Clinical and Experimental Immunology OR I GI NA L ART IC LE doi:10.1111/cei.

12663

The key roles of complement and tissue factor in Escherichia

coli-induced coagulation in human whole blood

A. Landsem,* H. Fure,* Summary

D. Christiansen,* E. W. Nielsen,
B. sterud, T. E. Mollnes***
and O. L. Brekke*
*Research Laboratory and Department of
Laboratory Medicine, Nordland Hospital,
Bod, Norway, Institute of Clinical Medicine,
UiT The Arctic University of Norway, Troms,
Norway, Department of Anesthesiology,
Nordland Hospital and University of Nordland,
Norway, K. G. Jebsen TREC, Institute of
Medical Biology, Faculty of Health Sciences,
UiT The Arctic University of Norway, Troms,
Norway, K.G. Jebsen TREC, UiT The Arctic
University of Norway, Troms, Norway,
**Department of Immunology, Oslo University
Hospital Rikshospitalet and K.G. Jebsen IRC,
University of Oslo, Norway, and Centre of
Molecular Inflammation Research, Norwegian
University of Science and Technology,
Trondheim, Norway
Accepted for publication 5 June 2015
Correspondence: A. Landsem, Research
Laboratory, Nordland Hospital,
N-8092 Bod, Norway.
E-mail: anne.landsem@nlsh.no
The complement system and the Toll-like (TLR) co-receptor CD14 play
important roles in innate immunity and sepsis. Tissue factor (TF) is a key
initiating component in intravascular coagulation in sepsis, and long
pentraxin 3 (PTX3) enhances the lipopolysaccharide (LPS)-induced
transcription of TF. The aim of this study was to study the mechanism by
which complement and CD14 affects LPS- and Escherichia coli (E. coli)-
induced coagulation in human blood. Fresh whole blood was anti-
coagulated with lepirudin, and incubated with ultra-purified LPS (100
ng/ml) or with E. coli (1 3 107/ml). Inhibitors and controls included the C3
blocking peptide compstatin, an anti-CD14 F(ab0 )2 antibody and a control
F(ab0 )2. TF mRNA was measured using quantitative polymerase chain
reaction (qPCR) and monocyte TF surface expression by flow cytometry. TF
functional activity in plasma microparticles was measured using an
amidolytic assay. Prothrombin fragment F 112 (PTF1.2) and PTX3 were
measured by enzyme-linked immunosorbent assay (ELISA). The effect of TF
was examined using an anti-TF blocking antibody. E. coli increased plasma
PTF1.2 and PTX3 levels markedly. This increase was reduced by 84>99%
with compstatin, 5597% with anti-CD14 and > 99% with combined
inhibition (P < 005 for all). The combined inhibition was significantly (P <
005) more efficient than compstatin and anti-CD14 alone. The LPS- and E.
coliinduced TF mRNA levels, monocyte TF surface expression and TF
functional activity were reduced by > 99% (P < 005) with combined C3
and CD14 inhibition. LPS- and E. coliinduced PTF1.2 was reduced by 76
81% (P < 005) with anti-TF antibody. LPS and E. coli activated the
coagulation system by a complement- and CD14-dependent up-regulation
of TF, leading subsequently to prothrombin activation.
Keywords: CD14, coagulation, complement, Escherichia coli, lipopolysac-
charide, sepsis, whole blood

Introduction cytokines, including tumour necrosis factor (TNF) and

Sepsis is a life-threatening disease that affects 750 000 peo-
ple in the United States each year [1], and may be associ-
ated with disseminated intravascular coagulation (DIC),
multi-organ failure and death. Sepsis is a systemic inflam-
matory process that involves immunocompetent cells, the
complement system and coagulation.
Tissue factor (TF) is not usually expressed by cells that
contact the blood, and TF is normally not found in blood,
although very low TF levels have been detected in plasma
microparticles in healthy people [2]. Endotoxin and several
interleukin (IL)21b, activate monocytes and enhance TF
surface expression [3]. Long pentraxin 3 (PTX3) is an
acute-phase protein that enhances lipopolysaccharide
(LPS)-induced TF transcription [3,4]. Immunothrombosis
is caused by thrombocyte activation and thrombus forma-
tion initiated by immune competent cells after activation
by pathogens [5]. Thrombosis may limit inflammation and
the spread of pathogens. Immunothrombosis dysregulation
leads to disseminated intravascular coagulation (DIC) [5].
Warr et al. showed that enhanced TF surface expression is

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A. Landsem et al.
important in sepsis-induced DIC [6]. Therefore, TF surface
expression inhibition may reduce the occurrence of DIC
during sepsis.
LPS is the main cell wall component in Gram-negative
bacteria. LPS binds to pattern recognition receptors
(PRRs), including Toll-like receptor (TLR)-4 [7]. Myeloid
differentiation protein 2 (MD-2) binds to the extracellular
region of TLR-4 and is required for intracellular signalling
[8]. CD14 transports LPS from LPS binding protein (LBP)
to TLR-4 [9], and is also a co-receptor for TLR-1, -2, -3, -6,
-7 and -9 [10].
Complement is activated by LPS only at very high con-
centrations in human whole blood compared with whole
Escherichia coli (E.coli) bacteria [11]. E. coli bacteria acti-
vate both the classical and alternative pathways of comple-
ment [12]. We have shown previously that the E. coli-
induced TF expression in human whole blood monocytes
is mainly complement- and CD14-dependent [13]. Com-
plement and CD14 inhibition may therefore reduce the E.
coli-induced coagulation mediated by TF. The role of com-
plement and CD14 on pure LPS-induced TF has, however,
not been studied previously in the human whole blood
model using lepirudin as anti-coagulant.
Because complement and CD14 are key molecules and
co-operate in innate immunity [14,15], we hypothesized
previously that the combined upstream inhibition of com-
plement and CD14 may limit sepsis-induced inflammation
and coagulation [14]. Combined complement and CD14
inhibition inhibits E. coli-induced cytokine release effi-
ciently [16,17]. Other studies have shown that combined
inhibition also inhibits efficiently complement receptor 3
(CR3) up-regulation, phagocytosis and oxidative burst
[11,16]. The aim of this study was to examine the effect of
selective or combined inhibition of complement and CD14
in LPS- or E. coli-induced coagulation and PTX3 release in
fresh human whole blood.
Materials and methods
Whole blood experiments
Whole blood experiments were performed with blood from
10 donors, as described previously [12]. The study was
approved by the regional ethics committee in Northern
Norway Regional Health Authority. All equipment, tips
and solutions were endotoxin-free. Polypropylene tubes
(45 ml; Nunc, Roskilde, Denmark) with lepirudin
R
(RefludanV; Celgene, Uxbridge, UK; 50 mg/l) were used as
anti-coagulant. Fresh blood (five parts) was distributed
immediately into polypropylene tubes containing Dulbec-
cos phosphate-buffered saline (PBS, one part), inhibitors
or controls (one part). The samples were preincubated for
8 min at 378C. Immediately after the preincubation, PBS
with CaCl2 and MgCl2 (Sigma-Aldrich, St Louis, MO,
USA), heat-inactivated E. coli (strain LE392, ATCC 33572;
82 C 2015 British Society for Immunology, Clinical and Experimental Immunology, 182: 8189
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American Type Culture Collection, Manassas, VA, USA) or
ultra-pure LPS (100 ng/ml) from E. coli 0111 (LPS-EB
Ultrapure; InvivoGen, Eugene, OR, USA) was added. The
time zero (T0) sample was processed immediately after
blood sampling. After 2 h of incubation at 378C, the blood
was distributed into three different tubes. Citrate solution
[32%, 1 : 9 (v/v)] was added immediately to the tubes
before flow cytometric analysis. Ethylenediamine tetraace-
tic acid (EDTA, 10 mM) was added to the tubes for
enzyme-linked immunosorbent assay (ELISA) and mRNA
analysis. No additive was used in the tubes prior to TF
functional analysis in plasma microparticles. The tubes
were centrifuged for 15 min at 3220 g at 48C. The plasma
was stored at 2808C until it was analysed. The cell pellets
were lysed using 13 Nucleic Acid Purification Lysis Solu-
tion (Applied Biosystems, Warrington, UK), and the lysates
were stored at 2808C until mRNA analysis was performed.
Inhibitors and antibodies
Anti-CD14 F(ab0 )2 (LPS concentration < 39 EU/ml) was
obtained from Diatec Monoclonals (Oslo, Norway). The
C3 convertase inhibitor compstatin (lot CP20) and its cor-
responding control peptide, synthesized as described previ-
ously [18], was a kind gift from Professor John Lambris.
Compstatin was used at a final concentration of 20 mM.
The fluorescein isothiocyanate (FITC)-conjugated anti-
human TF antibody (product no. 4508CJ, clone VD8) was
obtained from American Diagnostica, Inc. (Stamford, CT,
USA). The isotype-matched control anti-HIV-1 gp120
(clone G3-519) was a kind gift from M. Fung (Tanox Inc.,
Houston, TX, USA). The monoclonal mouse immunoglob-
ulin (Ig)G1 blocking antibody (Sekisui 4509) against
human TF [a-TF monoclonal antibody (mAb)] was
obtained from American Diagnostica, Inc.
Enzyme-linked immunosorbent assays
Prothrombin fragment F 112 (PTF12) plasma levels were
R
measured using the EnzygnostV F1 1 2 (monoclonal) kit
(Dade Behring, Marburg GmbH, Germany). Human PTX3
was analysed using an ELISA kit from R&D Systems (Min-
neapolis, MN, USA). Soluble TCC levels (sC5b-9) were
measured using a mAb against a specific C9 neoepitope in
the TCC complex, as described previously [19]. An MRX
microplate reader (Dynex Technologies, Denkendorf, Ger-
many) was used to measure optical densities. Cytokines
were analysed using the Bio-Plex Human Cytokine 27-plex
cytokine multiplex panel from Bio-Rad Laboratories (Her-
cules, CA, USA).
Real-time-quantitative polymerase chain reaction
(RTqPCR) of tissue factor mRNA levels
Total RNA was isolated from cell lysates using total RNA
chemistry and the AB6100 nucleic acid prep station
(Applied Biosystems, Foster City, CA, USA). The RNA
 LPS- and E. coli-induced complement activation and coagulation
concentrations were analysed using a NanoDrop 2000c
(Thermo Fisher Scientific, Wilmington, DE, USA). cDNA
was synthesized from 50 ng of total RNA using a High
Capacity cDNA Reverse Transcription kit and a 2720
Thermal cycler (Applied Biosystems) and was stored at
2808C. The TF mRNA levels were measured using the
7500 Fast Real-Time PCR System (Applied Biosystems),
TaqMan Fast Universal PCR Master Mix reagents and
R
predeveloped TaqManV gene expression assays. TF
(Hs00175225_m1) was the target gene, and human beta-2-
microglobulin (B2M, assay ID 4326319E; Applied Biosys-
tems) was used as a reference gene. We used 3 ml cDNA for
RTqPCR, and the samples were analysed in triplicate. The
relative TF mRNA levels were measured using the compar-
ative delta-delta Ct method. The TF mRNA levels in the
samples after 2-h incubation with PBS only were set to 1
and used to calibrate the results.
Flow cytometric analysis of TF surface expression
Monocyte TF surface expression was analysed using a BD
LSR II flow cytometer (Becton Dickinson, San Jose, CA,
USA). Whole blood (125 ml) was stained with FITC-
conjugated anti-human TF (product no. 4508CJ, clone
VD8; American Diagnostica, Inc.) and phycoerythrin (PE)-
conjugated anti-CD14 (Becton Dickinson) antibodies.
IgG1 FITC (BD 345815) was used as an isotype-matched
control. The blood was incubated for 15 min at room tem-
perature in the dark. Easy lyse (S2364, Dako Cytomation,
Glostrup, Denmark) was added to the blood and incubated
for 15 min at room temperature to lyse the red blood cells.
PBS with 01% (w/v) bovine serum albumin was used to
wash and resuspend the leucocytes. Monocytes and granu-
locytes were gated in a PE/side-scatter (SSC) dot-plot. The
results are shown as the median fluorescent intensity
(MFI).
TF functional activity in plasma microparticles
TF functional activity in plasma microparticles was ana-
lysed as described previously by Engstad et al. [20].
Platelet-poor plasma was centrifuged at 40 000 g for 90
min (48C) to isolate the microparticles. Thereafter, 200 ml
of 015 M NaCl was used to resuspend the microparticles.
TF functional activity was measured in a two-stage amido-
lytic assay. The assay was based on the ability of TF to
accelerate the activation of FX by FVII. Further, FXa in the
presence of FVa activates prothrombin to form thrombin
[20].
Statistical analysis
GraphPad Prism version 60 from GraphPad Software (San
Diego, CA, USA) was used for the statistical analyses. Any
skewed results were transformed logarithmically. The
results were analysed using paired Students t-test between
baseline samples with PBS only and samples with LPS
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[panel (a) in the figures] or E. coli [panel (b) in the fig-
ures]. One-way repeated-measures analysis of variance
(ANOVA) with Dunnetts multiple comparisons test was used
to compare samples treated with inhibitors and their corre-
sponding LPS- or E. coli-positive controls. One-way
repeated-measures ANOVA with Tukeys multiple compari-
sons test was used to analyse differences between combined
and single inhibition with compstatin and anti-CD14. The
results were considered statistically significant when P
< 005. The background activation (negative control) was
subtracted from the positive control and the intervention
groups before calculating the percentage inhibition by the
different inhibitors.
Results
The effects of selective and combined complement
and CD14 inhibition on LPS- and E. coli-induced
PTF1.2 level
LPS- and E. coli-induced coagulation was assessed by meas-
uring PTF12 levels. Incubation with ultra-pure E. coli LPS
(100 ng/ml) for 2 h increased the PTF12 level by 86-fold,
from 065 nmol/l in the PBS control to 56 nmol/l (Fig.
1a). We then examined the role of complement by adding
the selective C3 convertase inhibitor compstatin. The role
of CD14 was evaluated by adding an anti-CD14 blocking
F(ab0 )2. The LPS-induced PTF12 levels were reduced effi-
ciently and significantly (P < 005) by selective comple-
ment and CD14 inhibition to 096 and 097 nmol/l,
respectively.
Incubation with E. coli (1 3 107/ml) for 2 h enhanced
PTF12 levels from 15 nmol/l in the PBS control to 120
nmol/l (Fig. 1b). Compstatin significantly (P < 005)
reduced PTF12 levels to 33 nmol/l, anti-CD14 signifi-
cantly (P < 005) reduced the PTF1.2 levels to 6.3 nmol/l
and combined complement and CD14 inhibition signifi-
cantly (P < 005) reduced PTF12 levels to 079 nmol/l.
The combined inhibition was significantly (P < 005) more
efficient than the inhibition with anti-CD14 and compsta-
tin alone.
The effect of selective and combined complement and
CD14 inhibition on LPS- and E. coli-induced TF
mRNA up-regulation
TF mRNA levels were measured by RTqPCR. The results
are given as the relative quantity (RQ) of mRNA to the
mRNA level in the control sample, which was incubated
with PBS only for 2 h and set to 1. LPS increased the TF
mRNA levels significantly (P < 005) by 59-fold. LPS-
induced TF mRNA up-regulation was reduced significantly
(P < 005) to 03 RQ by anti-CD14 and compstatin com-
bined (Fig. 2a).
 A. Landsem et al.
(a)
PTF1.2 (nmol/l)
Ctrl.
(b)
PTF1.2 (nmol/l)
Ctrl.
Fig. 1. The effect of selective complement and CD14 inhibition on
lipopolysaccharide (LPS)- and Escherichia coli (E.coli)-induced
coagulation, as assessed by measuring prothrombin fragment F 112
(PTF1.2) levels in the plasma. Fresh human whole blood was
incubated for 2 h with LPS (100 ng/ml) (a) or E. coli (1 3 107/ml)
(b) in the presence of phosphate-buffered saline (PBS), anti-CD14
F(ab0 )2 (aCD14, 10 mg/ml), compstatin (Comp., 20 mM), compstatin
and anti-CD14 F(ab)2 combined (Comp. 1 aCD14) or a control
peptide (Ctrl.). PTF1.2 levels were analysed using enzyme-linked
immunosorbent assay (ELISA) and are shown as nmol/l. The values
are given as the means with 95% confidence interval (CI) from
separate experiments with different blood donors (n56). #P < 005:
comparison between phosphate-buffered saline (PBS) alone and LPS
(a) or E. coli (b). * P < 005: comparison between LPS (a) or E. coli
(b) and the inhibitors/controls. P < 005: selective comparison
between single and combined inhibition with compstatin and anti-
CD-14. T0 represents sample obtained at time zero.
Incubation with E. coli increased the TF mRNA levels by
65-fold after 2 h of incubation (Fig. 2b). The combined
complement and CD14 inhibition was most efficient, reduc-
ing the TF mRNA level significantly (P < 005) to 09 RQ.
Compstatin reduced the E. coli-induced TF mRNA levels sig-
nificantly (P < 005) to 19 RQ. In comparison, anti-CD14
alone did not reduce E. coli-induced TF mRNA levels.
84 C 2015 British Society for Immunology, Clinical and Experimental Immunology, 182: 8189
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(a)
Ctrl.
(b)
Ctrl.
Fig. 2. The effect of selective complement and CD14 inhibition on
lipopolysaccharide (LPS)- and Escherichia coli (E.coli)-induced tissue
factor (TF) mRNA up-regulation. LPS (100 ng/ml) (a) or E. coli (1
3 107/ml) (b) was added to whole blood and incubated for 2 h. The
inhibitors and controls that were added are described in the legend
for Fig. 1. TF mRNA was measured using real-timequantitative
polymerase chain reaction (RTqPCR). TF mRNA levels in the
samples without stimulus and inhibitor [phosphate-buffered saline
(PBS) control] were set to 1 and used to calibrate the assay. The
results are given as the relative quantity (RQ) to the calibrator. The
values are given as the means with 95% confidence interval (CI)
from separate experiments with different blood donors (n 5 6).
#
P < 005: comparison between PBS alone and with LPS (a) or
E. coli, (b). *P < 005: comparison between LPS (a) or E. coli (b)
and the inhibitors/controls. P < 005: selective comparison between
single and combined inhibition with compstatin and anti-CD14.
The effect of selective and combined complement and
CD14 inhibition on LPS- and E. coli-induced
monocyte TF surface expression
Whole blood monocyte TF surface expression was meas-
ured by flow cytometry (Fig. 3). Incubation with LPS
increased TF surface expression by 31-fold, from 88 MFI
in the PBS control to 275 MFI after 2 h of incubation (Fig.
 LPS- and E. coli-induced complement activation and coagulation
(a)
Monocyte TF (MFI)
Ctrl.
(b)
Monocyte TF (MFI)
Ctrl.
(c)
3
Tissue factor (MFI)
Fig. 3. The effect of selective complement and CD14 inhibition on
lipopolysaccharide (LPS)- and Escherichia coli (E.coli)-induced
monocyte tissue factor (TF) surface expression. Fresh human whole
blood was incubated for 2 h with LPS (100 ng/ml) (a) or E. coli (1
3 107/ml) (b). The inhibitors and controls were added as described
in the legend for Fig. 1. Monocyte tissue factor (TF) surface
expression was analysed using flow cytometry, and the results are
expressed as the median fluorescence intensity (MFI). The values are
C 2015 British Society for Immunology, Clinical and Experimental Immunology, 182: 8189
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3a). The combined inhibition with compstatin and anti-
CD14 was most efficient, significantly (P < 005) reducing
LPS-induced TF surface expression to 76 MFI. Both anti-
CD14 and compstatin alone significantly (P < 005)
reduced the LPS-induced TF surface expression to 110 and
151 MFI, respectively.
Incubation with E. coli significantly (P < 005) increased
the TF surface expression by 37-fold, from 309 MFI in the
PBS control to 1152 MFI after 2 h of incubation (Fig. 3b).
Consistent with previous findings [13], compstatin and
anti-CD14 combined were most efficient, significantly (P
< 005) reducing E. coli-induced TF surface expression to
157 MFI. Compstatin alone significantly (P < 005)
reduced TF surface expression to 495 MFI, and anti-CD14
significantly (P < 005) reduced TF expression to 614 MFI.
An example of the histogram from the flow cytometric
analysis of the TF surface expression is shown in Fig. 3.
The effect of selective and combined complement and
CD14 inhibition on LPS- and E. coli-induced TF
functional activity in plasma microparticles
Incubation with LPS (100 ng/ml) for 2 h significantly (P <
005) increased the TF functional activity in plasma micro-
particles by 65-fold, from 006 to 039 mU/ml (Fig. 4a). The
combined inhibition with compstatin and anti-CD14 signifi-
cantly (P < 005) reduced LPS-induced TF functional activ-
ity to 002 mU/ml compared to compstatin alone.
Incubation with E. coli (1 3 107/ml) for 2 h significantly
(P < 005) increased TF functional activity in plasma
microparticles by 62-fold, from 013 to 081 mU/ml (Fig.
4b). Consistent with previous findings [13], combined
compstatin and anti-CD14 significantly (P < 005) reduced
the functional activity to 006 mU/ml. The combined inhi-
bition was significantly (P < 005) more efficient than inhi-
bition with anti-CD14 and compstatin alone.
The effect of an anti-TF blocking antibody
on LPS-and E. coli-induced coagulation
Incubation with LPS (100 ng/ml) for 2 h increased PTF12
levels by 23-fold, from 061 in the PBS control to 138
nmol/l (Fig. 5a). The anti-TF blocking mAb significantly
(P < 005) reduced PTF12 levels to 31 nmol/l, whereas
the control mAb did not reduce PTF12 levels.
given as the means with 95% confidence interval (CI) from separate
experiments with different blood donors (n 5 6). #P < 005:
comparison between phosphate-buffered saline (PBS) alone and LPS
(a) or E. coli (b). *P < 005: comparison between with LPS (a) or E.
coli (b) and the inhibitors/controls. P < 0.05: selective comparison
between single and combined inhibition with compstatin and anti-
CD14. (c) Histogram of TF surface expression on monocytes in one
of six donors analysed using flow cytometry after 2 h incubation
with PBS (light grey), E. coli (medium grey), E. coli plus compstatin
and anti-CD14F(ab0 )2 combined (dark grey).
 A. Landsem et al.
(a)
TF activity (mU/ml)
Ctrl.
(b)
TF activity (mU/ml)
Ctrl.
Fig. 4. The effect of selective complement and CD14 inhibition on
lipopolysaccharide (LPS)- and Escherichia coli (E.coli)-induced tissue
factor (TF) functional activity in plasma microparticles. Fresh human
whole blood was incubated for 2 h with LPS (100 ng/ml) (a) or E. coli
(1 3 107/ml) (b). The inhibitors and controls that were added are
described in the legend for Fig. 1. Plasma microparticles were isolated
using high-speed centrifugation, and TF functional activity was
analysed using a amidolytic assay and expressed as mU/ml. The values
are given as the means with 95% confidence interval (CI) from
separate experiments with different blood donors (n 5 6). #P < 005:
comparison between phosphate-buffered saline (PBS) alone and LPS
(a) or E. coli (b). *P < 005: comparison between LPS (a) or E. coli (b)
and the inhibitors/controls. P < 005: selective comparison between
single and combined inhibition with compstatin and anti-CD14.
Incubation with E. coli (1 3 107/ml) increased PTF12
levels by 23-fold, from 083 nmol/l in the PBS control to
187 nmol/l (Fig. 5b). Again, the anti-TF blocking mAb
reduced PTF12 levels significantly (P < 005) to 52 nmol/l.
The effect of selective and combined complement and
CD14 inhibition on LPS- and E. coli-induced PTX3
levels
Next, we examined the effect of compstatin and anti-CD14
on LPS- and E. coli-induced PTX3 release (Fig. 6). Incubation
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(a)
PTF1.2 (nmol/l)
Ctrl.
(b)
PTF1.2 (nmol/l)
Ctrl.
Fig. 5. The effect of an anti- tissue factor (TF) blocking antibody on
lipopolysaccharide (LPS)- and Escherichia coli (E.coli)-induced
coagulation, as assessed by measuring prothrombin fragment F 112
(PTF1.2) levels. LPS (100 ng/ml) (a) or E. coli (1 3 107/ml) (b)
were added to human whole blood and incubated for 2 h.
Phosphate-buffered saline (PBS), anti-TF (aTF, 5 mg/ml) or an
isotype control antibody (Ctrl., 5 mg/ml) were added before
stimulation with LPS or E. coli. Plasma PTF1.2 levels were measured
by enzyme-linked immunosorbent assay (ELISA) and expressed as
nmol/l. The values are given as the means with 95% CI from
separate experiments with different blood donors (n 5 7). #P < 005:
comparison between PBS alone and LPS (a) or E. coli (b). *P <
005: comparison between LPS (a) or E. coli (b) and anti-TF or the
isotype control antibody.
with LPS for 2 h increased PTX3 levels by 22-fold, from 34
in the PBS control to 75 ng/ml (Fig. 6a). Anti-CD14 and
compstatin alone reduced LPS-induced PTX3 release signifi-
cantly (P < 005) to 40 and 32 ng/ml, respectively. Com-
bined anti-CD14 and compstatin reduced LPS-induced
PTX3 levels significantly (P < 005) to 13 ng/ml. The com-
bined inhibition was significantly (P < 005) more efficient
than the inhibition with anti-CD14 and compstatin alone.
Incubation with E. coli (1 3 107/ml) for 2 h enhanced
PTX3 levels significantly (P < 005) by 17-fold, from 40
ng/ml in the PBS control to 68 ng/ml (Fig. 6b). Anti-CD14
 LPS- and E. coli-induced complement activation and coagulation
(a)
PTX3 (ng/ml)
Ctrl.
(b)
PTX3 (ng/ml)
Fig. 6. The effect of selective complement and CD14 inhibition on
lipopolysaccharide (LPS)- and Escherichia coli (E.coli)-induced long
pentraxin 3 (PTX3) release. Fresh human whole blood was incubated
for 2 h with LPS (100 ng/ml) (a) or E. coli (1 3 107/ml) (b). The
inhibitor and control concentrations are indicated in the legend of Fig.
1. Plasma PTX3 levels were measured by enzyme-linked
immunosorbent assay (ELISA) and expressed as ng/ml. The values are
given as the means with 95% confidence interval (CI) from separate
experiments with different blood donors (n 5 6). #P < 005:
comparison between phosphate-buffered saline (PBS) alone and LPS
(a) or E. coli (b). *P < 005: comparison between LPS (a) or E. coli (b)
and the inhibitors/controls. P < 005: selective comparison between
single and combined inhibition with compstatin and anti-CD14.
and compstatin alone reduced PTX3 levels significantly
(P < 005) to 41 and 35 ng/ml, respectively. The combined
inhibition with compstatin and anti-CD14 reduced PTX3
levels significantly (P < 005) to 15 ng/ml. The combined
inhibition was significantly (P < 005) more efficient than
the inhibition with anti-CD14 and compstatin alone.
Discussion
In this study we show for the first time, to our knowledge,
that combined complement and CD14 inhibition inhibited
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LPS- and E. coli-induced coagulation efficiently, as assessed
by measuring PTF12 levels in human whole blood. Com-
bined complement and CD14 inhibition efficiently inhib-
ited LPS-induced TF mRNA levels, monocyte TF surface
expression and TF functional activity in plasma micropar-
ticles. Consistent with previous data using other E. coli
concentrations [13], we confirmed that this was also the
case for E. coli-induced TF induction. Most importantly,
we documented that a blocking mAb against TF nearly
abolished both the LPS and E. coli-induced increase in
PTF12 level, indicating that prothrombin activation is TF-
dependent and that the upstream activation was largely
complement- and CD14-mediated. These findings might
have important implications for the treatment of DIC in
sepsis.
The significant effect of the C3 convertase inhibitor
compstatin on LPS- and E. coli-induced coagulation indi-
cates a key role for complement activation in coagulation.
It is well known that E. coli bacteria activate both the classi-
cal and alternative complement pathways [12]. E. coli bac-
teria phagocytosis can be reduced significantly by
complement inhibition at the C3 level [12]. Reduced phag-
ocytosis of E. coli due to compstatin most probably reduces
immune stimulation and cytokine release in whole blood
monocytes and other phagocytes [11,16]. Additionally,
reduced E. coli-induced C5a formation and cytokine release
by compstatin could explain why complement inhibition
was more effective than selective CD14 inhibition at reduc-
ing E. coli-induced coagulation [21]. Our results further
indicate that complement activation is involved early on in
TF-mediated LPS- and E. coli-induced coagulation.
Although previous studies on E. coli-induced complement
activation detected TCC after 10 min of incubation [12],
monocyte TF surface expression does not appear for 23 h
[13]. Therefore, we speculate that the initial complement
activation up-regulates TF mRNA, followed by enhanced
monocyte TF surface expression and subsequent coagula-
tion. Although LPS added to whole blood only modestly
activates complement [11], complement inhibition with
compstatin reduced LPS-induced coagulation efficiently.
Thus, it is tempting to speculate that blocking even a low
degree of LPS-induced complement activation is sufficient
to attenuate TF expression.
CD14 is especially important for LPS-induced inflam-
mation [22]. Therefore, the impressive effect of anti-CD14
on LPS-induced coagulation and TF expression was
expected. The CD14 antibody hinders LPSLBP complex
binding to and activation of the TLR-4/CD14/MD2 com-
plex [23]. However, anti-CD14 had only a minor effect on
E. coli-induced coagulation. Hellerud et al. demonstrated
that by increasing the concentration of an LPS-deficient
Neisseria meningitidis strain 10-fold, the inflammatory
response was the same as for the wild-type, LPS-containing
bacteria. Clearly, molecules on Gram-negative bacteria
A. Landsem et al.
other than LPS are able to activate the inflammatory
responses and coagulation [24].
LPS- and E. coli-induced PTX3 release was reduced sig-
nificantly by both complement- and CD14 inhibition alone
and in combination. PTX3 has a number of functions in
innate immunity in general, and in complement in particu-
lar [25]. On one hand, high PTX3 levels during sepsis are
associated with mortality [26]. PTX3 levels are increased by
certain cytokines, including TNF and pathogen-associated
molecular patterns (PAMPs) such as LPS and microorgan-
isms [27]. Interestingly, PTX3 also enhances TF surface
expression [28], binds to antigens or other activating surfa-
ces and activates both the classical and lectin complement
pathways [27]. On the other hand, PTX3 seems to be pro-
tective, indicated by LPS-induced inflammation in PTX3
knock-out mice [29], as a previous study indicated that the
interaction between PTX3 and P-selectin might reduce leu-
cocyte recruitment to the inflammatory site, leading to
reduced local inflammation [30], and that between PTX3
and complement factor H may inhibit the amplification
loop to the alternative pathway [27]. Therefore, PTX3 may
both enhance and reduce complement activation. Because
PTX3 release in this study was partly complement-
dependent, we speculate that a loop with a reciprocal effect
exists between complement and PTX3. Notably, CD14
inhibition also attenuated PTX3 release induced both by
LPS and E. coli. Finally, the effective reduction of PTX3
release by combined complement and CD14 inhibition
indicates a synergistic effect of complement and CD14
(and TLRs) on PTX3 release, which might have implica-
tions for the dual therapeutic regimen.
A limitation of the whole blood model is that lepirudin
prevents thrombin activation and effects of thrombin can
therefore not be evaluated. Thrombin is involved in both
the coagulation and complement cascades and participates
in the amplification aspect of coagulation. Huber-Lang
et al. demonstrated that thrombin may cleave C5 directly
[31]. Compstatin inhibits at the C3-level and would there-
fore not interfere with a direct thrombin-mediated C5 acti-
vation. However, whole blood experiments cannot be
performed without an anti-coagulant. Lepirudin has the
advantage of not affecting complement activation; citrate,
heparin and EDTA cannot be used because these anti-
coagulants affect complement activation [12]. Thus,
although the model does not include endothelial cells, it is
the closest possible to physiological conditions at present.
The fact that prothrombin was activated implies a subse-
quent activation of thrombin, and therefore our data sup-
port a strong indication that thrombin would have been
activated and lead to a fulminant coagulation in the
absence of lepirudin.
In summary, combined complement and CD14 inhibi-
tion was most effective for all of the parameters studied in
this model of LPS- and E.coli-induced coagulation in
human whole blood. The results indicate a very early and
88 C 2015 British Society for Immunology, Clinical and Experimental Immunology, 182: 8189
V
pivotal co-operation between complement and CD14/TLRs
in immunothrombosis. Combined inhibition of comple-
ment and CD14 is suggested to prevent TF up-regulation
and the subsequent prothrombin activation and coagula-
tion leading to DIC in sepsis.
Acknowledgements
This study was supported by grants from The Northern
Norway Regional Health Authority and the Somatic
research fund at Nordland Hospital, Bod, Norway, The
Research Council of Norway, The Norwegian Council on
Cardiovascular Disease, The Southern and Eastern Norway
Regional Health Authority, Odd Fellow Foundation and the
European Communitys Seventh Framework Programme
under grant agreement no. 602699 (DIREKT).
Disclosure
The authors declare that they have no competing interests.
References
1 Skrupky LP, Kerby PW, Hotchkiss RS. Advances in the manage-
ment of sepsis and the understanding of key immunologic
defects. Anesthesiology 2011; 115:134962.
2 Mackman N. The many faces of tissue factor. J Thromb Hae-
most 2009; 7 (Suppl. 1):1369.
3 Napoleone E, di Santo A, Peri G et al. The long pentraxin
PTX3 up-regulates tissue factor in activated monocytes: another
link between inflammation and clotting activation. J Leukoc
Biol 2004; 76:2039.
4 Mantovani A, Garlanda C, Doni A, Bottazzi B. Pentraxins in
innate immunity: from C-reactive protein to the long pentraxin
PTX3. J Clin Immunol 2008; 28:113.
5 Engelmann B, Massberg S. Thrombosis as an intravascular effec-
tor of innate immunity. Nat Rev Immunol 2013; 13:3445.
6 Warr TA, Rao LV, Rapaport SI. Disseminated intravascular coag-
ulation in rabbits induced by administration of endotoxin or
tissue factor: effect of anti-tissue factor antibodies and measure-
ment of plasma extrinsic pathway inhibitor activity. Blood 1990;
75:14819.
7 Chow JC, Young DW, Golenbock DT, Christ WJ, Gusovsky F.
Toll-like receptor-4 mediates lipopolysaccharide-induced signal
transduction. J Biol Chem 1999; 274:1068992.
8 Shimazu R, Akashi S, Ogata H et al. MD-2, a molecule that
confers lipopolysaccharide responsiveness on Toll-like receptor
4. J Exp Med 1999; 189:177782.
9 Ohto U, Fukase K, Miyake K, Satow Y. Crystal structures of
human MD-2 and its complex with antiendotoxic lipid IVa. Sci-
ence 2007; 316:16324.
10 Zanoni I, Granucci F. Role of CD14 in host protection against
infections and in metabolism regulation. Front Cell Infect
Microbiol 2013; 3:32.
11 Brekke OL, Christiansen D, Fure H, Fung M, Mollnes TE. 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 2007; 81:140413.
 LPS- and E. coli-induced complement activation and coagulation
12 Mollnes TE, Brekke OL, Fung M et al. Essential role of the C5a
receptor in E. coli-induced oxidative burst and phagocytosis
revealed by a novel lepirudin-based human whole blood model
of inflammation. Blood 2002; 100:186977.
13 Brekke OL, Waage C, Christiansen D et al. The effects of selec-
tive complement and CD14 inhibition on the E. coli-induced
tissue factor mRNA upregulation, monocyte tissue factor
expression, and tissue factor functional activity in human whole
blood. Adv Exp Med Biol 2013; 735:12336.
14 Mollnes TE, Christiansen D, Brekke OL, Espevik T. Hypothesis:
combined inhibition of complement and CD14 as treatment
regimen to attenuate the inflammatory response. Adv Exp Med
Biol 2008; 632:25363.
15 Barratt-Due A, Pischke SE, Brekke OL et al. Bride and groom
in systemic inflammation the bells ring for complement and
Toll in cooperation. Immunobiology 2012; 217:104756.
16 Brekke OL, Christiansen D, Fure H et al. Combined inhibition
of complement and CD14 abolish E. coli-induced cytokine-,
chemokine- and growth factor-synthesis in human whole blood.
Mol Immunol 2008; 45:380413.
17 Lappegard KT, Riesenfeld J, Brekke OL, Bergseth G, Lambris JD,
Mollnes TE. Differential effect of heparin coating and comple-
ment inhibition on artificial surface-induced eicosanoid produc-
tion. Ann Thorac Surg 2005; 79:91723.
18 Morikis D, Assa-Munt N, Sahu A, Lambris JD. Solution struc-
ture of Compstatin, a potent complement inhibitor. Protein Sci
1998; 7:61927.
19 Mollnes TE, Redl H, Hogasen K et al. Complement activation
in septic baboons detected by neoepitope-specific assays for
C3b/iC3b/C3c, C5a and the terminal C5b-9 complement com-
plex (TCC). Clin Exp Immunol 1993; 91:295300.
20 Engstad CS, Lia K, Rekdal O, Olsen JO, Osterud B. A novel biolog-
ical effect of platelet factor 4 (PF4): enhancement of LPS-induced
tissue factor activity in monocytes. J Leukoc Biol 1995; 58:57581.
21 Lappegard KT, Christiansen D, Pharo A et al. Human genetic
deficiencies reveal the roles of complement in the inflammatory
C 2015 British Society for Immunology, Clinical and Experimental Immunology, 182: 8189
V 89
network: lessons from nature. Proc Natl Acad Sci USA 2009;
106:158616.
22 Verbon A, Dekkers PE, Ten HT et al. IC14, an anti-CD14 anti-
body, inhibits endotoxin-mediated symptoms and inflammatory
responses in humans. J Immunol 2001; 166:3599605.
23 Weinstein SL, June CH, DeFranco AL. Lipopolysaccharide-
induced protein tyrosine phosphorylation in human macro-
phages is mediated by CD14. J Immunol 1993; 151:382938.
24 Hellerud BC, Stenvik J, Espevik T, Lambris JD, Mollnes TE,
Brandtzaeg P. Stages of meningococcal sepsis simulated in vitro,
with emphasis on complement and Toll-like receptor activation.
Infect Immun 2008; 76:41839.
25 Jaillon S, Bonavita E, Gentile S et al. The long pentraxin PTX3
as a key component of humoral innate immunity and a candi-
date diagnostic for inflammatory diseases. Int Arch Allergy
Immunol 2014; 165:16578.
26 Huttunen R, Hurme M, Aittoniemi J et al. High plasma level of
long pentraxin 3 (PTX3) is associated with fatal disease in bac-
teremic patients: a prospective cohort study. PLOS ONE 2011;
6:e17653.
27 Inforzato A, Doni A, Barajon I et al. PTX3 as a paradigm for
the interaction of pentraxins with the complement system.
Semin Immunol 2013; 25:7985.
28 Napoleone E, di Santo A, Bastone A et al. Long pentraxin PTX3
upregulates tissue factor expression in human endothelial cells:
a novel link between vascular inflammation and clotting activa-
tion. Arterioscler Thromb Vasc Biol 2002; 22:7827.
29 Han B, Haitsma JJ, Zhang Y et al. Long pentraxin PTX3 defi-
ciency worsens LPS-induced acute lung injury. Intens Care Med
2011; 37:33442.
30 Deban L, Russo RC, Sironi M et al. Regulation of leukocyte
recruitment by the long pentraxin PTX3. Nat Immunol 2010;
11:32834.
31 Huber-Lang M, Sarma JV, Zetoune FS et al. Generation of C5a
in the absence of C3: a new complement activation pathway.
Nat Med 2006; 12:6827.