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PHAGOCYTES
From the Institut für Mikrobiologie und Hygiene,
Universitätsklinikum Charité, Medizinische Fakultät
der Humboldt-Universität zu Berlin, and Klinik für
Anaesthesiologie und operative Intensivmedizin,
Universitätsklinikum Benjamin Franklin, Freie Universität
Berlin, Germany, and the Institut für Statistik und
Ökonometrie, Universität Hamburg, Germany.
Lipopolysaccharide-binding protein (LBP), an
acute-phase protein recognizing lipopolysaccharide (LPS), catalyzes in
low concentrations its transfer to the cellular LPS receptor consisting
of CD14 and Toll-like receptor-4. It has recently been shown
that high concentrations of recombinant LBP can protect mice in a
peritonitis 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 septic shock and the ability of these sera to
modulate LPS effects in vitro was assessed employing different assays. Transfer of fluorescein isothiocyanate-labeled LPS to human monocytes was assessed by a fluorescence-activated cell sorter-based method, and
activation of monocytes was investigated by measuring LPS-induced tumor
necrosis factor- 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 inflammatory response can be a cause of organ
dysfunction remote from the primary site of infection, hypotension, or
shock For the initiation of the innate immune response, monocytes and
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- LBP is constitutively synthesized in hepatocytes and is present in
serum at concentrations of 5 to 15 µg/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
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 decreased 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 LBP
in patients with severe sepsis or 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 LBP
are a key factor for inhibition of LPS activity by acute-phase sera.
Patients
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 sera also 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
LBP and sCD14 assays Serum LBP concentrations were assessed by an enzyme-linked immunosorbent assay (ELISA) employing the monoclonal antibodies 1E8 and 2B5 (kindly provided by Dr A. 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 × 105 PMBCs per milliliter, corresponding to 1.5 × 105 monocytes per milliliter. The cells were incubated with 30 µL acute-phase serum of patients with severe sepsis/septic shock, or with serum from healthy controls, in a total volume of 200 µL. 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 fluorescence units and recorded on a logarithmic scale. LPS-induced TNF-  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 µL LAL reagent (Endo KTA-LAL, Charles River Endosafe, Charleston, SC) was added to 50 µL 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 µL substrate (Perfachrome LAL, Pentapharm, Basel, Switzerland). For quantification 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 or SEM, 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 coefficient 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.
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], respectively; 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 concentrations could be observed in survivors as compared with nonsurvivors 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). Multivariate analyses including severity of infection as a potential confounder 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 =
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 employing those severe sepsis sera containing peak LBP concentrations 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 monocytes 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 =
The mean fluorescence units representing the LPS transfer to
monocytes in the presence of sera of patients with severe sepsis or
septic 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 93.7 µg/mL LBP and of a healthy control serum with an LBP
concentration of 9.88 µg/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 µg/mL (Figure 4B). However, higher rhLBP concentrations led to a marked down-regulation of LPS binding. Next, we depleted severe sepsis sera from 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 µg/mL. LBP depletion significantly enhanced the LPS transfer activity and the LPS-induced TNF- secretion of this serum (Table
2). A representative 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 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 applied in concentrations above 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 concentration 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 of monocytes. To this end
freshly isolated PBMCs of healthy donors were stimulated with 10 ng/mL
LPS preincubated with severe sepsis or control sera. LPS-induced
monocytic TNF- secretion was significantly reduced 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 concentrations, a similar reduction in
TNF- secretion was observed (Figure 7B).
To evaluate the dose-dependent effect of LBP, rhLBP was added in
increasing amounts to control sera, and the TNF- 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 µg/mL,
were enhancing LPS-induced TNF- Inhibition of LPS effects in the absence of serum Finally, to address whether LBP can inhibit LPS activity independently 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 concentrations of up to 100 µg/mL gradually increased LPS binding to monocytes, the addition of rhLBP resulting in a concentration of 150 µg/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 µg/mL rhLBP also enhanced TNF- secretion, while acute-phase concentrations of more than 50 µg/mL led to a gradual decrease of LPS-induced TNF- secretion (Figure 8B).
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 important 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 down-regulation,
however, has remained unclear. In severe sepsis it has been
demonstrated that IL-10, IL-4, and transforming growth factor 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- 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 lipoproteins 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 mechanism.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 and reconstitution experiments 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 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, 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 concentrations added experimentally were always in at least 10-fold to 100-fold excess. In the serum-free assay described in this study, higher concentrations
of rhLBP were needed to achieve a decrease of LPS binding and a reduced
TNF- 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 compared 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.
We are very grateful to Fränzi Creutzburg for outstanding technical support and for performing LBP depletion and reconstitution experiments. Nicole Siegemund is acknowledged for excellent technical support. We are furthermore grateful to Ariane Asmus 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.
Submitted February 12, 2001; accepted August 2, 2001.
Supported in part by grants from the German Research Foundation (DFG, grant no. Schu 828/1-5) and by the Bundesministerium für Bildung und Forschung (BMBF, grants no. 01KV98067 and 01KI9855/0).
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.
Reprints: Ralf R. Schumann, Institut für Mikrobiologie und Hygiene, Universitätsklinikum Charité, Dorotheenstr 96, D-10117 Berlin, Germany; e-mail: ralf.schumann{at}charite.de.
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M. Majetschak, U. Krehmeier, M. Bardenheuer, C. Denz, M. Quintel, G. Voggenreiter, and U. Obertacke Extracellular ubiquitin inhibits the TNF-alpha response to endotoxin in peripheral blood mononuclear cells and regulates endotoxin hyporesponsiveness in critical illness Blood, March 1, 2003; 101(5): 1882 - 1890. [Abstract] [Full Text] [PDF]
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M. Gumenscheimer, I. Mitov, C. Galanos, and M. A. Freudenberg Beneficial or Deleterious Effects of a Preexisting Hypersensitivity to Bacterial Components on the Course and Outcome of Infection Infect. Immun., October 1, 2002; 70(10): 5596 - 5603. [Abstract] [Full Text] [PDF]
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Top
Abstract
Introduction
a syndrome termed severe sepsis or septic shock.
; LBP serum
concentration, 7.98 ± 1.03 µg/mL) and of 5 randomly chosen severe
sepsis patients (
; LBP serum concentration, 107 ± 32 µg/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.
have
pronounced anti-inflammatory effects on mononuclear
cells.
cells such as endothelial
and epithelial cells.
treatment.
Nat Med.
1997;3:678-681
B activation.
Eur J Immunol.
2000;30:211-216