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PHAGOCYTES
From the Institute of Immunology, The National
Hospital, University of Oslo, Norway; Department of Immunology and
Transfusion Medicine, Department of Clinical Chemistry, and Medical
Department, Nordland Central Hospital and University of Tromsø;
Department of Immunology and Transfusion Medicine, Institute of
Laboratory Medicine, Norwegian University of Science and Technology,
Trondheim, Norway; Institute of Medical Microbiology, Medical School,
Hannover, Germany; Tanox, Houston, TX; and Laboratory of Protein
Chemistry, Department of Pathology and Laboratory Medicine, University
of Pennsylvania, PA.
Complement plays an essential role in inflammation and tissue
damage. However, it is largely unknown to what extent the system acts
as a primary inducer of secondary mediator systems in the inflammatory
network of human whole blood. Here we describe a novel in vitro model
using the thrombin-specific hirudin analog lepirudin as anticoagulant,
which, in contrast to heparin, did not interfere with complement
activation. The model was used to study the role of complement in
Escherichia coli-induced inflammatory responses.
Granulocyte and monocyte oxidative burst was complement dependent as it
was reduced by 85% and 70%, respectively, by the CD3 binding peptide
compstatin. A similar reduction was found by inhibition of C5, C5a, and
C5a receptor (C5aR). Furthermore, anti-CR3 antibodies were as efficient
as the C5aR antagonist in reducing granulocyte oxidative burst, whereas
blocking CD14 or C3aR had no effect. Up-regulation of granulocyte CR3
was virtually abolished by a C5aR antagonist. Opsonization and
phagocytosis was completely inhibited by blocking of C5aR or CR3,
whereas blocking of the Fc Complement protects the host against invading
microorganisms. However, it is a double-edged sword in the sense that
it may induce undesirable inflammation if activated improperly or
uncontrolled. Numerous studies have confirmed that complement
contributes to tissue damage in conditions like
septicemia,1,2 ischemia-reperfusion injury,3,4 capillary leak syndrome,5,6
connective tissue disease,7,8 neurologic
disease,9 nephritis,10,11 and transplant
rejection.12,13 Many of the mechanisms by which complement
activates blood cells have been elucidated. These include binding of
activated complement fragments to their corresponding receptors14 and effects induced by sublytic attack by the
terminal C5b-9 complex.15-17
Complement-mediated activation of granulocytes and monocytes causes
oxidative burst with release of reactive oxygen species; production of
cytokines; release of arachidonic acid metabolites, histamine, and
platelet activating factor; and altered expression of adhesion
molecules. However, these various effects of complement have
mainly been studied in vitro by using isolated cells stimulated with
purified proteins. To investigate the role of complement in the complex
inflammatory network, all potential cellular and fluid-phase mediators
need to be present and able to interact simultaneously. In vitro, such
cross talk can only be achieved by using whole blood. However, whole
blood models have been hampered by the adverse effects by
anticoagulants on complement activation. Calcium chelators like
ethylenediaminetetraacetic acid (EDTA) and citrate inhibit complement
activation as well as a number of other biologic processes. Due to lack
of other suitable anticoagulants, heparin has been widely used,
although it has a number of effects on complement.18
Heparin inhibits complement activation at high concentrations,
whereas it enhances the activation at low
concentrations.19,20 Heparin also has various direct
effects on platelets21,22 and leukocytes,23 which excludes it as an optimal
anticoagulant in models to study the inflammatory network.
The aim of the present study was to investigate the role of human
complement as primary inducer of inflammation by developing an in vitro
whole blood model using an anticoagulant without adverse effects on
complement. This was achieved by the recombinant hirudin analog
lepirudin, a highly specific thrombin inhibitor.24 The C3
inhibitor compstatin, an anti-C5 neutralizing antibody, an anti-C5a
antibody, and a small-molecular C5aR antagonist peptide were used to
selectively inhibit complement activation. The results indicate that
Escherichia coli-induced oxidative burst and phagocytosis was complement mediated and virtually completely dependent on C5aR-mediated up-regulation of CR3. In contrast, E
coli-induced cytokine release was largely complement independent
and was efficiently inhibited by blocking CD14.
All equipment (eg, tubes, tips) and solutions used in the model
were endotoxin-free according to information from the manufacturers. Polypropylene tubes were used to obtain low background activation of complement.
Reagents
Complement inhibitors
Effect of lepirudin and heparin on complement Serum from 6 healthy voluntary blood donors was incubated for 60 minutes at 37°C in the presence of lepirudin (5, 50, and 500 µg/mL), heparin (0.2, 2, and 20 IU/mL), or PBS (control). After incubation, EDTA was added (10 mM final concentration) and the samples were stored at 70°C until being analyzed. Furthermore, whole blood
(4 mL) from 6 voluntary donors (written informed contest was obtained)
was collected into sterile polypropylene tubes (4.5-mL NUNC cryotubes;
Nalge Nunc International, Roskilde, Denmark). Immediately after
venipuncture, the sample was split and 500 µL blood added to sterile
polypropylene tubes (1.8-mL NUNC cryotubes) containing lepirudin (5, 50, or 500 µg/mL) or heparin (0.2, 2, or 20 IU/mL). Then E
coli (2 × 107/mL, endotoxin 140 ng/mL) or PBS was
added and the tubes rotated (Rock-n-Roller; Labinco, The Netherlands)
for 60 minutes at 37°C. Thereafter, further complement activation was
blocked by adding EDTA (10 mM). The tubes were centrifuged for 15 minutes at 4000g at 4°C. Plasma was stored at 70°C
until being analyzed for complement activation. The samples with
lepirudin 5 µg/mL and heparin 0.2 IU/mL clotted during incubation and
were excluded.
Standardization of the whole blood model Based on the results above, lepirudin 50 µg/mL was used as anticoagulant, and a standard temperature of 37°C was chosen in order to approach physiologic conditions. In the present study, the effect of complement on E coli-induced oxidative burst and phagocytosis was investigated in detail. However, to design the model to study several arms of the inflammatory network, changes in leukocyte and platelet cell surface markers, release of their granular proteins, and cytokine production was examined. Incubation up to 4 hours revealed changes in all mediator systems studied, except for interleukin-10 (IL-10), which required 18 hours of incubation. As a control, changes in the basic physiologic conditions were examined using a standard blood gas instrument. During the first 60 minutes pH did not change (stable between 7.3 and 7.4) and was above 7.2 even after 4 hours. The pCO2 did not change significantly during 4 hours of incubation, and base excess was unchanged the first 15 minutes. There were no differences in these parameters when the samples were incubated in a CO2 incubator using 5% (vol/vol) CO2 with the cap open or in a conventional incubator with the cap closed. However, pO2 increased more in the CO2 incubator (from 3 to 14 kPa) than in the conventional incubator (from 3 to 8 kPa) after 4 hours. The increase in pO2 after 15 minutes in the conventional incubator was minor (from 3 to 4 kPa), and this incubator was therefore chosen.Experimental design The effect of a panel of different inhibitors on the oxidative burst, phagocytosis, complement activation, and myeloperoxidase (MPO) release was examined as follows: All incubations were performed at 37°C. Whole blood anticoagulated with lepirudin 50 µg/mL was collected and distributed immediately into tubes containing PBS, inhibitor, or control. The samples were preincubated for 4 minutes until PBS (baseline samples) or 1 × 108 E coli per milliliter of blood was added (the E coli concentration was 2 × 107/mL blood in the experiments using compstatin as blocking agent). The T0 baseline sample was processed immediately, and further incubation was performed for the actual time periods as described for the different experiments. After removal of 100 µL blood for the flow cytometric assays, EDTA (10 mM) was added and the tubes were centrifuged for 15 minutes at 4000g (4°C). The plasma was stored at70°C until analyzed.
Flow cytometry The oxidative burst and the phagocytic activity was measured using Burst test and the Phago-test, respectively. Immediately after incubation, 100 µL blood was added to sterile polypropylene tubes (Falcon, Becton Dickinson, Franklin Lakes, NJ) and treated according to the kit procedures. Incubation with E coli was 10 minutes for oxidative burst and 20 minutes for phagocytosis. The cells were resuspended in PBS and analyzed on a FACSCalibur flow cytometer (Becton Dickinson, San José, CA). In a forward/side scatter (FSC/SSC) dot plot, gates were set on granulocytes and monocytes to analyze each population with regard to median fluorescence intensity (MFI). Leukocyte surface-bound bacteria in the phagocytosis test were neutralized using quenching solution. In experiments where phagocytosis was blocked, additional experiments omitting quenching solution were performed to evaluate the degree of opsonization.CD11b and Fc  R expression was measured principally in the
same manner using PE-conjugated anti-CD16/FcRIII,
anti-CD32/FcRII, and anti-CD64/FcRI antibodies.
Deposition of C3d and IgG on E coli Preopsonized E coli (1 × 108/mL) was preincubated with PBS or lepirudin plasma for 10 minutes at 37°C and washed twice with PBS containing 0.1% (wt/vol) bovine albumin. Nonoposonized E coli was used as negative control. Samples were then incubated 15 minutes at room temperature with FITC-labeled anti-C3d or anti-IgG (dilution 1:4). Bacteria were gated in an SSC/FSC dot plot, and deposition of C3d or IgG was determined using MFI. At least 10 000 bacteria were counted in each sample.Enzyme immunoassays C1rs-C1inhibitor complexes (C1rs-C1inh). Activation of the classical complement pathway was quantified in an enzyme immunoassays (EIA) described in detail elsewhere,32 using the mAb Kok 12 specific to a neoepitope exposed only when C1inh is in complex with its substrates. This antibody was a kind gift from Prof C. E. Hack, Amsterdam, The Netherlands. C3b-Bb-properdin complexes (C3bBbP).
Activation of the alternative pathway was detected by quantifying the
alternative convertase C3bBbP in an EIA. Microtiter plates (Maxisorp;
Nunc) were incubated at 4°C overnight with mouse antihuman
factor P, clone no. 2 (Quidel, San Diego, CA) diluted 1/1000 in 0.05 M
carbonate buffer, pH 9.6. Between each further incubation the plates
were washed thrice with PBS containing 0.1% (vol/vol) Tween 20. All
incubations were made with 50 µL per well, except for the substrate
(100 µL). Standard (see below) was diluted 2-fold from 1/100 to
1/3200, and test samples (containing 10 mM EDTA final concentration)
were diluted 1/25 in PBS containing 0.2% Tween 20 and 10 mM EDTA. The
plates were incubated for 60 minutes at room temperature. Detection
antibody was anti-C3c (Behringwerke, Marburg, Germany) diluted 1/1000
in PBS containing 0.2% (vol/vol) Tween 20. After 45 minutes of
incubation at 37°C, horseradish peroxidase-conjugated donkey
antirabbit Ig (NA9349; Amersham International, Little Chalfont, United
Kingdom), diluted 1/1000 in PBS containing 0.2% Tween 20, was added.
After 45 minutes of incubation at 37°C, substrate was added: ABTS
(2,2'-azino-di-(3-ethylbenzthiazoline sulfonic acid)), 180 mg/L, diluted in 0.15 M sodium acetate buffer, pH 4.0. H2O2 (10 µL of 3%) was added to 12.5 mL
substrate solution immediately before use. The standard was a
zymosan-activated human serum pool (ZAS) made by incubating serum with
10 mg/mL zymosan (Sigma Chemical, St Louis, MO) for 60 minutes at
37°C. After centrifugation the supernatant was split and stored in
aliquots at C3bc. Activation of the final common pathway was quantified in an EIA using the mAb bH6 specific for a neoepitope exposed in C3b, iC3b, and C3c as previously described.33 C5a. Activation of C5 was quantified in a C5a EIA using the neoepitope-specific mAb 4A2E10E2 as capture antibody and 3G3C4 as secondary antibody as previously described.34 The antibodies were a kind gift from Prof Kåre Bergh, Trondheim, Norway. The terminal sC5b-9 complex (TCC). Activation of the terminal pathway was quantified in an EIA using the mAb aE11 specific for C9 incorporated in the sC5b-9 complex as described previously.33 MPO. MPO was quantified using an EIA as described previously.23 TNF- Complement hemolytic activity The inhibitory activity of mAb 137-30 against human C5 was tested in a classical pathway hemolytic assay using sensitized chicken red blood cells as described earlier.35Colony forming units One or 10 µL of blood obtained immediately after adding E coli and after 20 minutes of incubation was seeded on microbiologic Petri dishes containing blood agar and further incubated 24 hours at 37°C. Bacterial growth was expressed as colony-forming units (CFUs) per milliliter of blood.Statistics Data are given as medians with 95% nonparametric confidence intervals (CIs) if otherwise not stated. The Friedman test with subsequent formulas for multiple comparisons was used to compare the interventions.36 A 2-tailed P < .01 was considered statistically significant due to the number of tests performed.
A lepirudin-based human whole blood model for complement studies Effect of heparin and lepirudin on spontaneous complement
activation in serum.
Because anticoagulation may have adverse effects on complement
activation, we investigated the thrombin-specific inhibitor lepirudin
with respect to effect on complement and compared it with heparin.
Serum experiments were first performed including a true negative
control (PBS with no anticoagulant) to reveal any effects on
spontaneous in vitro complement activation. Heparin and lepirudin were
tested in equal doses with respect to their known anticoagulant effect
(Figure 1). Complement activation as measured by TCC increased from 9.2 AU/mL (range, 6.0-12.6 AU/mL) at
baseline to 77 AU/mL (range, 57-99 AU/mL) after 60 minutes in the PBS
control (P < .001). Lepirudin at 3 different doses had no
effect (Figure 1, left panel), while heparin 0.2 IU/mL and 2 IU/mL
enhanced activation and 20 IU/mL inhibited activation compared with the
PBS control (P < .001) (Figure 1, right panel). Similar
results were found for C3bBbP and C3bc, whereas heparin increased
C1rs-C1inh complexes dose dependently, consistent with the known
enhancing effect of heparin on C1inh binding to C1s.
Effect of heparin and lepirudin on complement activation in whole
blood.
The effect on complement activation was studied by incubating blood for
10 minutes either with PBS (Figure 2,
upper panels) or with E coli (2 × 107
bacteria per milliliter) (Figure 2, lower panels). Heparin (20 IU/mL)
increased C1rs-C1inh complexes markedly, whereas lepirudin had no
effect (Figure 2, left panel). TCC formation was substantially inhibited by heparin, whereas lepirudin had no effect (Figure 2, right
panel). Similar results as those obtained for TCC were also found for
C3bBbP and C3bc. Lepirudin 50 µg/mL was therefore used as
anticoagulant in the further experiments. This concentration corresponded to approximately 2 times the dose required to avoid coagulation after 24 hours of incubation.
Role of complement in E coli-induced oxidative burst in whole blood Effect of compstatin on E coli-induced oxidative
burst.
The oxidative burst in granulocytes and monocytes increased rapidly and
time dependently with a maximal response after 10 to 15 minutes and
thereafter declined. Thus, 10 minutes of incubation was used in the
further burst experiments. The oxidative burst in granulocytes
increased from baseline MFI 4.5 (range, 3.2-5.3) to MFI 28 (range,
14-58) after incubation with E coli (2 × 107
bacteria per milliliter) (Figure 3, upper
panel). Compstatin virtually abolished this increase, reducing the
oxidative burst to MFI 7.4 (range, 5.2-11), whereas no effect was seen
using the control peptide (MFI 28 [range, 16-54]). The difference
between compstatin and the control peptide was highly significant
(P < .0001). The monocyte oxidative burst also increased
from baseline MFI 3.9 (range, 2.8-4.8) to MFI 13 (range, 6.5-16) after
incubation with E coli (Figure 3, lower panel). Compstatin
again reduced the oxidative burst to MFI 6.7 (range, 4.9-8.0) in
contrast to the control peptide, which had no effect (MFI 13.4 [range,
7.7-19]). The difference between compstatin and the control peptide
was highly significant (P < .0001). During the 10 minutes
of incubation, neither granulocytes nor monocytes showed any
spontaneous oxidative burst (Figure 3).
Effect of compstatin on complement activation.
Compstatin efficiently reduced the E coli-induced
complement activation in whole blood as measured by C3bBbP, C3bc, C5a,
and TCC (Figure 4). The differences
between compstatin and the control peptide were highly significant
(P < .001). Even the modest spontaneous activation
observed for C3bBbP and C3bc during the incubation was completely
abolished by compstatin. The effect on the terminal pathway was also
substantial and highly significant, although not as complete as for C3
activation. In contrast to the other activation products, only minor
changes were found for C1rs-C1inh complexes, which were not inhibited
by compstatin.
Effect of neutralizing C5 on the oxidative burst.
The effect of C5 activation on the oxidative burst was investigated
using an anti-C5 antibody (clone 137-30) shown to completely inhibit
lysis of antibody-sensitized chicken erythrocytes by human serum (Figure 5, left panel). Clone
137-30 (final concentration 0.7 µM) inhibited E
coli-induced granulocyte oxidative burst by 70% (Figure 5, right
panel). In comparison, monocyte oxidative burst was inhibited by
approximately 50%.
Effect of C5a and C5aR on E coli-induced oxidative
burst.
A monoclonal anti-C5a antibody (clone 561) inhibited granulocyte
oxidative burst dose dependently and to a degree comparable with
compstatin (Figure 6, upper panels).
Monocyte oxidative burst was also inhibited by the same reagents but to
a lower degree as compared with granulocytes (Figure 6, lower panels).
The synthetic cyclic hexapeptide AcF[OPdChaWR], a previously
described C5aR antagonist, inhibited granulocyte oxidative burst in a
dose-dependent manner (Figure 7, left
panel). By detailed titration the threshold for maximal effect of the
peptide was found at approximately 1 µM. The relative inhibition was
independent of the amount of E coli used (Figure 7, right
panel). A similar effect, although less pronounced, was seen in
monocytes. The C3aR antagonist had no effect on the oxidative burst.
Thus, the complement-mediated E coli-induced oxidative
burst, particularly in human granulocytes but also in monocytes, is
highly dependent on the C5a-C5aR interaction.
Role of complement in E coli-induced MPO release Spontaneous MPO release increased from 185 µg/L (range, 112-246 µg/L) at baseline to 404 µg/L (range, 267-636 µg/L) after 10 minutes of incubation (P < .01). E coli (2 × 107 bacteria per milliliter) significantly increased MPO release to 1002 µg/L (range, 605-1372 µg/L) (P < .0001). Compstatin reduced MPO to 687 µg/L (range, 461-1052 µg/L), whereas no effect was seen for the control peptide (1176 µg/L [range, 798-1601 µg/L]). The difference between compstatin and the control peptide was highly significant (P < .0001).Role of complement in E coli-induced CR3 and
Fc Effect of C5aR antagonist on CD11b expression.
E coli (1 × 108 bacteria per milliliter)
substantially increased granulocyte CD11b expression. This increase was
efficiently and dose dependently inhibited by the C5aR antagonist
(Figure 8, left panel). A modest E
coli-induced monocyte CD11b expression was also found.
Effect of C5aR antagonist on Fc Role of CR3 (CD11b/CD18) in E coli-induced oxidative burst The powerful effect of blocking C5aR both on the oxidative burst and on CR3 expression led us to investigate a possible role for CR3 in induction of E coli-induced burst. F(ab')2 fragments of anti-CD18 and anti-CD11b inhibited granulocyte oxidative burst dose dependently (Figure 8, right panel). The inhibition with anti-CD11b was as efficient as for the C5aR antagonist. Similar results were obtained for monocytes, although less pronounced. Inhibition of the oxidative burst was not seen by blocking CD14, although anti-CD14 efficiently reduced cytokine secretion (see below).Role of complement in phagocytosis of E coli Effect of C5aR antagonist on phagocytosis.
Addition of E coli (1 × 108 bacteria per
milliliter) led to an immediate and efficient phagocytosis. Samples
obtained just after addition of the bacteria to the whole blood
revealed 5 × 105 CFUs per milliliter, whereas after 20 minutes of incubation CFUs could not be detected. C5aR antagonist
completely abolished both granulocyte and monocyte phagocytosis as
measured by flow cytometry (Figure
9).
Effect of anti-CD11b and anti-Fc
Role of complement in E coli-induced cytokine formation Plasma concentrations of TNF- , IL-6, and IL-8
increased substantially after 2 hours and IL-10 after 18 hours of
incubation with E coli. Cytokine production was induced by
smaller amounts of E coli than required for oxidiative burst
(Figure 11). Spontaneous cytokine
release during incubation with PBS was not seen for TNF-, IL-6, or IL-10, whereas a modest release of IL-8 was
observed. Inhibition of complement had no effect on E
coli-induced secretion of TNF-, IL-6, or IL-10, whereas IL-8
formation was partly complement dependent. In contrast, anti-CD14 very
efficiently inhibited TNF-, IL-6, and IL-10 secretion, whereas the
effect on IL-8 was less pronounced. By combining compstatin and
anti-CD14, IL-8 secretion was abolished. Inhibition of E
coli-induced IL-6, IL-8, and IL-10 secretion by compstatin and
anti-CD14 is shown in Figure 11. The results for TNF- were virtually
identical to those for IL-6. The C5aR antagonist had the same effect as
compstatin on the cytokines.
The inappropriate effects of commonly used anticoagulants on the various inflammatory systems have been a major limitation when using whole blood to study inflammatory reactions. A main goal for an optimal whole blood model is to keep all potential effector systems functional and able to cross talk by mutual interaction and still avoid coagulation. Anticoagulants containing calcium chelators (eg, EDTA and citrate) inhibit complement activation as well as a number of other plasma and cell inflammation markers and therefore cannot be applied. Heparin has been used, although it is known that various effects on complement may influence the results. We confirm in the present study that low concentrations of heparin enhance, whereas high concentrations inhibit, complement activation.18-20 The known enhancing effect of heparin on C1inh binding to C1r and C1s18,37 was also evident from the present study. Heparin binds to a number of plasma proteins and also has adverse effects on leukocytes21,22 and platelets,23 disqualifying it as a suitable anticoagulant for whole blood studies on inflammation. In contrast to heparin, hirudin is a specific thrombin inhibitor.24 Thus, the coagulation cascade is kept functional upstream to thrombin formation. Hirudin has been used successfully in a whole blood model to study monocyte and platelet activation,21 and lepirudin per se has no effect on IL-6 or tissue factor formation.38 We demonstrate for the first time that the hirudin analog lepirudin has no adverse effects on complement activation and can be recommended as anticoagulant in models to study the role of complement in inflammation. The limitation of the model is the effect of lepirudin on thrombin. Thrombin is known to be an inflammatory mediator,39 and hirudin-based peptides were found to inhibit thrombin-induced inflammatory effects on endothelial cells.40 Because whole blood models need anticoagulation, it is impossible to circumvent this problem completely. However, we suggest that lepirudin presently is the best alternative in complement activation studies because its effect is limited to thrombin inhibition. The oxidative burst reflects formation of reactive oxygen species such
as the superoxide anion and hydrogen peroxide. Flow cytometric
detection of the oxidative burst has the advantage that single-cell
populations can be examined individually in whole blood. This assay was
found to be very stable and reproducible. No spontaneous oxidative
burst took place during the incubation period. In all individuals
tested the oxidative burst response was largely dependent on
complement. The oxidative burst of granulocytes was generally stronger
than for monocytes; however, granulocyte burst was also more
efficiently reduced by complement inhibition. Reactive oxygen species
are highly toxic and may damage host tissue and interfere with
homeostasis. They are known to activate the transcription factor
nuclear factor- Phagocytosis and oxidative burst are 2 closely linked leukocyte defense
events. Blocking the C5aR inhibited phagocytosis virtually completely
both in granulocytes and monocytes. Furthermore, opsonization of the
bacteria to the cell surface was also abolished. The opsonization and
subsequent phagocytosis was fully dependent on C5aR-induced CR3
up-regulation because blocking of CR3 also abolished phagocytosis. The
E coli-induced marked down-regulation of granulocyte CD16 (Fc The concentrations of E coli used in this study are similar to lethal and sublethal doses used in primate models of septicemia.45 Furthermore, our data confirm that the CFU count greatly underestimates the bacterial load in human whole blood,46 most likely due to rapid phagocytosis of bacteria by leukocytes, as demonstrated in the present study by the rapid decline in CFUs during E coli incubation in human whole blood. This suggests that the experimental conditions in the present in vitro model may be pathophysiologically relevant for an in vivo situation. Since Weisman et al3 in 1990 demonstrated that complement inhibition by recombinant soluble complement receptor 1 (sCR1) markedly reduced the tissue damage during experimental myocardial infarction, numerous studies have highlighted complement inhibition as an important issue in various pathophysiologic and clinical conditions. Thus, studies of specific inhibition of complement using either human regulatory proteins, antibodies to complement components or peptides to block activation or receptors, have demonstrated that complement is a key mediator of tissue injury and inflammation.47 However, limited data are available concerning the role of complement as a primary inducer of the other branches of the inflammatory network. The C5aR antagonist we used in the present study has been shown to reduce the adverse effects of endotoxic shock and immune complex-mediated tissue damage (reverse-passive arthus reaction).48 Similarly, antibodies neutralizing C5a have been found to protect against multiorgan failure and death in a rat cecal ligation and puncture (CLP) model of septicemia.49,50 These favorable effects may be explained by preservation of neutrophil function, attenuation of CR3 expression, and reduced production of reactive oxygen species, although inhibition of other secondary mediators triggered by complement activation may also be involved. Complement activation is known to induce cytokine production in models
using isolated cells, and the present C5aR antagonist was recently
shown to inhibit lipopolysaccharide (LPS)-dependent C5aR-induced IL-1 Taken together, our results show that the lepirudin-based human whole blood model is useful for studying the roles of complement activation within the inflammatory network. We have demonstrated that E coli-induced oxidative burst and phagocytosis is dependent on C5aR-mediated up-regulation of CR3. Inhibition of complement-mediated oxidative burst could be a therapeutic approach to attenuate tissue damage.
The authors thank Lynn Spruce for peptide synthesis and Dr William T. Moore for mass spectrometric analysis of the peptides. Berit Brusletto is greatly acknowledged for quantifying endotoxin in the E coli suspension and Arumugam Kugendradas for performing the MPO assays.
Submitted November 1, 2001; accepted May 6, 2002.
Supported by the Norwegian Council on Cardiovascular Disease; the Health and Rehabilitation Foundation; Tanox, Houston, TX; Odd Fellow Foundation; Gythfeldt's Legacy; and NIH grant GM62134.
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: Tom Eirik Mollnes, Institute of Immunology, The National Hospital, N-0027 Oslo, Norway; e-mail: t.e.mollnes{at}labmed.uio.no.
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  K. T. Lappegard, D. Christiansen, A. Pharo, E. B. Thorgersen, B. C. Hellerud, J. Lindstad, E. W. Nielsen, G. Bergseth, D. Fadnes, T. G. Abrahamsen, et al. Human genetic deficiencies reveal the roles of complement in the inflammatory network: Lessons from nature PNAS, September 15, 2009; 106(37): 15861 - 15866. [Abstract] [Full Text] [PDF]
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 E. B. Thorgersen, A. Pharo, K. Haverson, A. K. Axelsen, P. Gaustad, G. J. Kotwal, G. Sfyroera, and T. E. Mollnes Inhibition of Complement and CD14 Attenuates the Escherichia coli-Induced Inflammatory Response in Porcine Whole Blood Infect. Immun., February 1, 2009; 77(2): 725 - 732. [Abstract] [Full Text] [PDF]
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 A. Schreiber, H. Xiao, J. C. Jennette, W. Schneider, F. C. Luft, and R. Kettritz C5a Receptor Mediates Neutrophil Activation and ANCA-Induced Glomerulonephritis J. Am. Soc. Nephrol., February 1, 2009; 20(2): 289 - 298. [Abstract] [Full Text] [PDF]
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 M. A. Flierl, D. Rittirsch, B. A. Nadeau, D. E. Day, F. S. Zetoune, J. V. Sarma, M. S. Huber-Lang, and P. A. Ward Functions of the complement components C3 and C5 during sepsis FASEB J, October 1, 2008; 22(10): 3483 - 3490. [Abstract] [Full Text] [PDF]
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 H. Nitta, T. Imamura, Y. Wada, A. Irie, H. Kobayashi, K. Okamoto, and H. Baba Production of C5a by ASP, a Serine Protease Released from Aeromonas sobria J. Immunol., September 1, 2008; 181(5): 3602 - 3608. [Abstract] [Full Text] [PDF]
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M. Saethre, M. K. J. Schneider, J. D. Lambris, P. Magotti, G. Haraldsen, J. D. Seebach, and T. E. Mollnes Cytokine Secretion Depends on Gal{alpha}(1,3)Gal Expression in a Pig-to-Human Whole Blood Model J. Immunol., May 1, 2008; 180(9): 6346 - 6353. [Abstract] [Full Text] [PDF]
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 B. Salvesen, M. Fung, O. D. Saugstad, and T. E. Mollnes Role of Complement and CD14 in Meconium-Induced Cytokine Formation Pediatrics, March 1, 2008; 121(3): e496 - e505. [Abstract] [Full Text] [PDF]
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 S.-Y. Wang, E. Racila, R. P. Taylor, and G. J. Weiner NK-cell activation and antibody-dependent cellular cytotoxicity induced by rituximab-coated target cells is inhibited by the C3b component of complement Blood, February 1, 2008; 111(3): 1456 - 1463. [Abstract] [Full Text] [PDF]
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A. W. Pawluczkowycz, M. A. Lindorfer, J. N. Waitumbi, and R. P. Taylor Hematin Promotes Complement Alternative Pathway-Mediated Deposition of C3 Activation Fragments on Human Erythrocytes: Potential Implications for the Pathogenesis of Anemia in Malaria J. Immunol., October 15, 2007; 179(8): 5543 - 5552. [Abstract] [Full Text] [PDF]
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 I. Jongerius, J. Kohl, M. K. Pandey, M. Ruyken, K. P.M. van Kessel, J. A.G. van Strijp, and S. H.M. Rooijakkers Staphylococcal complement evasion by various convertase-blocking molecules J. Exp. Med., October 1, 2007; 204(10): 2461 - 2471. [Abstract] [Full Text] [PDF]
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 J. Lattin, D. A. Zidar, K. Schroder, S. Kellie, D. A. Hume, and M. J. Sweet G-protein-coupled receptor expression, function, and signaling in macrophages J. Leukoc. Biol., July 1, 2007; 82(1): 16 - 32. [Abstract] [Full Text] [PDF]
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O.-L. Brekke, D. Christiansen, H. Fure, M. Fung, and T. E. Mollnes The role of complement C3 opsonization, C5a receptor, and CD14 in E. coli-induced up-regulation of granulocyte and monocyte CD11b/CD18 (CR3), phagocytosis, and oxidative burst in human whole blood J. Leukoc. Biol., June 1, 2007; 81(6): 1404 - 1413. [Abstract] [Full Text] [PDF]
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 K. N. Ekdahl, D. Norberg, A. A. Bengtsson, G. Sturfelt, U. R. Nilsson, and B. Nilsson Use of Serum or Buffer-Changed EDTA-Plasma in a Rapid, Inexpensive, and Easy-To-Perform Hemolytic Complement Assay for Differential Diagnosis of Systemic Lupus Erythematosus and Monitoring of Patients with the Disease Clin. Vaccine Immunol., May 1, 2007; 14(5): 549 - 555. [Abstract] [Full Text] [PDF]
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C. D. Wrann, N. A. Tabriz, T. Barkhausen, A. Klos, M. van Griensven, H. C. Pape, D. O. Kendoff, R. Guo, P. A. Ward, C. Krettek, et al. The Phosphatidylinositol 3-Kinase Signaling Pathway Exerts Protective Effects during Sepsis by Controlling C5a-Mediated Activation of Innate Immune Functions J. Immunol., May 1, 2007; 178(9): 5940 - 5948. [Abstract] [Full Text] [PDF]
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 N. Di Simone, P.L. Meroni, M. D'Asta, F. Di Nicuolo, M.C. D'Alessio, and A. Caruso Pathogenic role of anti-{beta}2-glycoprotein I antibodies on human placenta: functional effects related to implantation and roles of heparin Hum. Reprod. Update, March 1, 2007; 13(2): 189 - 196. [Abstract] [Full Text] [PDF]
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S. von Gunten, A. Schaub, M. Vogel, B. M. Stadler, S. Miescher, and H.-U. Simon Immunologic and functional evidence for anti-Siglec-9 autoantibodies in intravenous immunoglobulin preparations Blood, December 15, 2006; 108(13): 4255 - 4259. [Abstract] [Full Text] [PDF]
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D. S. Daniel, G. Dai, C. R. Singh, D. R. Lindsey, A. K. Smith, S. Dhandayuthapani, R. L. Hunter Jr, and C. Jagannath The Reduced Bactericidal Function of Complement C5-Deficient Murine Macrophages Is Associated with Defects in the Synthesis and Delivery of Reactive Oxygen Radicals to Mycobacterial Phagosomes J. Immunol., October 1, 2006; 177(7): 4688 - 4698. [Abstract] [Full Text] [PDF]
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Y. Shi, Y. Tohyama, T. Kadono, J. He, S. M. Shahjahan Miah, R. Hazama, C. Tanaka, K. Tohyama, and H. Yamamura Protein-tyrosine kinase Syk is required for pathogen engulfment in complement-mediated phagocytosis Blood, June 1, 2006; 107(11): 4554 - 4562. [Abstract] [Full Text] [PDF]
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 A. E. Fiane, T. Ueland, S. Simonsen, H. Scott, K. Endresen, L. Gullestad, O. R. Geiran, G. Haraldsen, L. Heggelund, A. K. Andreassen, et al. Low mannose-binding lectin and increased complement activation correlate to allograft vasculopathy, ischaemia, and rejection after human heart transplantation Eur. Heart J., August 2, 2005; 26(16): 1660 - 1665. [Abstract] [Full Text] [PDF]
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A. G. Therien, R. Baelder, and J. Kohl Agonist Activity of the Small Molecule C3aR Ligand SB 290157 J. Immunol., June 15, 2005; 174(12): 7479 - 7480. [Full Text] [PDF]
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D. J. Allendorf, J. Yan, G. D. Ross, R. D. Hansen, J. T. Baran, K. Subbarao, L. Wang, and B. Haribabu C5a-Mediated Leukotriene B4-Amplified Neutrophil Chemotaxis Is Essential in Tumor Immunotherapy Facilitated by Anti-Tumor Monoclonal Antibody and {beta}-Glucan J. Immunol., June 1, 2005; 174(11): 7050 - 7056. [Abstract] [Full Text] [PDF]
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 K. T. Lappegard, J. Riesenfeld, O.-L. Brekke, G. Bergseth, J. D. Lambris, and T. E. Mollnes Differential Effect of Heparin Coating and Complement Inhibition on Artificial Surface-Induced Eicosanoid Production Ann. Thorac. Surg., March 1, 2005; 79(3): 917 - 923. [Abstract] [Full Text] [PDF]
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K. T. Lappegard, M. Fung, G. Bergseth, J. Riesenfeld, and T. E. Mollnes Artificial surface-induced cytokine synthesis: effect of heparin coating and complement inhibition Ann. Thorac. Surg., July 1, 2004; 78(1): 38 - 44. [Abstract] [Full Text] [PDF]
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T. Sprong, A.-S. W. Moller, A. Bjerre, E. Wedege, P. Kierulf, J. W. M. van der Meer, P. Brandtzaeg, M. van Deuren, and T. E. Mollnes Complement Activation and Complement-Dependent Inflammation by Neisseria meningitidis Are Independent of Lipopolysaccharide Infect. Immun., June 1, 2004; 72(6): 3344 - 3349. [Abstract] [Full Text] [PDF]
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H. Boshra, J. Li, R. Peters, J. Hansen, A. Matlapudi, and J. O. Sunyer Cloning, Expression, Cellular Distribution, and Role in Chemotaxis of a C5a Receptor in Rainbow Trout: The First Identification of a C5a Receptor in a Nonmammalian Species J. Immunol., April 1, 2004; 172(7): 4381 - 4390. [Abstract] [Full Text] [PDF]
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F. Lin, D. Spencer, D. A. Hatala, A. D. Levine, and M. E. Medof Decay-Accelerating Factor Deficiency Increases Susceptibility to Dextran Sulfate Sodium-Induced Colitis: Role for Complement in Inflammatory Bowel Disease J. Immunol., March 15, 2004; 172(6): 3836 - 3841. [Abstract] [Full Text] [PDF]
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S. Shankar-Sinha, G. A. Valencia, B. K. Janes, J. K. Rosenberg, C. Whitfield, R. A. Bender, T. J. Standiford, and J. G. Younger The Klebsiella pneumoniae O Antigen Contributes to Bacteremia and Lethality during Murine Pneumonia Infect. Immun., March 1, 2004; 72(3): 1423 - 1430. [Abstract] [Full Text] [PDF]
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K. T. Lappegard, M. Fung, G. Bergseth, J. Riesenfeld, J. D. Lambris, V. Videm, and T. E. Mollnes Effect of complement inhibition and heparin coating on artificial surface-induced leukocyte and platelet activation Ann. Thorac. Surg., March 1, 2004; 77(3): 932 - 941. [Abstract] [Full Text] [PDF]
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T. M. Woodruff, T. V. Arumugam, I. A. Shiels, R. C. Reid, D. P. Fairlie, and S. M. Taylor A Potent Human C5a Receptor Antagonist Protects against Disease Pathology in a Rat Model of Inflammatory Bowel Disease J. Immunol., November 15, 2003; 171(10): 5514 - 5520. [Abstract] [Full Text] [PDF]
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T. Sprong, P. Brandtzaeg, M. Fung, A. M. Pharo, E. A. Hoiby, T. E. Michaelsen, A. Aase, J. W. M. van der Meer, M. van Deuren, and T. E. Mollnes Inhibition of C5a-induced inflammation with preserved C5b-9-mediated bactericidal activity in a human whole blood model of meningococcal sepsis Blood, November 15, 2003; 102(10): 3702 - 3710. [Abstract] [Full Text] [PDF]
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M. Kirschfink and T. E. Mollnes Modern Complement Analysis Clin. Vaccine Immunol., November 1, 2003; 10(6): 982 - 989. [Full Text] [PDF]
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 A. E. Fiane, V. Videm, P. S. Lingaas, L. Heggelund, E. W. Nielsen, O. R. Geiran, M. Fung, and T. E. Mollnes Mechanism of Complement Activation and Its Role in the Inflammatory Response After Thoracoabdominal Aortic Aneurysm Repair Circulation, August 19, 2003; 108(7): 849 - 856. [Abstract] [Full Text] [PDF]
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Top
Abstract
Introduction
indicates anti-C5 (clone 137-30); 
,
isotype-matched control antibody (clone G3-519); 
, whole blood
incubated 10 minutes with E coli (1 × 108
bacteria per milliliter); and 
, baseline value (T0).
indicates baseline
value (T0); 
, whole blood incubated 10 minutes with E
coli (1 × 108 bacteria per milliliter), 
B (NF-
, TNF-