Download Critical Role of Lipopolysaccharide-Binding Protein

Critical Role of Lipopolysaccharide-Binding
Protein and CD14 in Immune Responses
against Gram-Negative Bacteria
This information is current as
of June 18, 2017.
Didier Le Roy, Franco Di Padova, Yoshiyuki Adachi, Michel
Pierre Glauser, Thierry Calandra and Didier Heumann
J Immunol 2001; 167:2759-2765; ;
doi: 10.4049/jimmunol.167.5.2759
http://www.jimmunol.org/content/167/5/2759
Subscription
Permissions
Email Alerts
This article cites 36 articles, 23 of which you can access for free at:
http://www.jimmunol.org/content/167/5/2759.full#ref-list-1
Information about subscribing to The Journal of Immunology is online at:
http://jimmunol.org/subscription
Submit copyright permission requests at:
http://www.aai.org/About/Publications/JI/copyright.html
Receive free email-alerts when new articles cite this article. Sign up at:
http://jimmunol.org/alerts
The Journal of Immunology is published twice each month by
The American Association of Immunologists, Inc.,
1451 Rockville Pike, Suite 650, Rockville, MD 20852
Copyright © 2001 by The American Association of
Immunologists All rights reserved.
Print ISSN: 0022-1767 Online ISSN: 1550-6606.
Downloaded from http://www.jimmunol.org/ by guest on June 18, 2017
References
Critical Role of Lipopolysaccharide-Binding Protein and CD14
in Immune Responses against Gram-Negative Bacteria1
Didier Le Roy,* Franco Di Padova,† Yoshiyuki Adachi,‡ Michel Pierre Glauser,*
Thierry Calandra,* and Didier Heumann2*
P
lasma LPS-binding protein (LBP)3 and monocyte CD14
are central molecules of the innate immune system, in response to LPS. Due to its essential role in the mediation of
the presentation of LPS to CD14 (1, 2), LBP contributes to enhance LPS-induced lethality, as shown in animals with a LBPdisrupted gene (3) or in wild-type mice treated with anti-LBP Abs
(4 – 6). Similarly, mice with a CD14-disrupted gene were resistant
to LPS-induced toxicity (7). Abs to CD14 provided therapeutic
benefit after in vivo exposure to endotoxin in rabbits and primates
(8, 9).
The role of LBP and CD14 in bacterial infections is not clearly
defined. LBP and CD14 have been postulated to be a prerequisite
in the mechanisms involved in initiation of host defense against
Gram-negative bacteria, alarming the host to the presence of
minute amounts of LPS (1). In favor of this hypothesis, LBP⫺/⫺
mice, although resistant to LPS, were susceptible to Salmonella
typhimurium (3), and the intestinal mucosa of rabbits treated with
a neutralizing anti-CD14 mAb exhibited a 50-fold increase in Shigella invasion and more severe injury compared with controls (10).
The role of excess of proinflammatory cytokines in pathogenic
events triggered by systemic injections of LPS or high numbers of
bacteria has been well documented (11, 12). Yet, in the presence
of a low inoculum of bacteria, endogenous production of cytokines
and of TNF-␣ appears pivotal for normal innate immune responses
against an invading organism. Ab-mediated blockade of TNF-␣ or
disruption of the TNF-␣ gene or of the TNFp55 receptor gene was
*Division of Infectious Diseases, Centre Hospitalier Universitaire Vaudois-Lausanne,
Lausanne, Switzerland; †Pharma Research, Novartis, Basel, Switzerland; and ‡Laboratory of Immunopharmacology of Microbial Products, Tokyo University of Pharmacy and Life Science, Tokyo, Japan
Received for publication January 29, 2001. Accepted for publication June 27, 2001.
The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked advertisement in accordance
with 18 U.S.C. Section 1734 solely to indicate this fact.
1
This work was supported by Swiss National Science Foundation Grants 3200055529.98/1 (to D.H.) and 32-49129.96 (to T.C.). T.C. is a recipient of a career award
from the Swiss National Foundation (32-48916-96).
2
Address correspondence and reprint requests to Dr. Didier Heumann, Division of
Infectious Diseases, BH19, CHUV 1011 Lausanne, Switzerland. E-mail address:
[email protected]
3
Abbreviations used in this paper: LBP, LPS-binding protein; PMN, polymorphonuclear neutrophil.
Copyright © 2001 by The American Association of Immunologists
detrimental in models of intracellular facultative bacteria (13–16).
This was also shown for extracellular bacteria, in models of cecal
ligation puncture (17), of pneumonia (18 –20), or of peritonitis
(21). Conversely, treatment with TNF-␣ improved survival of
mice injected with S. typhimurium or undergoing cecal ligation
puncture (22, 23). Importantly, LPS-hyporesponsive C3H/HeJ
mice were found to be more susceptible to lethal infection with
Escherichia coli than normal LPS-responsive mice, a defect that
was corrected by administration of TNF-␣ (24).
Thus a body of data emphasizes the need for an intact mechanism of recognition of LPS leading to cytokine production in initiating host defense mechanisms against Gram-negative infections.
In the present study, we investigated the contribution of LBP,
CD14, and TNF-␣ in bacterial sepsis. We hypothesized that blocking of the innate immune responses with Abs to LBP, CD14, or
TNF-␣ may impair the development of normal innate responses to
low doses of bacteria, and thus augment lethality.
Materials and Methods
Bacteria
An encapsulated strain of Klebsiella pneumoniae isolated from a bacteriemic patient was used in the present experiments (25). This strain was
selected among other Klebsiella strains for its high virulence in mice. Bacteria were grown for 2 h in tryptic soy broth (Difco, Detroit, MI) and
collected in the exponential growth phase at an OD of 0.18, corresponding
to 5.5 ⫻ 107 ⫾ 0.15 CFU/ml. Bacteria were then diluted to the desired
concentration in saline before injection into mice. E. coli O111:B4 was
cultivated as described (26).
Reagents and Abs
Two rat mAbs to mouse LBP described in (6) were studied: 1) the neutralizing mAb (clone M330-9, referred thereafter as to anti-LBP mAb),
preventing the binding of LPS to LBP, suppressing LPS-induced TNF-␣
production and blocking LBP activity in vivo up to 7 h after injection in
mice; and 2) the control anti-LBP mAb (clone M306-5, referred thereafter
as to control mAb), which does not neutralize LBP activity. 4C1 is a rat
mAb that neutralizes mouse CD14 (27). MAbs were purified by protein G
chromatography, dialyzed into PBS, and stored at ⫺80°C. Anti-TNF Abs
were raised in rabbits and polyclonal IgG from immunized rabbits or control rabbits isolated by protein G chromatography. Recombinant murine
TNF-␣ was a gift from G. Grau (University of Marseille, Marseille,
France). Presence of LPS in reagents was determined with the Limulus
assay (Chromogenix, Embrach, Switzerland). The LPS content of the
mAbs and of the IgG was 1 pg/␮g of protein.
0022-1767/01/$02.00
Downloaded from http://www.jimmunol.org/ by guest on June 18, 2017
LPS-binding protein (LBP) and CD14 potentiate cell activation by LPS, contributing to lethal endotoxemia. We analyzed the
contribution of LBP/CD14 in models of bacterial infection. Mice pretreated with mAbs neutralizing CD14 or LBP showed a delay
in TNF-␣ production and died of overwhelming infection within 24 h, after a challenge with 250 CFU of virulent Klebsiella
pneumoniae. Blockade of TNF-␣ also increased lethality, whereas pretreatment with TNF-␣ protected mice, even in the presence
of LBP and CD14 blockade. Anti-LBP or anti-CD14 mAbs did not improve or decrease lethality with a higher inoculum (105 K.
pneumoniae) and did not affect outcome following injections of low or high inocula of Escherichia coli O111. These results point
to the essential role of LBP/CD14 in innate immunity against virulent bacteria. The Journal of Immunology, 2001, 167: 2759 –
2765.
2760
LBP AND CD14 IN BACTERIAL SEPSIS
Galactosamine model
Because 4C1 Ab has never been evaluated in vivo, we assessed whether
4C1 prevented endotoxemia in mice sensitized with D-galactosamine. Mice
were injected i.v. with 200 ␮g of the neutralizing anti-CD14 mAb 4C1 2 h
before an i.p. challenge of 50 ng of E. coli O111 LPS (Sigma, St. Louis,
MO) given in combination with 20 mg D-galactosamine (Sigma). Mice
were bled 1.5 h after LPS challenge to measure plasma TNF-␣ concentration. The injection of 4C1 reduced TNF-␣ production, and prevented death
(0 deaths/5 mice) compared with injection of saline (5/5) (data not shown).
Similarly to neutralizing anti-LBP mAb, anti-CD14 mAb was effective at
blocking LPS-induced cell activation and death for 5–7 h (data not shown).
Bacterial challenge
OF1 female mice, 5- to 6-wk old, were purchased from IFFA Credo (Lyon,
France). OF1 mice were injected i.v. with saline or with 100 ␮g/mouse of
rat mAbs in a volume of 250 ␮l of saline, and with K. pneumoniae or E.
coli suspended in 250 ␮l saline. Plasma was obtained via the tail vein to
determine plasma TNF-␣ concentrations, bacterial counts, and neutrophil
counts. To avoid excess bleeding, most of the mice were bled only once
(50-␮l aliquots), occasionally twice.
TNF-␣ was measured in plasma by bioassay using WEHI clone 13 as
targets, as previously described (28).
Neutrophil count determination
Türck Blue (Sigma) was added to whole blood samples, and the total number of white blood cell counts was determined by microscopy. For neutrophil count determination, plasma samples were lysed on ice with 0.1 M
NH4Cl/0.1 M KHCO3 and cytocentrifuged. The percentage of polymorphonuclear neutrophils (PMN) was determined with May-Grünwald-Giemsa staining. The number of PMN per milliliter of blood was determined
taking into account the total number of white blood cells and the percentage of neutrophils.
Statistics
The ␹2 test was used to assess the significance of differences between
treatment groups. The ANOVA test on ranks was used to assess the significance of intergroup differences for the various markers (bacteria, PMN
cell, TNF-␣).
Results
LBP and CD14 blockade is associated with delayed production
of TNF-␣ in mice injected i.v. with a low inoculum of K.
pneumoniae
In preliminary experiments, the LD50 for K. pneumoniae was
found to be between 50 and 250 CFU/mouse. We thus used this
inoculum to investigate the role of LBP and CD14 in the early
steps of infection leading to death.
To investigate the effect of LBP activity blockade on TNF-␣
production induced by bacterial sepsis, mice were injected with
anti-LBP mAbs before a challenge with ⬍250 CFU of K. pneumoniae (Fig. 1). Bioactive TNF-␣ was not detectable in the blood
within the first 5 h, irrespective of treatment (control or anti-LBP
mAb). Five hours after infection, TNF-␣ levels were 0.8 ⫾ 0.4
ng/ml in mice treated with control mAb, and undetectable in mice
treated with anti-LBP mAb. There was a 1- to 2-h delay in TNF-␣
production in mice treated with anti-LBP mAb compared with
mice treated with control mAb. Six hours after infection, TNF-␣
levels were 1 ⫾ 0.5 ng/ml in mice treated with control mAb, and
0.2 ⫾ 0.1 ng/ml in mice treated with anti-LBP mAb. Yet, 7 h after
infection, TNF-␣ levels were higher thereafter in mice treated with
anti-LBP mAb.
A similar delay in TNF-␣ production was obtained in mice
treated with anti-CD14 mAb (Fig. 1). Yet, TNF-␣ levels were still
higher in mice treated with anti-CD14 mAb than in mice treated
with anti-LBP mAb, 7 h after infection.
LBP and CD14 blockade is associated with impairment of
neutrophil recruitment in mice injected i.v. with a low inoculum
of K. pneumoniae
In the early hours after infection with K. pneumoniae, neutrophil
counts doubled in the first 30 min, irrespective of treatment (Fig.
2). In mice treated with control mAb, neutrophil counts continued
to increase to a 4-fold level over basal levels, peaking at 1.5 h, then
progressively decreasing to basal levels at 6 h. In contrast, animals
treated with anti-LBP mAb did not show any increase of neutrophils from 30 min to 6 h. The same observation was made during
the first 3 h after infection, in mice treated with anti-CD14 mAb.
Yet, in those mice, numbers of bacteria were similar to controls
from 3 to 6 h.
LBP and of CD14 blockade is associated with uncontrolled
bacterial proliferation in mice injected i.v. with a low inoculum
of K. pneumoniae
Death was associated with elevated bacteremia, and moribund
mice had bacterial counts higher than 107 CFU/ml of blood (Fig.
3). Kinetics of bacterial counts were similar in mice receiving saline or control mAb for the first 24 h after bacterial challenge. Fig.
3A illustrates the kinetics of bacterial counts in mice treated with
control mAb. Blood bacterial counts increased in survivors and
nonsurvivors from 100 to 250 CFU/ml to ⬃104 CFU/ml during the
first 12 h, then plateaued until 48 h, with slightly higher numbers
FIGURE 2. Effect of anti-LBP and anti-CD14 mAbs on blood neutrophil counts of mice injected i.v. with a low inoculum (from 60 to 260
CFU/mouse) of K. pneumoniae. Mice were injected with 100 ␮g/mouse of
control mAb (E), anti-LBP mAb (■), or anti-CD14 mAb (Œ) 5 min before
i.v. infection. Data are the mean of five different experiments (42 mice/
group). p ⬍ 0.05, by ANOVA comparing anti-LBP mAb vs control mAb
(ⴱ) or anti-CD14 mAb vs control mAb (#).
Downloaded from http://www.jimmunol.org/ by guest on June 18, 2017
TNF bioassay
FIGURE 1. Effect of anti-LBP and anti-CD14 mAbs on blood TNF-␣
levels of mice injected i.v. with a low inoculum (from 60 to 260 CFU/
mouse) of K. pneumoniae. Mice were injected with 100 ␮g/mouse of control mAb (circles), anti-LBP mAb (squares), or anti-CD14 mAb (triangles)
5 min before i.v. infection. Data are the mean of five different experiments
(42 mice/group). p ⬍ 0.05, by ANOVA comparing anti-LBP mAb vs control mAb (ⴱ) or anti-CD14 mAb vs control mAb (#).
The Journal of Immunology
2761
FIGURE 4. Correlation between blood TNF-␣ and bacterial counts. f,
Control mAb; E, anti-LBP mAb.
in nonsurvivors than in survivors. From day 3, bacteria were
cleared from the circulation in survivors, whereas blood bacterial
counts progressively increased until death in nonsurvivors. Fig. 3B
shows the kinetics of blood bacterial counts in mice injected with
anti-LBP mAb (nonsurvivors). Blood bacterial counts were similar
(no statistically significant difference) to those measured in survivors or nonsurvivors receiving control mAb for the first 6 h. However, between 7 and 12 h, mice that were moribund all had increased numbers of circulating bacteria than survivors found either
in this group of mice or in the group of mice receiving saline or
control mAb. Under this condition, bacterial counts remained elevated until death. Kinetics of blood bacterial counts was even
more accelerated when mice were treated with anti-CD14 mAb
(Fig. 3C, nonsurvivors). All mice had similar numbers of bacteria
until 4 h (survivors and nonsurvivors). However, 7 h after bacterial
challenge, bacterial numbers were at least 2 logs higher than those
of controls (103 vs 105 CFU/ml, p ⬍ 0.0001). Bacterial numbers
corresponding to those observed in moribund mice were found in
all mice, 14 h after challenge.
Effect of LBP and of CD14 blockade on survival of mice
injected i.v. with a low inoculum of K. pneumoniae
The Klebsiella strain was very virulent, because 50% in 5 days in
control mice receiving saline (Fig. 5). Injection with control mAb
had no effect on survival rates. In contrast, injection of anti-LBP or
anti-CD14 mAb increased lethality, with a striking increase during
the first 24 h.
Ab-mediated blockade of LBP and CD14 could be delayed up to
6 h after the onset of infection, leading to accelerated death rates
over controls, indicating that LBP and CD14 are necessary over a
sustained period of time to trigger host defense mechanisms (Table
I). In most mice, TNF-␣ measured at 6 h after infection was not
detected; TNF-␣ was only detectable in mice presenting bacterial
counts 105 CFU/ml at 6 h (data not shown).
Correlation between blood bacterial counts and TNF-␣ levels in
mice treated with anti-LBP or anti-CD14 mAb and injected i.v.
with a low inoculum of K. pneumoniae
We next analyzed whether blood TNF-␣ levels were related to
numbers of bacteria (Fig. 4). TNF-␣ was not detected in samples
containing ⬍104 CFU/ml, irrespective of treatment. Mice treated
with control mAb had detectable levels of blood TNF-␣, from 60
to 10,000 pg/ml, irrespective of the number of circulating bacteria
detected in these samples (from 104 to 106 CFU/ml). Yet, in mice
treated with anti-LBP mAb (or anti-CD14 mAb, data not shown),
TNF-␣ was not detectable or barely detectable in samples in which
bacterial counts were ⬍105 CFU/ml. However, when blood bac-
FIGURE 5. Effect of anti-LBP and anti-CD14 mAbs on the survival of
mice injected i.v. with a low inoculum (from 60 to 260 CFU/mouse) of K.
pneumoniae. Mice were injected with saline (‚), with 100 ␮g/mouse of
control mAb (F), anti-LBP mAb (f), or anti-CD14 mAb (䡺) 5 min before
i.v. infection. Data are the mean of five different experiments (42 mice/
group). No deaths were recorded after 6 days. Survival was assessed by the
␹2 test.
Downloaded from http://www.jimmunol.org/ by guest on June 18, 2017
FIGURE 3. Effect of anti-LBP and anti-CD14 mAbs on the blood bacterial counts of mice injected i.v. with a low inoculum (from 60 to 260
CFU/mouse) of K. pneumoniae. Mice were injected with 100 ␮g/mouse of
control mAb (A, Survivors, E; nonsurvivors, F), anti-LBP mAb (B), or
anti-CD14 mAb (C) 5 min before i.v. infection. Data are the mean of five
different experiments (42 mice/group). ⴱ, p ⬍ 0.05, by ANOVA compared
to CFU of survivors receiving the control mAb.
terial counts exceeded 5 ⫻ 105 CFU/ml, blood TNF-␣ levels were
higher than those found in animals receiving control mAb ( p ⬍
0.001). Thus, it appeared that LBP or CD14 blockade induced a
delay in early TNF-␣ production that was associated both with
time and number of bacteria (⬍105 CFU/ml). However, as soon as
bacteria reached 5 ⫻ 105 CFU/ml in the blood, TNF-␣ was produced in higher amounts in mice treated with anti-LBP or antiCD14 mAb, suggesting LBP- and CD14-independent mechanisms
of TNF-␣ production at high bacterial loads.
2762
LBP AND CD14 IN BACTERIAL SEPSIS
Table I. Effect of delayed treatment with neutralizing anti-LBP and
anti-CD14 mAbs on the lethality of mice injected with 100 CFU of K.
pneumoniaea
Treatment
Saline
Anti-LBP mAb
Anti-CD14 mAb
Time of Administration
of mAb (h)
0
⫹2
⫹4
⫹6
0
⫹4
⫹6
Lethality (%) 24 h
Postchallenge
12 (1/8)
75 (6/8)*
62 (5/8)*
75 (6/8)*
62 (5/8)*
100 (8/8)*
100 (8/8)*
50 (4/8)*
Mice were injected i.v. with bacteria and 100 ␮g/mouse of Abs given at the same
time (t0) or at 2, 4, or 6 h after bacterial challenge. Early deaths observed at 24 h (%,
in parenthesis, dead/total mice) were recorded and compared to those of mice injected
with bacteria and saline given at t0. *, p ⬍ 0.05 by the Fisher exact test, comparing
lethality of anti-LBP and anti-CD14 groups vs saline group.
a
Experiments with a low inoculum of bacteria indicated a delay in
TNF-␣ production in animals receiving anti-LBP or anti-CD14
mAb, compared with that of controls. This was observed during
the initial steps of infection, when bacterial numbers were low.
Thus we hypothesized that the absence of TNF-␣ production may
be associated with a defective innate immune response. We investigated the role of TNF-␣ in that model. As shown in Fig. 6, pretreatment of mice with neutralizing anti-TNF-␣ Abs augmented
early deaths.
TNF-␣ treatment reverses the effect of LBP or CD14 blockade
in mice challenged with a low inoculum of K. pneumoniae
We next investigated whether pretreatment with rTNF-␣ would
improve survival. As shown in Fig. 7, administration of rTNF-␣,
given at the time of bacterial challenge, induced a good degree of
protection in mice challenged with a low inoculum of K. pneumoniae. Yet, TNF has to be given in the early hours after infection,
with a failure to protect mice when administered 6 or 10 h after
infection (Table II).
Taken together, these experiments suggested that LBP or CD14
blockade, by suppressing TNF-␣, induced a defective immune re-
sponse leading to uncontrolled bacterial multiplication. Thus, we
investigated whether pretreatment with rTNF-␣ would also improve outcome in mice treated with anti-LBP or anti-CD14 mAb.
Fig. 8 indicates that administration of rTNF-␣ almost fully protected mice treated with anti-LBP mAb, that otherwise died rapidly
(80% survival compared with 0% survival). Addition of TNF-␣
also partially restored outcome in mice receiving anti-CD14 mAb
(Fig. 9), but protection was less than in mice treated with
anti-LBP mAb.
Role of LBP and CD14 in bacterial sepsis induced by a high
inoculum of K. pneumoniae
We next investigated how LBP and CD14 blockade affected outcome in mice challenged with high inocula of bacteria. Data obtained ex vivo indicated LBP- and CD14-independent mechanisms
of TNF-␣ production with 105 heat-killed K. pneumoniae (data not
shown). Mice were pretreated with the three mAbs and injected
with 105 live K. pneumoniae. Lethality was extremely rapid with
this high inoculum. Yet, death rates and bacterial numbers were
similar in all groups of mice (Table III), indicating that LBP and
CD14 did not contribute to the worsening of infection under these
conditions. Plasma TNF-␣ levels were also not different in the
three groups of mice.
Role of LBP and CD14 in bacterial sepsis induced by a low and
a high inoculum of E. coli
Finally, we investigated whether LBP and CD14 played a similar
role in a systemic challenge of mice with the less virulent Gramnegative bacterium E. coli O111. The LD50 for this particular
strain is ⬃108 CFU/mouse. Mice were injected with a nonlethal
inoculum (105/mouse) to assess whether blockade of the innate
system would impair the natural defense mechanisms of mice. As
Table II. Effect of delayed treatment with rTNF␣ on the lethality of
mice injected with 80 CFU of K. pneumoniaea
Treatment
FIGURE 6. Effect of anti-TNF Abs on survival of mice injected i.v.
with a low inoculum (from 60 to 260 CFU/mouse) of K. pneumoniae. Mice
were injected with 500 ␮g/mouse of control rabbit IgG (■) or anti-TNF
IgG (E) 5 min before i.v. infection. Data are the mean of two pooled
experiments (16 mice/group). No deaths were recorded after 6 days. Survival was assessed by the ␹2 test.
Saline
TNF-␣
Time of Administration
of TNF-␣ (h)
Lethality
0
⫹6
⫹10
4/8
1/8*
3/8
5/8
a
Mice were injected i.v. with bacteria and 50 ng of TNF-␣ given at the same time
(t0) or at 6 or 10 h after bacterial challenge. Deaths were recorded until 6 days. *, p ⬍
0.05 by the Fisher exact test.
Downloaded from http://www.jimmunol.org/ by guest on June 18, 2017
Role of TNF-␣ in mice challenged i.v with a low inoculum of K.
pneumoniae
FIGURE 7. Effect of rTNF-␣ on survival of mice injected i.v. with a
low inoculum (from 60 to 260 CFU/mouse) of K. pneumoniae. Mice were
injected with 500 ng TNF-␣ (E), 100 ng TNF-␣ (Œ), or saline (■) 5 min
before i.v. infection. Data are the mean of two pooled experiments (16
mice/group). No deaths were recorded after 6 days. Survival was assessed
by the ␹2 test.
The Journal of Immunology
2763
Table III. Plasma TNF-␣ levels, blood bacterial counts, and death
rates in mice injected with 105 CFU of K. pneumoniaea
TNF-␣ (ng/ml) at
Log CFU/ml at
Deaths at
Treatment
1 h 30 min
7h
7h
7h
12 h
Control mAb
Anti-LBP mAb
Anti-CD14 mAb
8.5 ⫾ 8.2
10.2 ⫾ 7.5
9.3 ⫾ 6.4
1.2 ⫾ 0.7
1.5 ⫾ 2.6
2.1 ⫾ 2.4
8.7 ⫾ 1.5
8.8 ⫾ 0.6
8.9 ⫾ 0.4
3/8
2/8
4/8
8/8
8/8
8/8
a
Mice were injected i.v. with 100 ␮g/mouse of the indicated mAbs given 5 min
prior to an i.v. challenge of 105 K. pneumoniae. Data of log CFU per milliliter in
blood and of blood TNF-␣ levels are mean ⫾ SD. No statistical difference for all
parameters between the groups.
shown in Table IV, in mice receiving the nonlethal inoculum,
blockade of LBP or CD14 with Abs did not worsen outcome, nor
did it alter bacterial clearance. We also injected mice with a high
inoculum (109), which we knew from ex vivo experiments was
LBP- and CD14- independent. In mice receiving the high and lethal inoculum, death was very rapid. Anti-LBP or anti-CD14 mAb
did not modify TNF-␣ levels, did not reduce the number of circulating bacteria, and did not prevent or delay death.
Discussion
The results demonstrate the significance of coordinated blockade
of proximal molecules CD14 and LBP in innate immunity against
Gram-negative bacteria. This is the first report of treatment of mice
with a neutralizing mAb to mouse CD14. A virulent strain of K.
pneumoniae and a less virulent strain of E. coli were selected to
assess whether similar innate responses were triggered for these
two strains. The results indicated that LBP/CD14 contributed to
control infection induced by virulent K. pneumoniae, not by less
virulent E. coli, suggesting different mechanisms in the elimination
FIGURE 9. Effect of rTNF-␣ on survival of mice treated with antiCD14 mAb and injected i.v. with a low inoculum (from 60 to 260 CFU/
mouse) of K. pneumoniae. Mice were injected with 100 ng/mouse of
rTNF-␣ (‚), 100 ␮g/mouse of anti-CD14 mAb (F), or 100 ␮g/mouse of
anti-CD14 mAb together with 100 ng/mouse of rTNF-␣ (f) 5 min prior to
i.v. infection. Data are the mean of two pooled experiments (16 mice/
group). No deaths were recorded after 6 days. Survival was assessed by the
␹2 test.
of virulent and avirulent bacteria. The two strains display very
different characteristics of virulence. E. coli O111 is the prototype
of a Gram-negative organism lacking an invasive phenotype (11).
The LD50 for this strain is 108 CFU. Yet, E. coli O111, when
injected in low numbers, are likely to be killed by complementmediated bacteriolysis or opsonophagocytosis and killing by neutrophils, not requiring the necessary contribution of cytokine production mediated by LPS. In fact, upon exposure to low numbers
of E. coli O111, LBP- or CD14 blockade did not affect normal
bacterial clearance, so that mice survived as control mice. This was
not the case for virulent K. pneumoniae, for which the LD50 is K.
pneumoniae in mice treated with control mAb, anti-LBP-, or antiCD14 mAbs, up to 4 – 6 h following injection. This indicated that
bacteria were not eliminated but multiplied from the original inoculum of 200 CFU/ml to 104–105 CFU/ml of blood after 4 – 6 h.
This also indicated that CD14 and LBP blockade did not affect
clearance of bacteria in the initial steps of infection. After 6 h,
bacteria multiplied dramatically in mice treated with anti-LBP or
anti-CD14 mAb, and mice died of overwhelming infection. This
implies a critical role for LBP/CD14 in the triggering of host responses that requires time, because the first measurable effects of
blockade of the innate system were not observed before 6 –7 h of
infection.
Mechanisms responsible for aggravation of the disease imply
that a vigorous inflammatory response likely triggered by LPS
through LBP and CD14 was necessary to eliminate low numbers
of virulent bacteria. For this response, TNF-␣ stands as a key cytokine, and neutrophil activation and recruitment is of paramount
importance. In the Klebsiella model with a low inoculum, TNF-␣
was determinant, as 1) treatment with anti-LBP or anti-CD14 mAb
delayed TNF-␣ appearance in the blood; 2) treatment of mice with
anti-TNF-␣ Abs was found to aggravate infection; 3) treatment of
mice with rTNF-␣ was protective; 4) treatment of mice with
rTNF-␣ almost totally protected mice treated with anti-LBP mAb,
and partially restored survival in mice treated with anti-CD14
mAb, indicating that blockade of CD14 induced more profound
alterations of host defense than the sole prevention of TNF-␣ production; and 5) TNF-␣ had to be present early to prevent death,
because administration of rTNF-␣ was not effective when given
5–10 h after bacterial challenge.
Bioactive TNF-␣ was not detectable when bacterial numbers
were ⬍104 CFU/ml in the blood, and not before 4 h following
infection. Determinant mechanisms for elimination of bacteria
(that remain to be defined) happen between 5 and 6 h after infection, and are dependent on host activation by TNF-␣. During this
2-h period, anti-CD14- and anti-LBP-treated mice had no detectable bioactive TNF-␣. Yet, 1 h later, at 7 h, TNF-␣ levels were
higher in anti-LBP- and anti-CD14-treated mice than in controls,
in relation with the fact that bacterial counts higher than in controls
were present in these mice. This suggests that TNF-␣ production
Downloaded from http://www.jimmunol.org/ by guest on June 18, 2017
FIGURE 8. Effect of rTNF-␣ on survival of mice treated with anti-LBP
mAb and injected i.v. with a low inoculum (from 60 to 260 CFU/mouse)
of K. pneumoniae. Mice were injected with saline (Œ), 100 ␮g/mouse of
anti-LBP mAb (E), or 100 ␮g/mouse of anti-LBP mAb together with 100
ng/mouse rTNF-␣ (f) 5 min before i.v. infection. Data are the mean of two
pooled experiments (16 mice/group). No deaths were recorded after 6 days.
Survival was assessed by the ␹2 test.
2764
LBP AND CD14 IN BACTERIAL SEPSIS
Table IV. Plasma TNF-␣ levels, blood bacterial counts, and death rates in mice injected with 105 or 109
CFU of E. coli O111a
TNF-␣ (ng/ml) at
Treatment
Log CFU/ml at
Deaths at
1 h 30 min
1 h 30 min
6h
5h
12 h
72 h
ND
ND
ND
4.12 ⫾ 0.11
4.07 ⫾ 0.09
4.07 ⫾ 0.12
1.26 ⫾ 1.18
1.63 ⫾ 1.22
1.18 ⫾ 1.18
0/8
0/8
0/8
0/8
0/8
0/8
0/8
0/8
0/8
9.7 ⫾ 2.8
11.9 ⫾ 2.9
13.6 ⫾ 2.3
8.38 ⫾ 0.18
8.41 ⫾ 0.16
8.42 ⫾ 0.17
ND
ND
ND
6/8
4/8
6/8
8/8
8/8
8/8
5
Inoculum: 10 CFU/mouse
Control mAb
Anti-LBP mAb
Anti-CD14
Inoculum: 109 CFU/mouse
Control mAb
Anti-LBP mAb
Anti-CD14 mAb
a
Mice were injected i.v. with 100 ␮g/mouse of the indicated mAbs given 5 min prior to an i.v. challenge with a low (105)
or a high inoculum (109) of E. coli O111. Data of log CFU per milliliter in blood and of blood TNF-␣ levels are mean ⫾ SD.
No statistical difference for all parameters between the groups.
spite triggering host response, bacteria were not eliminated and
death occurred rapidly. The present study indicates that under
these conditions, administration of anti-LBP or anti-CD14 mAb
did not decrease the proinflammatory response.
The detrimental contribution of LBP in models of endotoxemia
has been clearly defined (3– 6). Its role in infection has been examined in only one study so far. It was reported that LBP was
necessary to combat a low-dose infection induced by an i.p. challenge of S. typhimurium, presumably because LBP-deficient animals were unable to trigger an adequate response mediating
phagocytosis and killing of the microorganism (3). S. typhimurium
is an intracellular organism, and the present study now extends this
observation to extracellular Gram-negative bacteria. With regard
to CD14, the present study confirms and extends the conclusion of
a recent report showing that blockade of CD14 aggravates experimental shigellosis in rabbits (10). These data as well as the present
data may appear at variance with an earlier study that showed that
CD14 knockout mice had a better survival than wild-type mice
following challenge with E. coli (7). There is no explanation for
these discrepant results. In the aforementioned study (7), CD14deficient mice survived to an i.p. inoculum of 5 ⫻ 106 E. coli
O111, which was lethal to control mice. In the present study, administration of anti-LBP or anti-CD14 mAbs did not alter outcome
with a similar inoculum of the same E. coli strain given systemically. Studies using gene-deficient animals or animals treated with
Abs are not similar.
To conclude, the present study is in agreement with the concept
proposed many years ago by Ulevitch and colleagues (1, 2, 36),
that LPS shed from Gram-negative bacteria binds to LBP and that
the LPS/LBP complexes are presented to CD14 to trigger monocyte activation, leading among other mechanisms to cytokine synthesis, necessary for the host response. This study also stresses the
need for critical evaluation of novel therapeutic approaches for the
management of patients with severe sepsis or shock.
References
1. 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.
2. 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.
3. Jack, R. S., X. L. Fan, M. Bernheiden, G. Rune, M. Ehlers, A. Weber, G. Kirsch,
R. Mentel, B. Fürll, M. Freudenberg, et al. 1997. Lipopolysaccharide-binding
protein is required to combat a Gram-negative bacterial infection. Nature 389:
742.
4. 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.
Downloaded from http://www.jimmunol.org/ by guest on June 18, 2017
is dependent on LBP/CD14 in the initial steps, when LPS-triggered
monocyte activation occurs. Prophylactic blockade of LBP and
CD14 led to the most severe effects. Yet, experiments with a delayed blockade of LBP or CD14 up to 6 h after the start of infection also indicate that these molecules were required during a sustained period of time to protect the mice. From 6 h on, two
pathways may be responsible for cell activation, either 1) LPSdependent (but LBP- and CD14-independent, because concentrations of LPS are too high) mechanisms; or 2) mechanisms dependent on other bacterial products.
Mechanisms responsible for aggravation of the disease following LBP/CD14 blockade are likely not due to prevention of phagocytosis by the Abs, because bacterial numbers were similar in control mice or in mice treated with anti-LBP or anti-CD14 mAbs in
the initial steps of infection, whatever the strain of bacteria used.
Earlier reports have suggested that phagocytosis of Gram-negative
bacteria may occur via a CD14-dependent pathway (29, 30). Yet,
these experiments have been performed with heat-killed bacteria,
a treatment that breaks capsules and likely allows binding of LBP
or CD14 to Gram-negative bacteria. We previously reported that
LBP bound efficiently to heat-killed Gram-negative bacteria, but
did not bind to most live bacteria, including Klebsiella species,
whereas it binds to rough live bacteria (31). Binding to CD14 was
not observed for the Klebsiella strain used in the present study
(data not shown). Thus indirect mechanisms, other than prevention
of phagocytosis, are responsible for the failure of anti-LBP- and
anti-CD14-treated mice to control infection.
Blockade of innate immune responses by anti-LBP or antiCD14 mAb was associated with an impairment of optimal neutrophil recruitment during the initial steps of infection, which is necessary for host defenses. Concentrations of chemokines have not
been measured, but one may speculate that they would also be
diminished. Cytokines and chemokines are known to potentiate the
function of neutrophils and macrophages, resulting in activation of
microbial mechanisms, including generation of superoxide production, reactive oxygen and nitrogen intermediates, or enhancement
of phagocytosis, with an increased ability to kill microorganisms
(32–35).
It has been largely documented that activation of myelomonocytic cells by LPS via LBP/CD14 occurs only at low doses of LPS,
and that LBP or CD14 blockade does not prevent cell activation by
high doses of LPS. We approached this question by comparing the
effect of CD14 or LBP blockade in mice challenged with a low vs
a high bacterial inoculum. The present study indicated a role for
LBP and CD14 almost exclusively for low inocula of virulent bacteria. With a high inoculum of bacteria, virulent or not, cytokine
synthesis occurred that was LBP- and CD14-independent. Yet, de-
The Journal of Immunology
22. Nakano, Y., K. Onozuka, Y. Terada, H. Shinomiya, and M. Nakano. 1990. Protective effect of recombinant tumor necrosis factor-␣ in murine salmonellosis.
J. Immunol. 144:1935.
23. Alexander, H. R., B. C. Sheppard, J. C. Jensen, H. N. Langstein, C. M. Buresch,
D. Venzon, E. C. Walker, D. L. Fraker, M. C. Stovroff, and J. A. Norton. 1991.
Treatment with recombinant human tumor necrosis factor-␣ protects rats against
the lethality, hypotension, and hypothermia of Gram-negative sepsis. J. Clin.
Invest. 88:34.
24. Cross, A. S., J. C. Sadoff, E. Bernton, and P. Gemski. 1989. Pretreatment with
recombinant murine tumor necrosis factor ␣/cachectin and murine interleukin 1
␣ protects mice from lethal bacterial infection. J. Exp. Med. 169:2021.
25. Le Roy, D., C. Crouzier, M. Dho-Moulin, A. S. Dumont, A. Bouchet,
J. P. Lafont, and A. Andremont. 1995. Results of passive and active immunization directed against ferric aerobactin in experimental enterobacterial infections
in mice and chickens. Res. Microbiol. 146:167.
26. Zanetti, G., D. Heumann, J. Gérain, J. Kohler, P. Abbet, C. Barras, M. P. Glauser,
and J. D. Baumgartner. 1992. Cytokine production after intravenous or peritoneal
Gram negative bacterial challenge in mice: comparative protective efficacy of
antibodies to TNF␣ and to LPS. J. Immunol. 148:1890.
27. 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.
28. 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 LPSinduced TNF secretion in human peripheral blood monocytes. J. Immunol. 148:
3505.
29. Schiff, D. E., L. Kline, K. Soldau, J. D. Lee, J. Pugin, P. S. Tobias, and
R. J. Ulevitch. 1997. Phagocytosis of Gram-negative bacteria by a unique CD14dependent mechanism. J. Leukocyte Biol. 62:786.
30. Grunwald, U., X. Fan, R. S. Jack, G. Workalemahu, A. Kallies, F. Stelter, and C.
Schütt. 1996. Monocytes can phagocytose Gram-negative bacteria by a CD14dependent mechanism. J. Immunol. 157:4119.
31. 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.
32. Müller, M., R. Althaus, D. Fröhlich, K. Frei, and H. P. Eugster. 1999. Reduced
antilisterial activity of TNF-deficient bone marrow-derived macrophages is due to
impaired superoxide production. Eur. J. Immunol. 29:3089.
33. Berkow, R. L., D. Wang, J. V. Larrick, R. W. Dodson, and T. J. Howard. 1987.
Enhancement of neutrophil superoxide production by preincubation with recombinant human tumor necrosis factor. J. Immunol. 139:3783.
34. Yonemaru, M., K. E. Stephens, A. Ishizaka, H. Zheng, R. S. Hogue,
J. J. Crowley, J. R. Hatherill, and T. A. Raffin. 1989. Effects of tumor necrosis
factor on PMN chemotaxis, chemiluminescence, and elastase activity. J. Lab.
Clin. Med. 114:674.
35. Bogdan, C., M. Röllinghoff, and A. Diefenbach. 2000. Reactive oxygen and
reactive nitrogen intermediates in innate and specific immunity. Curr. Opin. Immunol. 12:64.
36. 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.
Downloaded from http://www.jimmunol.org/ by guest on June 18, 2017
5. 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.
6. 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.
7. 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.
8. Schimke, J., J. Mathison, J. Morgiewicz, and R. J. Ulevitch. 1998. Anti-CD14
mAb treatment provides therapeutic benefit after exposure to endotoxin. Proc.
Natl. Acad. Sci. USA 95:13875.
9. Leturcq, D., A. M. Moriarty, G. Talbott, R. K. Winn, T. R. Martin, and
R. J. Ulevitch. 1996. Antibodies against CD14 protect primates from endotoxininduced shock. J. Clin. Invest. 98:1533.
10. Wenneras, C., P. Ave, M. Huerre, J. Arondel, R. J. Ulevitch, J. C. Mathison, and
P. Sansonetti. 2000. Blockade of CD14 increases Shigella-mediated invasion and
tissue destruction. J. Immunol. 164:3214.
11. Cross, A. S., S. M. Opal, J. C. Sadoff, and P. Gemski. 1935. Choice of bacteria
in animal models of sepsis. Infect. Immun. 61:2741.
12. Oberholzer, A., C. Oberholzer, and L. L. Moldawer. 2000. Cytokine signaling:
regulation of the immune response in normal and critically ill states. Crit. Care
Med. 28(Suppl. 4):N3.
13. Havell, E. A. 1989. Evidence that tumor necrosis factor has an important role in
antibacterial resistance. J. Immunol. 143:2894.
14. van Furth, R., T. L. Van Zwet, A. M. Buisman, and J. T. Van Dissel. 1994.
Anti-tumor necrosis factor antibodies inhibit the influx of granulocytes and
monocytes into an inflammatory exudate and enhance the growth of Listeria
monocytogenes in various organs. J. Infect. Dis. 170:234.
15. Rothe, J., W. Lesslauer, H. Lötscher, Y. Lang, P. Koebel, F. Köntgen, A. Althage,
R. Zinkernagel, M. Steinmetz, and H. Bluethmann. 1993. Mice lacking the tumour necrosis factor receptor 1 are resistant to TNF-mediated toxicity but highly
susceptible to infection by Listeria monocytogenes. Nature 364:798.
16. Eugster, H. P., M. Müller, U. Karrer, B. D. Car, B. Schnyder, V. M. Eng,
G. Woerly, M. Le Hir, F. Di Padova, M. Aguet, et al. 1996. Multiple abnormalities in tumor necrosis factor and lymphotoxin-␣ double-deficient mice. Int. Immunol. 8:23.
17. Echtenacher, B., W. Falk, D. N. Männel, and P. H. Krammer. 1990. Requirement
of endogenous tumor necrosis factor/cachectin for recovery from experimental
peritonitis. J. Immunol. 145:3762.
18. van der Poll, T., C. V. Keogh, W. A. Buurman, and S. F. Lowry. 1997. Passive
immunization against tumor necrosis factor-␣ impairs host defense during pneumococcal pneumonia in mice. Am. J. Resp. Crit. Care Med. 155:603.
19. Gosselin, D., J. DeSanctis, M. Boulé, E. Skamene, C. Matouk, and D. Radzioch.
1995. Role of tumor necrosis factor ␣ in innate resistance to mouse pulmonary
infection with Pseudomonas aeruginosa. Infect. Immun. 63:3272.
20. Laichalk, L. L., S. L. Kunkel, R. M. Strieter, J. M. Danforth, M. B. Bailie, and
T. J. Standiford. 1996. Tumor necrosis factor mediates lung antibacterial host
defense in murine Klebsiella pneumonia. Infect. Immun. 64:5211.
21. O’Brien, D. P., D. E. Briles, A. J. Szalai, A. H. Tu, I. Sanz, and M. H. Nahm.
1999. Tumor necrosis factor ␣ receptor I is important for survival from Streptococcus pneumoniae infections. Infect. Immun. 67:595.
2765