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doi:10.1182/blood.V98.13.3800
2001 98: 3800-3808
Janine Zweigner, Hans-Joachim Gramm, Oliver C. Singer, Karl Wegscheider and Ralf R. Schumann
response in human monocytes
patients with severe sepsis or septic shock inhibit the lipopolysaccharide
High concentrations of lipopolysaccharide-binding protein in serum of
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PHAGOCYTES
High concentrations of lipopolysaccharide-binding protein in serum of
patients with severe sepsis or septic shock inhibit the lipopolysaccharide
response in human monocytes
Janine Zweigner, Hans-Joachim Gramm, Oliver C. Singer, Karl Wegscheider, and Ralf R. Schumann
Lipopolysaccharide-binding protein (LBP),
an acute-phase protein recognizing lipo-
polysaccharide (LPS), catalyzes in low
concentrations its transfer to the cellular
LPS receptor consisting of CD14 and
Toll-like receptor-4. It has recently been
shownthathighconcentrations ofrecom-
binant LBP can protect mice in a peritoni-
tis model from the lethal effects of LPS.
To determine whether in humans the
acute-phase rise of LBP concentrations
can inhibit LPS binding to monocytes and
induction of proinflammatory cytokines,
LBP concentrations were analyzed in 63
patients meeting the American College of
Chest Physicians/Society of Critical Care
Medicine criteria of severe sepsis or sep-
tic shock and the ability of these sera to
modulate LPS effects in vitro was as-
sessedemploying differentassays.Trans-
fer of fluorescein isothiocyanate–labeled
LPS to human monocytes was assessed
by a fluorescence-activated cell sorter–
based method, and activation of mono-
cyteswas investigatedbymeasuring LPS-
inducedtumor necrosisfactor-␣ secretion
in the presence of the sera. Anti-LBP
antibodies and recombinant human LBP
wereinstrumental for depletionand recon-
stitution of acute-phase sera and subse-
quent assessment of their modulating
effects on LPS activity. Sera of patients
with severe sepsis/septic shock exhib-
ited a diminished LPS transfer activity
and LPS-induced tumor necrosis factor-␣
secretion as compared with sera from
healthy controls. LBP depletion of sepsis
sera and addition of rhLBP resulting in
concentrations found in severe sepsis
confirmed that LBP was the major serum
component responsible for the observed
effects. In summary, the inhibition of LPS
effects by high concentrations of LBP in
acute-phase serum, as described here,
may represent a novel defense mecha-
nism of the host in severe sepsis and
during bacterial infections. (Blood. 2001;
98:3800-3808)
© 2001 by TheAmerican Society of Hematology
Introduction
Recognition of bacterial components such as lipopolysaccharide
(LPS) by the innate immune system is an early and key event for
triggering the inflammatory host response necessary for clearance
of invading microorganisms.
1,2
However, uncontrolled, the inflam-
matory response can be a cause of organ dysfunction remote from
the primary site of infection, hypotension, or shock—a syndrome
termed severe sepsis or septic shock.
3,4
Despite highly effective
antimicrobial chemotherapy and powerful supportive treatment
strategies, no significant reduction of mortality attributable to
severe sepsis has been achieved in the last 2 decades. The incidence
of severe sepsis and septic shock is 1% to 2% of hospital and 9% to
22% of intensive care unit admissions, with a crude mortality rate
of 35% to 60%.
5,6
Although novel approaches employing anti-
inflammatory agents have been successfully applied for the control
of severe sepsis and septic shock in experimental settings, only
limited efficacy has been demonstrated in clinical trials.
7,8
Thus, a
better understanding of the molecular mechanisms of the host
response and its regulation is needed to be able to develop
successful innovative treatment strategies for patients with severe
sepsis or septic shock.
For the initiation of the innateimmune response, monocytesand
macrophages play an essential role. These cells have the ability to
recognize bacterial compounds and to activate the innate immune
system by the release of a large number of different mediators, such
as tumor necrosis factor-␣ (TNF-␣), interleukin-1 (IL-1), and
IL-6.
9
Furthermore, as professional antigen-processing cells they
play a crucial role in the activation of the adaptive immune
system.
10
In infections caused by Gram-negative microorganisms,
the principal bacterial constituent recognized by the innate immune
system is LPS, a glycolipid in the outer bacterial membrane.
11
The
acute-phase protein initiating recognition and monomerization of
LPS aggregates and its transfer to mononuclear cells is lipopolysac-
charide-binding protein (LBP).
12
LBP binds to the amphipathic
lipid A moiety of LPS and in low concentrations catalyzes its
transfer to membrane-bound CD14, a glycosylphosphatidylinositol
(GPI)–linked protein that is part of the cellular receptor for LPS.
13
Recently, the Toll-like receptor-4 has been identified as the
signal-transducing element of the LPS receptor.
10,14
LBP is constitutively synthesized in hepatocytes and is present
in serum at concentrations of 5 to 15 g/mL. During the acute-
phase response, IL-1 and IL-6 synergize in inducing LBP synthesis,
leading to an increase of LBP serum concentrations.
15-18
It has
From the Institut fu¨r Mikrobiologie und Hygiene, Universita¨tsklinikum Charite´,
Medizinische Fakulta¨t der Humboldt-Universita¨t zu Berlin, and Klinik fu¨r
Anaesthesiologie und operative Intensivmedizin, Universita¨tsklinikum Benjamin
Franklin, Freie Universita¨t Berlin, Germany, and the Institut fu¨r Statistik und
O
¨
konometrie, Universita¨t Hamburg, Germany.
Submitted February 12, 2001; acceptedAugust 2, 2001.
Supported in part by grants from the German Research Foundation (DFG,
grant no. Schu 828/1-5) and by the Bundesministerium fu¨r Bildung und
Forschung (BMBF, grants no. 01KV98067 and 01KI9855/0).
Reprints: Ralf R. Schumann, Institut fu¨r Mikrobiologie und Hygiene,
Universita¨tsklinikum Charite´, Dorotheenstr 96, D-10117 Berlin, Germany;
e-mail: ralf.schumann@charite.de.
The publication costs of this article were defrayed in part by page charge
payment. Therefore, and solely to indicate this fact, this article is hereby
marked ‘‘advertisement’’in accordance with 18 U.S.C. section 1734.
© 2001 by TheAmerican Society of Hematology
3800 BLOOD, 15 DECEMBER 2001
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recently been discovered that epithelial cells of the intestines and
the lungs may represent additional sources of LBP.
19,20
Besides its
ability to amplify the immune response by recognizing small
concentrations of LPS, LBP has also been shown in vitro to
catalyze the transfer of LPS into high-density lipoprotein particles,
resulting in LPS neutralization.
21,22
This function of LBP may be
protective during severe sepsis, as suggested by animal studies by
both others and ourselves.
23,24
Recently, 2 clinical studies were
published, one of which showed a positive correlation between
high initial serum LBP concentrations and improved patient
outcome in severe sepsis.
16
The other report demonstrated a
correlation between high serum LBP concentrations and a de-
creased incidence of cardiovascular morbidity in patients with
end-stage renal disease and hemodialysis.
25
No experimental data, however, up to now have been available
regarding the biological activity of high concentrations of LBP in
the presence of acute-phase sera from patients. In recent years
several studies applying whole blood or isolated peripheral blood
mononuclear cells (PBMCs) from patients with severe sepsis have
shown a significant reduction of LPS-induced cytokine release and
impaired monocytic antigen presentation as compared with whole
blood or PBMCs of healthy controls.
26-29
The aim of the present
study was to find out whether elevated LBP is a key factor for
inhibition of LPS activity in acute-phase serum. We analyzed
serum concentrations of LBPin patients with severe sepsisor septic
shock and investigated in vitro whether acute-phase concentrations
of LBP can modulate the LPS-induced monocytic response. We
found that serum containing high concentrations of LBP clearly
reduce LPS activity, an effect that was reversed by LBP depletion.
Addition of recombinant human (rh)LBP to normal or LBP-
depleted sepsis serum led to a decrease of LPS effects. These
findings support the hypothesis that high concentrations of LBPare
a key factor for inhibition of LPS activity by acute-phase sera.
Patients, materials, and methods
Patients
Sixty-three patients meeting the criteria of severe sepsis or septic shock
were prospectively enrolled over a 14-month period in a 22-bed noncoro-
nary intensive care unit of a tertiary care university hospital. Severe sepsis
and septic shock were defined according to the criteria of the American
College of Chest Physicians/Society of Critical Care Medicine consensus
conference.
3
Briefly, severe sepsis was defined by evidence of infection
accompanied by at least 2 of the following criteria: fever or hypothermia
(temperature ⬎ 38.0°C or ⬍ 36.0°C), tachycardia (⬎ 90 ventricular beats/
min), tachypnea (respiratory rate ⬎ 20 breaths/min or Pa
CO
2
⬍ 4.3 kPa), or
changes in the white blood cell count (leukocyte count ⬎ 12 ⫻ 10
9
/L or
⬍ 4 ⫻ 10
9
/L or ⬎ 10% immature band forms) and, in addition, at least one
acute organ dysfunction remote from the site of infection as indicated by
mental disorientation, oliguria, arterial hypoxemia, thrombocytopenia,
unexplained metabolic acidosis, or hypotension. Septic shock was defined
as a systolic blood pressure below 90 mm Hg for at least 1 hour despite
adequate intravascular volume expansion or the necessity for vasopressor
therapy. A total of 60.3% of the study cohort fulfilled the criteria of severe
sepsis, and 39.7% were in septic shock. Another 52.4% developed septic
shock after the onset of severe sepsis. The median duration of severe
sepsis/septic shock was 4.8 days (range 2.3-31 days) in survivors and 5.5
days (range 1.0-35 days) in nonsurvivors. The severity of acute illness was
scored daily by means of the Acute Physiology, Age, and Chronic Health
Evaluation III (APACHE III) classification system and the Sequential
Organ Failure Assessment (SOFA) score.
30,31
The site of infection was
identified using criteria of the Centers for Disease Control and Prevention
except for sepsis.
32
The infections included diffuse peritonitis (n ⫽ 39),
pneumonia (n ⫽ 8), severe soft tissue infection (n ⫽ 6), catheter-associated
bloodstream infection (n ⫽ 1), urogenital infection (n ⫽ 2), and others
(n ⫽ 7). Documented bacteremia with Gram-negative microorganisms
occurred in 4 cases, Gram-positive microorganisms in 6 cases, and in 5
cases bacteremia was polymicrobial. Twenty-one infections were prospec-
tively classified as moderate (pneumonia, catheter-associated bloodstream
infection, and isolated intra-abdominal abscess) and 42 as severe (diffuse
peritonitis, severe soft tissue infection, deep organ abscess). One patient in
the study cohort had a relevant liver disease as defined by the criteria of the
APACHE III score. This patient with a biopsy-proven liver cirrhosis,
however, did not exhibit any impaired LBP synthesis with peak serum LBP
concentrations of 86.8 mg/Lduring severe sepsis. The patients’characteris-
tics are given in Table 1.
For determination of reference values for LBP and soluble CD14
(sCD14), blood samples were drawn from 40 healthy hospital employees
(median age 31 years; range 20-57 years).These seraalso served as controls
for the LPS binding and stimulation assays.
The investigation was carried out in agreement with the ethical
standards of the declaration of Helsinki/Tokyo. The study protocol was
approved by the Committee on Medical Ethics of the University Hospital
Benjamin Franklin, and informed consent was obtained from volunteers
and the patients or their nearest relatives before enrollment.
Sample collection
The first blood sample was drawn within 24 hours after onset of severe
sepsis and subsequently once daily until recovery or the patient died.
Depending on the duration of the severe sepsis, a median of 7 samples
(range 1-38) per patient was collected. Blood samples were obtained either
Table 1. Demographic and clinical characteristics of the study cohort
Severe sepsis/
septic shock
Demographic characteristics
Age, mean ⫾ SD, y 58.4 ⫾ 16.1
Sex, % female/male 43/57
Severity of the underlying disease, No.
(McCabe/Jackson classification)
Nonfatal 48
Fatal 12
Rapidly fatal 3
APACHE III score, mean ⫾ SD, points
Intensive care unit admission 61 ⫾ 20
Severe sepsis/septic shock onset 67 ⫾ 21
SOFA score, mean ⫾ SD, points
Severe sepsis onset/septic shock 12 ⫾ 4
Length of treatment, median (range), d
Intensive care unit 14 (1-71)
Hospital 28 (3-148)
Mortality, %
Severe sepsis-related 63.5
Severe sepsis-related less than 3 d 19.0
All cause 28 d 63.5
All cause intensive care unit 65.0
All cause hospital 69.8
Positive microbial culture, local/blood, % 55.6/23.8
Gram-positive, No. 12/6
Gram-negative, No. 7/4
Polymicrobial, No. 16/5
Sites of infection, No.
Diffuse peritonitis 39
Pneumonia 8
Soft tissue infection 6
Catheter-associated bloodstream
infection 1
Urogenital infection 2
Others 7
HIGH-LEVELLBP INHIBITSLPS RESPONSE 3801BLOOD, 15 DECEMBER 2001
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from an arterial line or by peripheral venous puncture and collected in
sterile 8-mL tubes with SST Gel and ClotActivator and in 4-mL tubes with
ethylenediaminetetraacetic acid (K
2
) 7.2 mg (Vacutainer) (Becton Dickin
-
son, Meylan, France). After centrifugation at 3000g for 15 minutes and
separation, serum and plasma samples were split into 6 aliquots and stored
at ⫺70°C until assayed.
LBP and sCD14 assays
Serum LBPconcentrations were assessed by an enzyme-linked immunosor-
bent assay (ELISA) employing the monoclonal antibodies 1E8 and 2B5
(kindly provided by DrA. Moriarty, Johnson & Johnson, La Jolla, CA).The
lower limit of detection of this assay is 1.0 ng/mL. The reference range of
serum LBP in healthy humans was found to be 1.85 to 17.4 mg/L (median
7.94 mg/L). Soluble CD14 was measured using a commercially available
ELISA according to the manufacturer’s instructions (sCD14 ELISA, IBL,
Hamburg, Germany).All assays were performed in duplicate.
LBP-dependent binding of LPS to monocytes
Blood was obtained from 6 healthy volunteers, collected in endotoxin-free
8 mL tubes containing heparine (Vacutainer CPT, Becton Dickinson,
Brussels, Belgium), and peripheral mononuclear cells subsequently were
isolated by density gradient centrifugation according to the manufacturer’s
instructions. Cells in the interphase were collected, washed twice, and
brought to a concentration of 5 ⫻ 10
5
PMBCs per milliliter, corresponding
to 1.5 ⫻ 10
5
monocytes per milliliter. The cells were incubated with 30 L
acute-phase serum of patients with severe sepsis/septic shock, or with
serum from healthy controls, in a total volume of 200 L. One microgram
per milliliter fluorescein isothiocyanate (FITC)–conjugated LPS from
Escherichia coli O111:B4 (Sigma, Deisenhofen, Germany) was added and
incubated with the cells for 1 hour at 37 °C. After centrifugation and
washing of the adherent cells with cold phosphate-buffered saline, cells
were subjected to flow cytometry analysis. In certain experiments rhLBP
(kindly provided by Dr S. F. Carroll, Xoma, Berkeley, CA) was added to the
sera as indicated. Partial LBP depletion from severe sepsis sera, as well as
depletion below the lower detection limit of the LBP ELISA, was
performed by immunoprecipitation with a rabbit monoclonal anti–human
LBP antibody (kindly provided by Dr S. F. Carroll) and protein A/G–
Sepharose (Santa Cruz Biotechnology, Santa Cruz, CA) according to the
manufacturer’s instructions.
FITC-LPS–labeled monocytes were analyzed by flow cytometry with
the FACScan analyser using the Cellquest software (Becton Dickinson, San
Jose, CA). Monocytes were gated according to their forward and side
scatter characteristics. The fluorescence signal was expressed in fluores-
cence units and recorded on a logarithmic scale.
LPS-induced TNF-␣ secretion by monocytes
PBMCs were adjusted to 5 ⫻ 10
5
/mL and incubated for 2 hours at 37°C in
96-well plastic plates containing RPMI 1640 cell culture medium (PAA
Laboratories, Linz,Austria).After removalof nonadherent cells by washing
with RPMI 1640, sera of patients with severe sepsis/septic shock or sera of
healthy controls preincubated for 15 hours with 10 ng/mL nonfluorescen-
ceinated LPS from E coli 0111:B4 (Sigma) were added. Supernatants were
taken after 4 hours of stimulation at 37°C and stored at ⫺70°C until
assayed. TNF-␣ was assessed employing a commercially available ELISA
with 2 monoclonal mouse anti–human TNF-␣ antibodies (Pharmingen,
Heidelberg, Germany).
Limulus-amebocyte-lysate assay
The chromogenic limulus-amebocyte-lysate (LAL) assay was performed in
a modified form of a published protocol.
33
Serum samples were not heated
in order to preserve binding of LPS to LBP or lipoproteins and to measure
unbound endotoxin. Samples were not sterile-filtered for elimination of
bacteria. A total of 50 L LAL reagent (Endo KTA-LAL, Charles River
Endosafe, Charleston, SC) was added to 50 L serum in a 96-well flat
bottom microtiter plate (Becton Dickinson, Brussels). After incubation at
room temperature for 25 minutes, samples were supplemented with 100 L
substrate (Perfachrome LAL, Pentapharm, Basel, Switzerland). For quanti-
fication of the endotoxin concentration, an E coli O111:B4 endotoxin was
used as standard according to the manufacturer’s instructions (Charles
River Endosafe). After 5 minutes the reaction was stopped with acetic acid
40% and the reaction was quantified by an ELISA reader (Spectra Fluor
plus, Tecan, Crailsheim, Germany).
Statistical analysis
Data are presented as absolute or relative frequencies for categorical
variables, mean ⫾ SD orSEM, or 25th, 50th,and 75th percentiles and, also,
range for continuous parameters. All assays were performed in duplicate,
and the mean value was calculated. Differences between groups were
evaluated using the Mann-Whitney U test or the Wilcoxon test, where
appropriate. The association between mean fluorescence units and LBP
serum concentrations was analyzed using the Pearson correlation coeffi-
cient and the corresponding test based on the bivariate normal distribution,
after double logarithmic transformation. Furthermore, the corresponding
ordinary least squares regression line was fitted to the data. The prognostic
values of onset LBP and sCD14 serum concentrations were assessed by
logistic regression modeling in the severe sepsis/septic shock cohort. All
tests were 2-sided, and P ⬍ .05 was considered statistically significant.
Results
Serum LBP concentrations in severe sepsis or septic shock
For the entire study cohort the median serum LBP concentration at
onset of severe sepsis/septic shock was 46.2 mg/L (range 3.74-
155), and the median sCD14 serum concentration 9.05 mg/L(range
3.64-37.1). These values were significantly different to the serum
LBP and sCD14 concentrations of the healthy volunteers (7.94
mg/L [range 1.85-17.4] and 3.16 mg/L [range 2.48-4.36], respec-
tively; P ⬍ .001). At onset of severe sepsis the serum LBP and
sCD14 concentrations were not significantly different in the
patients with severe sepsis as compared with patients with septic
shock. No significant difference in serum LBP and sCD14 concen-
trations could be observed in survivors as compared with nonsurvi-
vors at onset of severe sepsis or septic shock (44.2 mg/L [range
3.74-112] vs 55.5 mg/L [range 7.36-155] and 8.04 mg/L [range
3.64-15.4] vs 9.79 mg/L [range 4.98-37.1], respectively). Multivar-
iate analyses including severity of infection as a potential con-
founder revealed that neither LBP nor sCD14 concentrations were
independent significant as prognostic indicators for severe
sepsis-related mortality.
During the inflammatory host response in severe sepsis or septic
shock, peak serum LBP and sCD14 concentrations increased
10.5-fold and 4.7-fold (83.1 mg/L [range 11.8-275] and 14.7 mg/L
[range 5.18-39.4], respectively) as compared with the reference
values of the healthy controls. Peak LBP concentrations were
reached after a median time of 40 hours (range 1-120) from the
onset of severe sepsis (Figure 1). No significant differences in peak
serum LBP concentrations were observed in Gram-negative versus
Gram-positive bacteremia in the present study cohort (72.4 mg/L
[range 28.6-143] and 88.6 mg/L [range 33.3-133], respectively).
Furthermore, we failed to detect a significant correlation between
age and serum LBP concentrations (Pearson correlation coefficient
r ⫽⫺0.218, P ⫽ .088).
FITC-LPS binding to monocytes in the presence of different
concentrations of patient sera and sera of healthy controls
To investigate the ability of acute-phase sera to modulate LPS
binding and response of monocytes, 2 in vitro experiments
3802 ZWEIGNER etal BLOOD, 15 DECEMBER 2001
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employing those severe sepsis sera containing peak LBPconcentra-
tions and control sera from healthy volunteers were performed: A
fluorescence-activated cell sorter (FACS)–based assay was used to
assess the binding of FITC-labeled LPS to PBMCs from healthy
donors in the absence or presence of serum. In a second assay the
serum-dependent LPS-induced TNF-␣ secretion by these mono-
cytes was measured by ELISA. Peak LBP serum concentrations of
patients with severe sepsis were identified by serial measurements
as described above.
The correlation between LBP serum concentrations and mean
fluorescence units was statistically significant (Pearson correlation
coefficient: r ⫽⫺0.64, P ⬍ .001). The inverse relation between
log LBP concentrations and log mean fluorescence units is
demonstrated in Figure 2. Both subpopulations seem to follow a
common trend.
The mean fluorescence units representing the LPS transfer to
monocytes inthe presence ofsera of patientswith severe sepsis orseptic
shock were significantly lower as compared with the mean fluorescence
units obtained with the control group sera (Mann-Whitney U test,
P ⬍ .05; Figure 3A). An example of the FITC-LPS FACS assay
employing serum from a randomly chosen severe sepsis patient
containing 93.7g/mLLBPand ofa healthy control serumwith anLBP
concentration of 9.88 g/mL is shown in Figure 3B.
FITC-LPS binding to monocytes in the presence of
rhLBP-supplemented control sera and partially
LBP-depleted severe sepsis sera
When rhLBP was added to serum of healthy controls leading to
total LBP concentrations comparable to peak serum concentrations
of the severe sepsis/septic shock cohort, the LPS transfer activity of
these sera was diminished, comparable to the results obtained with
severe sepsis sera (Figure 4A). When increasing concentrations of
rhLBP were added, the transfer activity was slightly enhanced for
LBP concentrations up to 50 g/mL (Figure 4B). However, higher
rhLBP concentrations led to a marked down-regulation of LPS
binding. Next, we depleted severe sepsis serafrom LBP by using an
anti-LBP antibody resulting in serum LBP concentrations found in
healthy controls. This partial LBP depletion of severe sepsis sera
led to a mean LBP concentration of 16.7⫾ 10.1 g/mL. LBP
depletion significantly enhanced the LPS transfer activity and the
LPS-induced TNF-␣ secretion of this serum (Table 2). A represen-
tative patient’s example of this effect is shown in Figure 4C.
FITC-LPS binding to monocytes of healthy volunteers in the
presence of different concentrations of severe sepsis sera
and sera of healthy controls
To obtain information on whether other serum compounds in
addition to LBP might be responsible for LPS-inhibitory activity,
increasing concentrations of serum were used in the FITC-LPS
Figure 1. Kinetics of LBP serum concentrations in severe sepsis or septic
shock. Shown is the reference range of serum LBP concentrations of healthy
controls (n ⫽ 40) and the serum LBPkinetics of patients with severe sepsis or septic
shock (n ⫽ 63) beginning with the onset of severe sepsis (day 0) and ending with the
day of hospital discharge. Data are presented as minimum (lower o); 10th (lower
dash), 25th (lower end of box), 50th (median, line in box), 75th (upper end of box),
and 90
th
(upper dash) percentiles;and maximum (upper o).
Figure 2. Correlation between FITC-LPS binding to monocytes and LBP serum
concentrations. The scattergram shows the association between log serum LBP
concentrations of the severe sepsis and control cohort and the log mean fluores-
cence units as a measure of FITC-LPS binding to monocytes. The Pearson
correlation coefficient and ordinary least squares regression line are supplemented
(
r
⫽⫺0.64,
P
⬍ .001).
Figure 3. FITC-LPS binding to monocytes inthe presence of severesepsis sera
and healthy control sera. (A) Mean fluorescence of monocytes incubated with
FITC-LPS in the presence of 15% serum from patients with severe sepsis or from
healthy controls. Data are presented as 25th (box), 50th (median), and 75th
percentiles (box) and range (-). Statistical analysis was performed with the Mann-
Whitney
U
test; *
P
⬍ .05. (B) Patient example of the FITC-LPS FACS experiment:
Monocytes were incubated with FITC-LPS in the presence of 15% serum from a
healthy volunteer (light gray graph) and serum from a randomly chosen patient with
severe sepsis (darkgray graph).
HIGH-LEVELLBP INHIBITSLPS RESPONSE 3803BLOOD, 15 DECEMBER 2001
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FACS assay. The addition of severe sepsis and control sera in
concentrations up to 5% of the total volume per well enhanced
binding of FITC-LPS to monocytes. While acute-phase sera
showed an inhibitory activity when appliedin concentrationsabove
7.5%, the inhibitory activity of control sera could be demonstrated
at a concentration above 30% (Figure 5).
Endotoxin-neutralizing capacity of sera measured by the
LAL assay
Because LPS transfer to monocytes was significantly reduced by
severe sepsis sera, we next investigated whether these sera would
display an increased LPS binding resulting in a reduced concentra-
tion of “free” LPS. The median baseline LPS concentration found
in the sera of 8 randomly chosen patients was 86.5 pg/mL (range
36-150 pg/mL) as compared with a median concentration of 35
pg/mL(range 17-37 pg/mL) in sera of 6 healthy controls. While the
sera of sepsis patients thus exhibit elevated LPS levels, it can be
clearly ruled out that these concentrations would affect baseline
responsiveness in our biological assays.
Sera of the study cohort and of healthy controls were incubated
with 10 ng/mL LPS without heating and subjected to a modified
LAL assay as described in detail in “Patients, materials, and
methods” (Figure 6). Endotoxin concentrations measured in the
absence of serum were taken as 100%. Sera from healthy controls
were able to reduce LPS detected by LAL to a median value of
60%. Sera from severe sepsis patients, however, exhibited a
significantly more pronounced ability to bind LPS, resulting in a
median endotoxin concentration of 20% as measured by the LAL
assay (Mann-Whitney U test, P ⬍ .05).
LPS-induced TNF-␣ secretion in the presence of severe sepsis
sera, healthy control sera, LBP-depleted severe sepsis sera,
and rhLBP-supplemented control and LBP-depleted severe
sepsis sera
We next investigated whether sera from severe sepsis patients
would influence LPS-induced TNF-␣ secretion of monocytes. To
Figure 4. FITC-LPS binding to monocytes in the presence of healthy control
sera, rhLBP-supplemented control sera, and LBP-depleted severe sepsis sera.
(A) Mean fluorescence of monocytes incubated with FITC-LPS in the presence of
control sera and control sera supplemented with 100 g/mL rhLBP. Data are
presented as 25th (box), 50th (median), and 75th percentiles (box) and range (-).
Statistical analysis wasperformed withthe Mann-Whitney
U
test; *
P
⬍ .05. (B) Mean
fluorescence of monocytes incubated with FITC-LPS in the presence of control sera
supplemented with increasing concentrations of rhLBP assessed by FACS analysis.
Data are presented as mean ⫾ SEM of 5 independent experiments. (C) Patient
example of the FITC-LPS FACS experiment: mean fluorescence of monocytes
incubated with FITC-LPS in the presence of severe sepsis serum (dark gray graph)
and partially LBP-depletedsevere sepsis serum (light graygraph).
Table 2. Enhancement of LPS-induced monocytic response by LBPdepletion
of severe sepsis sera
LBP concentration,
mg/L*
LPS binding,
FU†
TNF-␣ secretion,
pg/mL‡
Severe sepsis sera
Native 60.5 ⫾ 38.2 36.7 ⫾ 17.9 544 ⫾ 228
Partially LBP-depleted 16.7 ⫾ 10.1 65.1 ⫾ 28.0§ 991 ⫾ 245§
Data are presented as mean ⫾ SD of 8 independent experiments with randomly
chosen sera ofsevere sepsis patients.
*LBP serum concentrations wereassessed by ELISA.
†Mean fluorescence units were obtained by FACS analysis of gated monocytes
incubated with FITC-LPS in the presence of 15% native or partially LBP-depleted
severe sepsis sera.
‡Monocytes were stimulatedwith 10ng/mLLPS in the presence of 15% native or
partially LBP-depleted severe sepsis sera,and TNF-␣ concentrations in the superna-
tants were assessedby ELISA.
§The statistical significance of the difference between groups was assessed by
the Mann-Whitney
U
test;
P
⬍ .05.
Figure 5. FITC-LPS binding to monocytes ofhealthy volunteers in thepresence
of different concentrations of severe sepsis sera and sera of healthy controls.
Binding of FITC-LPS to monocytes was assessed employing a FACS-based method
as described in “Patients, materials, and methods.” Five independent experiments
with sera of 5 healthy controls (F;LBPserum concentration,7.98 ⫾ 1.03 g/mL)and
of 5 randomly chosen severe sepsis patients (Œ;LBPserum concentration,107 ⫾ 32
g/mL) were performed. Different volumes of serum were added as indicated. Data
are presented as mean ⫾ SEM. Differences were analyzed by the Mann-Whitney
U
test; *
P
⬍ .05.
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this end freshly isolated PBMCs of healthy donors were stimulated
with 10 ng/mLLPS preincubated with severe sepsis or control sera.
LPS-induced monocytic TNF-␣ secretion was significantly re-
duced in the presence of severe sepsis sera as compared with
control sera (Mann-Whitney U test, P ⬍ .05; Figure 7A). When
rhLBP was added to control sera, resulting in acute-phase concen-
trations, a similar reduction in TNF-␣ secretion was observed
(Figure 7B).
To evaluate the dose-dependent effect of LBP, rhLBPwas added
in increasing amounts to control sera, and the TNF-␣ secretion
induced by 10 ng/mL LPS preincubated with these sera was
assessed (Figure 7C). Increasing LBP serum concentrations led to a
clearly reduced LPS-induced TNF-␣ secretion in an LBP dose-
dependent manner (Friedman test, P ⬍ .05). We also performed
this experiment using an LPS concentration of 1 ng/mLto stimulate
the monocytes and found that a 5-fold lesser concentration of
rhLBP was able to achieve a similar reduction of TNF-␣ secretion
as compared with stimulation with 10 ng/mL (data not shown).
To prove the specificity of LBP in inhibiting the LPS-induced
effects on monocytes, 6 randomly chosen severe sepsis sera were
depleted of LBP. Employing an anti-LBP antibody, we were able to
reduce LBP concentrations of severe sepsis sera to a concentration
below the lower detection limit of the LBP ELISA. Following this
depletion procedure, these sera were supplemented stepwise with
rhLBP. These sera were incubated with 10 ng/mL LPS, and the
stimulation experiment with monocytes was repeated. We found
that sera containing only traces of LBP, as well as those sera
reconstituted with rhLBP up to 1 g/mL, were enhancing LPS-
induced TNF-␣ secretion as compared with the sepsis sera.Adding
back rhLBP up to acute-phase concentrations restored the LPS-
inhibiting activity comparable to native severe sepsis sera (Fig-
ure 7D).
Inhibition of LPS effects in the absence of serum
Finally, to address whether LBP can inhibit LPS activity indepen-
dently of other serum factors, we established a serum-free in vitro
system: Monocytes were incubated with FITC-LPS in the presence
of increasing concentrations of rhLBP, and binding of LPS was
assessed by FACS as described above (Figure 8A). While concen-
trations of up to 100 g/mL gradually increased LPS binding to
monocytes, the addition of rhLBP resulting in a concentration of
150 g/mL significantly reduced LPS binding as measured by
fluorescence intensity. LPS bioactivity measured by LPS-induced
TNF-␣ secretion of monocytes was studied as described above. As
has been shown by others, low concentrations of LBP (10-100
ng/mL) increased LPS-induced TNF-␣ secretion of monocytes as
compared with stimulation in the absence of LBP(data not shown).
Addition of 1, 10, and 50 g/mL rhLBP also enhanced TNF-␣
secretion, while acute-phase concentrations of more than 50 g/mL
led to a gradual decrease of LPS-induced TNF-␣ secretion
(Figure 8B).
Discussion
Several lines of evidence are presented here indicating that
increased LBP concentrations found in serum of severe sepsis
patients inhibit proinflammatory activity of LPS. The acute-phase
increase of LBP concentrations therefore may represent an impor-
tant part of the antimicrobial defense system of the host. A
down-regulation of proinflammatory cytokine release upon in vitro
LPS stimulation has been demonstrated by others in whole blood
obtained from severe sepsis patients.
26-28
The mechanism of this
Figure 7. LPS-induced TNF-␣ secretion in the presence of severe sepsis sera,
control sera, LBP-depleted severe sepsis sera, and rhLBP-supplemented sera.
Monocytic TNF-␣ secretion in the presence of different sera preincubated with LPS.
(A) LPS-induced TNF-␣ secretion in the presence of 15% sera from patients with
severe sepsis and from healthy controls. (B) LPS-induced TNF-␣ secretion in the
presence of15% control sera and controlsera supplemented with100 g/mLrhLBP,
resulting in LBP concentrations comparable to those found in severe sepsis. Data of
panels A and B are presented as 25th (box), 50th (median), and 75th percentiles
(box) and range (-).Statistical analysiswas performedwith theMann-Whitney
U
test;
*
P
⬍ .05. (C) Monocytes were stimulated with 10 ng/mL LPS preincubated in the
presenceof seraofhealthy controls supplemented withincreasing amountsofrhLBP.
LPS-inducedTNF-␣ concentration in the presence ofnonsupplemented serumof the
healthy volunteers was set as 100%. Presented is the mean ⫾ SEM of 6 independent
experiments. (D) Monocytes were stimulated with 10 ng/mL LPS preincubated with
native severe sepsis sera, LBP-depleted severe sepsis sera, and LBP-depleted
severe sepsis sera supplemented with increasing concentrations of rhLBP starting
with 1g/mL. Each experiment was performed in duplicate employing 6 different
randomlychosen severe sepsissera. Data arepresented as mean⫾ SEM. Statistical
analysis was performedwith the Wilcoxon test; *
P
⬍ .05.
Figure 6. Endotoxin-neutralizing capacity of severe sepsis and control sera
measured by the LAL assay. The LAL assay was performed as described in
“Patients, materials, and methods,” employing either sera of healthy controls or sera
of patients with severe sepsis/septic shock. The concentration of LPS measured in
the absence of serum was set as 100%. Presented are the median; the 10th, 25th,
75th, and 90th percentiles; and the range. Statistical analysis was performed using
the Mann-Whitney
U
test; *
P
⬍ .05.
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down-regulation, however, has remained unclear. In severe sepsis it
has been demonstrated that IL-10, IL-4, and transforming growth
factor  have pronounced anti-inflammatory effects on mono-
nuclear cells.
34
It has remained unclear, however, whether hepatic
acute-phase proteins can modulate the monocytic secretion of
proinflammatory cytokines. In the present study we provide
evidence that LBP has this ability.
Several studies have shown that LBP at low concentrations
activates and amplifies the inflammatory host response to LPS, thus
potentially serving as a critical component in the initiation of the
innate immune response.
18
Blocking LBP with a polyclonal
antibody led to protection of mice after LPS application.
35,36
First
experiments with LBP-deficient mice supported these findings,
because deletion of the LBP gene was associated with suppression
of TNF-␣ induction and lethality in an LPS sepsis model.
23,37
After
injection of whole Gram-negative bacteria, however, an enhanced
mortality in LBP knock-out mice was observed due to an over-
whelming spread of bacteria.
24
This may have been caused either
by a lack of the early and effective activation of the innate immune
system or by a lack of the inhibitory function of LBP, as suggested
by the data shown here.
The mechanisms of the LPS-inhibitory activity of LBP are
currently not clear. LBP has been found to transfer LPS into
lipoproteins, thus inhibiting LPS effects.
21,22,38,39
We have recently
shown in a mouse sepsis model that high concentrations of LBP
generated by application of recombinant LBP can protect mice
against a lethal intraperitoneal injection of LPS or vital Gram-
negative bacteria.
24
The ability of LBP to transfer LPS to lipopro
-
teins may be the key mechanism for this protective role and for the
LPS-inhibitory activity of severe sepsis sera described in the
present paper. Although the early recognition of LPS may be
crucial for host defense, the spread of LPS monomers from a site of
infection via the bloodstream may be prevented by this mecha-
nism.
21,40
In addition, our results in a serum-free system point to a
second mechanism of inhibitory activity of high-dose LBP. We and
others have proposed a “silent uptake” of LPS that potentially is
mediated by higher concentrations of LBP.
41,42
Recent observations
by us suggest that these cellular mechanisms may occur both
CD14-dependently and CD14-independently (unpublished results,
2001), which is in line with recent observations by others on
CD14-independent effects of Gram-negative bacteria and LPS.
43-45
We demonstrate here that acute-phase sera of severe sepsis
patients are able to reduce LPS binding to monocytes and their
subsequent activation.The LBP depletion andreconstitution experi-
ments strongly suggest that these inhibitory effects can be mainly
attributed to LBP. Besides LBP, other serum proteins are known to
bind LPS and modulate LPS-induced monocytic activity: sCD14 is
released from neutrophil membranes by shedding and has been
shown to act as a coligand in the LPS-induced activation of CD14
⫺
cells such as endothelial and epithelial cells.
46,47
However, sCD14
is also able to inhibit LPS-induced TNF-␣ synthesis by monocytes
in the presence of LBP and high-density lipoprotein.
48,49
The
median of the peak serum sCD14 concentration measured in the
study cohort with severe sepsis was 14.7 g/mL (range 5.18-39.4
g/mL). These concentrations are well below the inhibiting
concentrations observed in the study by Haziot et al,
49
and sCD14
therefore can be ruled out as being mainly responsible for the
observed inhibitory effects of the severe sepsis sera. Nevertheless,
sCD14 and LBP might act synergistic. Another inhibitory LPS
binding protein is the bactericidal/permeability-increasing protein
(BPI) usually not detectable in plasma of healthy volunteers.
17,50,51
It has been demonstrated that in experimental endotoxemia as well
as in patients with severe sepsis, the peak LBP serum concentra-
tions were approximately 250-fold to 3000-fold higher than the
peak plasma BPI concentrations.
17,50,51
Although we did not
determine BPI plasma concentrations in the present study, we
assume that the BPI concentrations in severe sepsis sera were not
sufficient enough to be responsible for the modulatory effects on
the LPS activity observed. Other serum factors, such as serum
amyloid A and P, albumin, transferrin, and LDL, have been shown
to have the ability to bind LPS.
2,19,39,52
The composition of the
acute-phase sera is quite complex, and we cannot exclude these
factors from also being responsible for the LPS modulating effects
observed here. On the other hand, our LBP depletion and recon-
stitution experiments presented here present clear evidence that
LBP is a major factor for LPS detoxification during the acute-
phase response.
Results of others up to now mainly indicated LPS-enhancing
effects of LBP.
18,53,54
In these studies, however, always low
concentrations of LBP were employed. Enhancement of LPS
effects was demonstrated by the addition of either 0.1% to 1%
acute-phase serum of severe sepsis patients or by using up to 10%
of control serum. In the present study we confirmed these results
for the addition of low amounts of serum (Figure 5); however,
increasing volumes of serum, thus increasing LBP concentrations,
Figure 8. Inhibition of LPS effects in the absence of serum. (A) LPS transferwas
assessed by a FACS-based method as described in “Patients, materials, and
methods.” Monocytes were incubated with increasing concentrations of rhLBP
starting with 1g/mL as indicated and with 1 g/mL FITC-LPS. Presented are the
values of3 independent experiments.(B) Monocytes wereincubated with increasing
concentrations of rhLBP starting with 1g/mL as indicated and with 10 ng/mL LPS.
TNF-␣ concentrations were measured by ELISA. Presented are the values of 3
independent experiments.
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clearly down-regulated LPS transfer and monocytic activation.
Endotoxin present in patients’sera, although elevated, can be ruled
out as influencing our in vitro results, because the LPS concentra-
tions added experimentally were always in at least 10-fold to
100-fold excess.
In the serum-free assay described in this study, higher concentra-
tions of rhLBP were needed to achieve a decrease of LPS binding
and a reduced TNF-␣ secretion as compared with the assays
employing severe sepsis sera. Furthermore, we obtained evidence
that small concentrations of LPS are more easily inhibited by LBP
as compared with higher concentrations, in line with results
obtained with a murine macrophage cell line.
24
Our different assays
exhibited a different degree of inhibition, with the FITC-LPS
binding assay giving rise to lesser inhibition. This also may very
well be related to the higher LPS concentrations employed in this
assay. A more detailed analysis of serum components, their
concentrations, and LPS inhibition is subject of a separate ongoing
investigation in our laboratory. As the major mechanism of the
effects observed here in severe sepsis sera, we propose that high
serum LBP concentrations were able to down-regulate LPS activity
in the presence of lipoproteins. Several studies have demonstrated
reduced lipoprotein concentrations in severe sepsis.
55,56
It is
tempting to speculate that sera from patients with elevated LBP, yet
higher lipoprotein concentrations, are even more efficient in LPS
neutralization. Experiments addressing the synergistic role of
lipoproteins and LBP in inhibiting LPS activity are also currently
under way in our laboratory.
In the present prospective clinical study, we observed that the
up-regulation of LBP takes place both in Gram-negative as well as
in Gram-positive severe sepsis and is thus not specific for
infections with Gram-negative microorganisms. We could not
confirm in our study the findings of Opal et al that serum LBP
concentrations of nonsurvivors were significantly lower as com-
pared with survivors.
16
In the present study with a clearly defined
onset of severe sepsis and serial measurements, we did not find a
correlation between initial or peak serum LBP concentrations and
outcome in patients with severe sepsis or septic shock. This is
consistent with other reports.
17,18,50
In summary, our experiments with human severe sepsis sera
demonstrate an inhibitory role of high LBP concentrations in the
LPS-induced inflammatory response. Further experiments are
needed to completely elucidate this novel defense mechanism. A
complete understanding of the innate immune system during the
acute-phase response in severe sepsis or septic shock and its
regulation may provide a basis for new therapeutic approaches in
patients suffering from an uncontrolled systemic inflammation.
Acknowledgments
We are very grateful to Fra¨nzi Creutzburg for outstanding technical
support and for performing LBP depletion and reconstitution
experiments. Nicole Siegemund is acknowledged for excellent
technical support. We are furthermoregrateful toArianeAsmus and
Naser Qedra for assistance in blood sample collection and clinical
evaluation of the patients. We also thank Peter Germain for the
critical reading of this manuscript.
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