP- and CD14-independent mechanisms in the
activation of monocytes and macrophages by low concentrations of LPS
(up to 100 ng/mouse). However, they do not rule out the possibility
that cells may be activated by LBP- and CD14-independent mechanisms in
the presence of high concentrations of LPS (from 1 µg to 1 mg/mouse).
| |
ACKNOWLEDGMENTS |
This study was supported by grant no. 32-55829.98 to D.H. and
grants no. 32-489/6.96 and no. 32-49129.96 to T.C. from the Fonds
National Suisse de la Recherche.
We thank M. Knaup for technical assistance.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Division of
Infectious Diseases, CHUV, CH-1011 Lausanne, Switzerland. Phone: 41 21 314 10 25. Fax: 41 21 314 10 36. E-mail:
dheumann{at}hola.hospvd.ch.
Editor:
J. D. Clements
| |
REFERENCES |
| 1.
|
Adachi, Y.,
C. Satokawa,
M. Saeki,
N. Ohno,
H. Tamura,
S. Tanaka, and T. Yadomae.
1999.
Inhibition by a CD14 monoclonal antibody of lipopolysaccharide binding to murine macrophages.
J. Endotoxin Res.
5:139-146.
|
| 2.
|
Amura, C. R.,
L. C. Chen,
N. Hirohashi,
M. G. Lei, and D. C. Morrison.
1998.
Two functionally independent pathways for lipopolysaccharide-dependent activation of mouse peritoneal macrophages.
J. Immunol.
159:5079-5083[Abstract].
|
| 3.
|
Amura, C. R.,
T. Kamei,
N. Ito,
M. J. Soares, and D. C. Morrison.
1998.
Differential regulation of lipopolysaccharide (LPS) activation pathways in mouse macrophages by LPS-binding proteins.
J. Immunol.
161:2552-2560[Abstract/Free Full Text].
|
| 4.
|
Chow, J. C.,
D. W. Young,
D. T. Golenbock,
W. J. Christ, and F. Gusovsky.
1999.
Toll-like receptor-4 mediates lipopolysaccharide-induced signal transduction.
J. Biol. Chem.
274:10689-10692[Abstract/Free Full Text].
|
| 5.
|
Dean, D. F.,
P. N. Bochsler,
R. C. Caroll,
F. W. Olchowy,
N. R. Neilsen, and D. O. Slauson.
1998.
Signaling pathways for tissue factor expression in lipopolysaccharide-stimulated bovine alveolar macrophages.
Am. J. Vet. Res.
59:445-451[Medline].
|
| 6.
|
Dentener, M. A.,
V. Bazil,
E. J. U. Von Asmuth,
M. Ceska, and W. A. Buurman.
1993.
Involvement of CD14 in lipopolysaccharide-induced tumor necrosis factor-alpha, IL-6 and IL-8 release by human monocytes and alveolar macrophages.
J. Immunol.
150:2885-2891[Abstract].
|
| 7.
|
Gallay, P.,
S. Carrel,
M. P. Glauser,
C. Barras,
R. J. Ulevitch,
P. S. Tobias,
J. D. Baumgartner, and D. Heumann.
1993.
Purification and characterization of murine lipopolysaccharide-binding protein.
Infect. Immun.
61:378-383[Abstract/Free Full Text].
|
| 8.
|
Gallay, P.,
D. Heumann,
D. Le Roy,
C. Barras, and M. P. Glauser.
1993.
Lipopolysaccharide-binding protein as a major plasma protein responsible for endotoxemic shock.
Proc. Natl. Acad. Sci. USA
90:9935-9938[Abstract/Free Full Text].
|
| 9.
|
Gallay, P.,
D. Heumann,
D. Le Roy,
C. Barras, and M. P. Glauser.
1994.
Mode of action of anti-lipopolysaccharide binding protein (LBP) antibodies for prevention of endotoxemic shock in mice.
Proc. Natl. Acad. Sci. USA
91:7922-7926[Abstract/Free Full Text].
|
| 10.
|
Haziot, A.,
E. Ferrero,
F. Köntgen,
N. Hijiya,
S. Yamamoto,
J. Sliver,
C. L. Stewart, and S. M. Goyert.
1996.
Resistance to endotoxin shock and reduced dissemination of gram-negative bacteria in CD14-deficient mice.
Immunity
4:407-414[CrossRef][Medline].
|
| 11.
|
Heumann, D.,
P. Gallay,
C. Barras,
P. Zaech,
R. J. Ulevitch,
P. S. Tobias,
M. P. Glauser, and J. D. Baumgartner.
1992.
Control of LPS binding and LPS-induced TNF secretion in human peripheral blood monocytes.
J. Immunol.
148:3505-3512[Abstract].
|
| 12.
|
Hoshino, K.,
O. Takeuchi,
T. Kawai,
H. Sanjo,
T. Ogawa,
Y. Takeda,
K. Takeda, and S. Akira.
1999.
Toll-like receptor 4 (TLR4)-deficient mice are hyporesponsive to lipopolysaccharide: evidence for TLR4 as the Lps gene product.
J. Immunol.
162:3749-3752[Abstract/Free Full Text].
|
| 13.
|
Ingalls, R. R., and D. T. Golenbock.
1995.
CD11c/CD18, a transmembrane signaling receptor for lipopolysaccharide.
J. Exp. Med.
181:1473-1479[Abstract/Free Full Text].
|
| 14.
|
Jack, R. S.,
X. L. Fan,
M. Bernheiden,
G. Rune,
M. Ehlers,
A. Weber,
G. Kirsch,
R. Mentel,
B. Fürll,
M. Freudenberg,
G. Schmitz,
F. Stelter, and C. Schütt.
1997.
Lipopolysaccharide-binding protein is required to combat a Gram-negative bacterial infection.
Nature
389:742-745[CrossRef][Medline].
|
| 15.
|
Jahr, T. G.,
A. Sundan,
H. S. Lichenstein, and T. Espevik.
1995.
Influence of CD14, LBP and BPI in the monocyte response to LPS of different polysaccharide chain length.
Scand. J. Immunol.
42:119-127[CrossRef][Medline].
|
| 16.
|
Jungi, T. W.,
H. Sager,
H. Adler,
M. Brcic, and H. Pfister.
1997.
Serum factors, cell membrane CD14, and 2 integrins are not required for activation of bovine macrophages by lipopolysaccharide.
Infect. Immun.
65:3577-3584[Abstract].
|
| 17.
|
Khemlani, L. S.,
Z. Yang, and P. N. Bochsler.
1994.
Identification and characterization of a bovine lipopolysaccharide-binding protein.
J. Leukoc. Biol.
56:784-791[Abstract].
|
| 18.
|
Lamping, N.,
R. Dettmer,
W. J. Schröder,
D. Pfeil,
W. Hallatschek, and R. R. Schumann.
1998.
LPS-binding protein protects mice from septic shock caused by LPS or Gram-negative bacteria.
J. Clin. Investig.
101:2065-2071[Medline].
|
| 19.
|
Lengacher, S.,
C. V. Jongeneel,
D. Le Roy,
J. D. Lee,
V. Kravtchenko,
R. J. Ulevitch,
M. P. Glauser, and D. Heumann.
1996.
Reactivity of murine and human recombinant LPS-binding protein (LBP) with LPS and Gram negative bacteria.
J. Inflamm.
47:165-172.
|
| 20.
|
Le Roy, D.,
F. Di Padova,
R. Tees,
S. Lengacher,
R. Landmann,
M. P. Glauser,
T. Calandra, and D. Heumann.
1999.
Monoclonal antibodies to murine lipopolysaccharide (LPS)-binding protein (LBP) protect mice from lethal endotoxemia by blocking either the binding of LPS to LBP or the presentation of LPS/LBP complexes to CD14.
J. Immunol.
162:7454-7460[Abstract/Free Full Text].
|
| 21.
|
Leturcq, D.,
A. M. Moriarty,
G. Talbott,
R. K. Winn,
T. R. Martin, and R. J. Ulevitch.
1996.
Antibodies against CD14 protect primates from endotoxin-induced shock.
J. Clin. Investig.
98:1533-1538[Medline].
|
| 22.
|
Lynn, W. A.,
Y. Liu, and D. T. Golenbock.
1993.
Neither CD14 nor serum is absolutely necessary for activation of mononuclear phagocytes by bacterial lipopolysaccharide.
Infect. Immun.
61:4452-4461[Abstract/Free Full Text].
|
| 23.
|
Martin, T. R.,
J. C. Mathison,
P. S. Tobias,
D. J. Leturcq,
A. M. Moriarty,
R. J. Maunder, and R. Ulevitch.
1992.
Lipopolysaccharide binding protein enhances the responsiveness of alveolar macrophages to bacterial lipopolysaccharide. Implications for cytokine production in normal and injured lungs.
J. Clin. Investig.
90:2209-2219.
|
| 24.
|
Mathison, J. C.,
E. Wolfson,
S. Steinemann,
P. S. Tobias, and R. J. Ulevitch.
1993.
Lipopolysaccharide (LPS) recognition in macrophages. Participation of LPS-binding protein and CD14 in LPS-induced adaptation in rabbit peritoneal exudate macrophages.
J. Clin. Investig.
92:2053-2059.
|
| 25.
|
Perera, P. Y.,
N. Qureshi,
W. J. Christ,
P. Stütz, and S. N. Vogel.
1998.
Lipopolysaccharide and its analog antagonists display differential factor dependencies for induction of cytokine in murine macrophages.
Infect. Immun.
66:2562-2569[Abstract/Free Full Text].
|
| 26.
|
Perera, P. Y.,
S. N. Vogel,
G. R. Detore,
A. Haziot, and S. M. Goyert.
1997.
CD14-dependent and CD14-independent signaling pathways in murine macrophages from normal and CD14 knockout mice stimulated with lipopolysaccharide or taxol.
J. Immunol.
158:4422-4429[Abstract].
|
| 27.
|
Poltorak, A.,
X. He,
I. Smirnova,
M. Y. Liu,
C. V. Huffel,
X. Du,
D. Birdwell,
E. Alejos,
M. Silva,
C. Galanos,
M. Freudenberg,
P. Ricciardi-Castagnoli,
P. Layton, and B. Beutler.
1998.
Defective LPS signaling in C3H/HeJ and C57BL/10ScCr mice: mutations in Tlr4 gene.
Science
282:2085-2088[Abstract/Free Full Text].
|
| 28.
|
Schumann, R. R.,
S. R. Leong,
G. W. Flaggs,
P. W. Gray,
S. D. Wright,
J. C. Mathison,
P. S. Tobias, and R. J. Ulevitch.
1990.
Structure and function of lipopolysaccharide binding protein.
Science
249:1429-1431[Abstract/Free Full Text].
|
| 29.
|
Shimazu, R.,
S. Akashi,
H. Ogata,
Y. F. K. Nagai,
K. Miyake, and M. Imoto.
1999.
MD-2, a molecule that confers lipopolysaccharide responsiveness on Toll-like receptor 4.
J. Exp. Med.
189:1777-1782[Abstract/Free Full Text].
|
| 30.
|
Ulevitch, R. J., and P. S. Tobias.
1994.
Recognition of endotoxin by cells leading to transmembrane signaling.
Curr. Biol.
6:125-130.
|
| 31.
|
Wright, S. D.,
R. A. Ramos,
P. S. Tobias,
R. J. Ulevitch, and J. C. Mathison.
1990.
CD14, a receptor for complexes of lipopolysaccharide (LPS) and LPS binding protein.
Science
249:1431-1433[Abstract/Free Full Text].
|
| 32.
|
Wurfel, M. M.,
B. G. Monks,
R. R. Ingalls,
R. L. Dedrick,
R. Delude,
D. Zhou,
N. Lamping,
R. R. Schumann,
R. Thieringer,
M. J. Fenton,
S. D. Wright, and D. Golenbock.
1997.
Targeted deletion of the lipopolysaccharide (LPS)-binding protein gene leads to profound suppression of LPS responses ex vivo, whereas in vivo responses remain intact.
J. Exp. Med.
186:2051-2056[Abstract/Free Full Text].
|
Infection and Immunity, January 2001, p. 378-385, Vol. 69, No. 1
0019-9567/01/$04.00+0 DOI: 10.1128/IAI.69.1.378-385.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
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Abstract
Introduction
