Download Response during Sepsis Activation Pathway for the Innate Host

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Distinct Different Contributions of the
Alternative and Classical Complement
Activation Pathway for the Innate Host
Response during Sepsis
Katja Dahlke, Christiane D. Wrann, Oliver Sommerfeld,
Maik Soßdorf, Peter Recknagel, Svea Sachse, Sebastian W.
Winter, Andreas Klos, Gregory L. Stahl, Yuanyuan Xu Ma,
Ralf A. Claus, Konrad Reinhart, Michael Bauer and Niels C.
Riedemann
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Print ISSN: 0022-1767 Online ISSN: 1550-6606.
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J Immunol published online 24 January 2011
http://www.jimmunol.org/content/early/2011/01/23/jimmun
ol.1002741
Published January 24, 2011, doi:10.4049/jimmunol.1002741
The Journal of Immunology
Distinct Different Contributions of the Alternative and
Classical Complement Activation Pathway for the Innate
Host Response during Sepsis
Katja Dahlke,* Christiane D. Wrann,† Oliver Sommerfeld,* Maik Soßdorf,*
Peter Recknagel,* Svea Sachse,‡ Sebastian W. Winter,† Andreas Klos,x Gregory L. Stahl,{
Yuanyuan Xu Ma,‖ Ralf A. Claus,* Konrad Reinhart,* Michael Bauer,*
and Niels C. Riedemann*
D
uring acute inflammation, the complement system can be
activated by at least three well-known pathways: the
classical pathway, the lectin or mannose-binding lectin
(MBL) pathway, and the alternative pathway (1). The classical
pathway is activated, for example, by Ag–Ab complexes that react
with activated C1q. The lectin pathway is initiated by either serum
MBL or ficolins that recognize certain oligosaccharide moieties on
microbial surfaces. The alternative pathway can be activated either
by the presence of foreign surfaces such as LPSs or through C3b
*Department of Anesthesiology and Intensive Care Therapy, Jena University Hospital, Friedrich Schiller University Jena, 07747 Jena, Germany; †Institute for Functional and Applied Anatomy, Hannover Medical School, 30625 Hannover, Germany;
‡
Department of Medical Microbiology, Jena University Hospital, Friedrich Schiller
University Jena, 07747 Jena, Germany; xDepartment of Medical Microbiology, Hannover Medical School, 30625 Hannover, Germany; {Center for Experimental Therapeutics and Reperfusions Injury, Anesthesiology, Perioperative Medicine and Pain
Medicine, Brigham and Woman’s Hospital, Harvard Medical School, Boston, MA
02115; and ‖Division of Clinical Immunology and Rheumatology, Department of
Medicine, University of Alabama, Birmingham, AL 35294
Received for publication August 13, 2010. Accepted for publication December 14,
2010.
Address correspondence and reprint requests to Dr. Niels C. Riedemann, Department
of Anesthesiology and Intensive Care Therapy, Jena University Hospital, Friedrich
Schiller University Jena, Erlanger Allee 101, 07747 Jena, Germany. E-mail address:
[email protected]
Abbreviations used in this article: CBA, cytometric bead assay; CLP, cecal ligation
and puncture; GOT/AST, glutamate oxaloacetate transaminase/aspartate transaminase; LDH, lactate dehydrogenase; MAC, membrane attack complex; MBL,
mannose-binding lectin; MPO, myeloperoxidase; PAMP, pathogen-associated molecular pattern.
Copyright Ó 2011 by The American Association of Immunologists, Inc. 0022-1767/11/$16.00
www.jimmunol.org/cgi/doi/10.4049/jimmunol.1002741
generated by spontaneous hydrolyses, the so-called “tick-over”
(2). All three pathways merge at the level of C3, and activation
of either pathway ultimately results in generation of the potent
proinflammatory complement split products C3a and C5a as well
as the terminal membrane attack complex (MAC) (3). Excessive
generation of C5a during the onset of sepsis causes various
harmful effects mediated by C5aR (1, 4, 5), and blockade of either
C5a or C5aR leads to greatly improved survival of rodents in
experimental sepsis (6, 7). C5a has been shown to alter innate
immune functions, such as generation of inflammatory mediators
as well as neutrophil functions, leading to a status of immune
suppression (8). We recently demonstrated that C5a alters intracellular signaling pathways in neutrophils in vitro and during
the onset of sepsis in vivo (9–12), offering an explanation for the
above-mentioned suppression of innate immune functions.
Studies employing different complement knockout mice in a
model of streptococcal pneumonia first suggested that the classical activation pathway appeared to be dominant for clearance
of these bacteria (13). However, little is known about the contribution of the separate complement pathways during the onset
phase of sepsis. Initial experimental infection studies with knockout mice lacking all three complement pathways demonstrated
the importance of an intact complement activation system for
successful survival, and results with C1q knockout mice showed
involvement of the classical pathway (14, 15). Although
both reports suggested importance of the alternative and lectin
pathways, the individual contribution has not been dissected so
far. We therefore conducted cecal ligation and puncture (CLP)
studies with C1q2/2 mice, lacking the classical pathway, and
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Complement activation represents a crucial innate defense mechanism to invading microorganisms, but there is an eminent lack of
understanding of the separate contribution of the different complement activation pathways to the host response during sepsis. We
therefore investigated different innate host immune responses during cecal ligation and puncture (CLP)-induced sepsis in mice
lacking either the alternative (fD2/2) or classical (C1q2/2) complement activation pathway. Both knockout mice strains showed
a significantly reduced survival and increased organ dysfunction when compared with control mice. Surprisingly, fD2/2 mice
demonstrated a compensated bacterial clearance capacity as control mice at 6 h post CLP, whereas C1q2/2 mice were already
overwhelmed by bacterial growth at this time point. Interestingly, at 24 h after CLP, fD2/2 mice failed to clear bacteria in a way
comparable to control mice. However, both knockout mice strains showed compromised C3 cleavage during sepsis. Investigating
potential causes for this discrepancy, we were able to demonstrate that despite normal bacterial clearance capacity early during
the onset of sepsis, fD2/2 mice displayed increased inflammatory cytokine generation and neutrophil recruitment into lungs and
blood when compared with both control- and C1q2/2 mice, indicating a potential loss of control over these immune responses.
Further in vitro experiments revealed a strongly increased Nf-kB activation capacity in isolated neutrophils from fD2/2 mice,
supporting this hypothesis. Our results provide evidence for the new concept that the alternative complement activation
pathway exerts a distinctly different contribution to the innate host response during sepsis when compared with the classical
pathway. The Journal of Immunology, 2011, 186: 000–000.
2
CONTRIBUTION OF COMPLEMENT PATHWAYS IN SEPSIS
fD2/2 mice, lacking the alternative pathway, and investigated the
effect of impaired activation of these complement pathways on
outcome and innate immune responses in experimental sepsis.
after CLP and analyzed for urea, bilirubin, glutamate oxaloacetate
transaminase/aspartate transaminase (GOT/AST), and lactate dehydrogenase (LDH) using an automated clinical chemistry analyzer (Fuji DriChem 3500i; Sysmex).
Materials and Methods
Bacterial load in organs from CLP animals
Reagents
Six hours and 24 h after CLP blood, lung, liver, and kidneys were isolated
from wild-type, fD2/2, and C1q2/2 mice in a sterile manner. Then, the
organs were washed in a 70% ethanol bath for 10 s to avoid surface
bacterial contamination. Each organ was manually homogenized in 3 ml
sterile 0.9% NaCl. Then, 2 ml enriched brain-heart infusion was added.
Samples were plated on sheep blood agar or anaerobic blood agar and
incubated for 48 h at 37˚C under aerobic and anaerobic conditions. CFU
were then determined in all plates and multiplied by the dilution factor.
Data are presented as CFU per 50 ml nondiluted blood or CFU per 200 mg
nondiluted tissue homogenate.
All reagents were purchased from Sigma-Aldrich Chemie (Taufkirchen,
Germany) unless otherwise indicated.
CLP procedure in mice
Collection of plasma samples in mice
After induction of CLP, animals were sacrificed at the indicated time points
and blood was drawn using direct needle puncture of the heart. Blood
samples were analyzed immediately after collection by automated veterinary hematology analyzer (PocH-100iV Diff; Sysmex, Leipzig, Germany).
For plasma collection, the samples were stored at 4˚C, centrifuged at
4700 3 g for 10 min at 4˚C, and the plasma was collected and immediately
snap-frozen in liquid nitrogen and stored at 280˚C until used for further
analyses. For both plasma collection and whole blood analyses, EDTA or
heparin was used as anticoagulant as warranted for the particular test.
C5a ELISA
The C5a concentration in various plasma samples was analyzed by the
commercially available ELISA mouse complement component C5a DuoSet
according to the manufacturer’s instructions (R&D Systems, Minneapolis,
MN).
Quantitation of phagocytosis in whole blood cells
To determine the ability of blood granulocytes and monocytes to conduct phagocytosis, flow cytometric-based assay was used according to
the manufacturer’s instructions (Phagotest; Orpegen Pharma, Heidelberg,
Germany). Mouse whole blood samples were incubated with FITC-labeled
Escherichia coli bacteria (1.7 3 109 bacteria/ml) for 20 min in a 37˚C
warm water bath. Leukocyte surface-bound bacteria were neutralized using
quenching solution. Cells were analyzed in a FACSCalibur flow cytometer
(BD Biosciences, San Jose, CA). In a forward/side scatter dot plot, gates
were set on granulocytes and monocytes to analyze each population with
regard to their mean fluorescence intensity.
Quantitation of myeloperoxidase in the lung
Immediately after sacrificing the animals, organs were flushed with sterile
physiological sodium chloride solution retrograde through the caudal caval
vein, and the organs were harvested and snap-frozen in liquid nitrogen.
Tissue homogenates were prepared according to the manufacturer’s instructions and the myeloperoxidase (MPO) concentration was analyzed by
a commercially available ELISA (Hycult Biotechnology, Uden, Netherlands). The protein content of the homogenates was determined with
Bradford’s method.
Quantitation of MPO in plasma samples
Plasma samples were collected as outlined above. The MPO concentration
in various plasma samples was analyzed by a commercially available
ELISA according to the manufacturer’s instructions (Hycult Biotechnology).
Immunohistochemical staining for C3/C3b/C3c deposition in
kidney sections
Determination of histological organ damage during sepsis
in vivo
Immediately after sacrificing an animal, the organs were flushed with sterile
physiological sodium chloride solution retrograde through the caudal caval
vein, and the organs were harvested and snap-frozen in liquid nitrogen. The
organs were embedded in Tissue-Tek OCT compound (Sakura Finetek,
Heppenheim, Germany), and 6-mm tissue sections were prepared, air-dried,
and then fixed in acetone for 10 min at 220˚C. Next, the tissue sections
were washed with 0.01% TBST (Sigma-Aldrich Chemie), incubated with
methanol containing 3% H2O2 for 10 min in the dark, and washed with
TBST again. After 30 min blocking with ChemMate Ab diluent (Dako,
Hamburg, Germany), a polyclonal rabbit anti-human C3c Ab (Dako) in
a dilution of 1:800 of the stock (0.5 mg/ml) in Tween 20 and TBS with
1% BSA (PAA Laboratories, Coelbe, Germany) was applied on the sections and incubated for 1 h at room temperature. The anti-C3c Ab used
was specific for C3c, C3, and C3b with proven mouse cross-reactivity
according to the manufacturer’s manual. Thereafter, the tissue sections
were washed for 2 min with TBST before incubation with undiluted
EnVision+/HRP, Rb (Dako) for 30 min at room temperature. After washing
with TBST the sections were stained with diaminobenzidine solution
(0.05% diaminobenzidine/0.01% H2O2 in TBST) (Dako). Counterstaining
was achieved with Mayer’s hemalaun (Dako) for 10 min. Tissue sections
were fixed and cover slides were mounted with Vitro-Clud (Langenbrinck,
Emmendinge, Germany). Staining was documented using light microscopy
(Axiophot; Zeiss, Jena, Germany) and digital imaging (Spot Advanced
software; VisitronSystems, Puchheim, Germany).
To visualize organ damage on the histological level, we conducted CLP
studies in the various mouse strains and isolated fresh organs at 0, 6, and
24 h after CLP immediately after sacrificing the animals. Organs were fixed
for 24 h in 5% formaldehyde, embedded in paraffin (Leica TP 1020 tissue
processor, Leica EG1160 embedding center) and sectioned at a thickness of
3 mm, placed on glass slides, and stained with H&E according to the
following protocol: The tissue sections were deparaffinized and rehydrated
with xylene and decreasing graded ethanol (100, 100, 96, 80, 60, 50%),
dyed for 2 min with Mayer’s hemalaun, rinsed for 10 min in water, counterstained for 7 min with eosin, and rinsed with water again. All reagents
used for histology were purchased from Roth (Karlsruhe, Germany).
Staining was documented using light microscopy (AxioVision; Zeiss).
Quantitation of organ dysfunction during sepsis in vivo
To determine organ dysfunction of various organs in vivo, plasma samples
were collected from the various groups of mice at different time points
Quantitation of IL-6, MCP-, IFN-g, and TNF-a
Plasma samples were collected as outlined above. For quantification of
various mediators, a commercially available flow cytometric bead assay
(CBA) was performed according to the manufacturer’s instructions (mouse
inflammation kit; BD Biosciences, Heidelberg, Germany).
Isolation, culture, and activation of peripheral murine
leukocytes
Peripheral leukocytes from whole blood of all mouse strains were separated.
Whole blood was drawn from 24 untreated mice of each strain as described
above. In the first step platelet-rich plasma was demounted after centrifugation (15 min, 80 3 g, room temperature plus 12 min, 240 3 g, room
temperature). Cells were purified from erythrocytes using ammonium
chloride lysis buffer (0.155 M NH4Cl, 0.01 M KHCO3, and 0.1233 M
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Mice were anesthetized by i.p. injection of ketamine (Ketamin; DeltaSelect,
Dreieich, Germany) and xylazine (Rompun; Bayer, Leverkusen, Germany).
The cecum was exposed through a 2-cm abdominal midline incision and
about two-thirds of the cecum was ligated. The ligated part of the cecum was
punctured through and through with a 21-gauge needle. After repositioning
the bowel, the abdomen was closed in layers, using a 5.0 surgical suture
(Ethicon, Norderstedt, Germany). Mice were monitored for various signs of
sickness every 6 h for 7 d.
C1q2/2 mice were generated on the background of C57BL/6 mice as
described previously by Botto et al. (16). Factor D2/2 mice were generated
as described previously by Xu et al. (17), also on the background of
C57BL/6 mice. Specific pathogen-free, C57BL/6 mice were used for
control studies and obtained from Charles River, Sulzfeld, Germany. All
animal studies were reviewed and approved by the local ethic committee of
the state of Lower Saxony, Germany.
The Journal of Immunology
3
EDTA [pH 7.27]), and the remaining leukocytes were cultured for 2 or
24 h (RPMI 1640 medium containing 10% mouse serum and 1% GlutaMax, 37˚C, 5% CO2). Afterwards, the leukocytes were left untreated or
were stimulated with 200 ng/ml LPS for 30 min. After 24 h incubation the
cells were lysed with NETN buffer (10 mM Tris [pH 8.0], 100 mM NaCl, 1
mM EDTA, 10% glycerol, 1 mM DTT, 0.5% Nonidet P-40, 0.5 mM PMSF,
proteinase inhibitor mixture 1:100) with sonification following. The protein content of the homogenates was determined with Bradford’s method.
NF-kB generation in isolated mouse leukocytes: avidin-biotin
complex DNA assay and Western blot
Statistical analysis
All values were expressed as the mean 6 SEM. Significance was assigned
where p , 0.05. Significances of normal distributed data were identified
using one-way ANOVA followed by post hoc comparisons using Bonferroni–Holm correction. Statistical analyses of data that did not follow
normal distribution were conducted by the nonparametric Kruskal–Wallis
test followed by a Dunn’s post test. The software used was GraphPad
Prism 5.0 (GraphPad Software).
FIGURE 1. Outcome during CLP-induced sepsis in wild-type, fD2/2,
and C1q2/2 mice. CLP was induced with a 21-gauge needle and a twothirds ligation of the cecum. Animals were monitored for signs of sickness
and mortality on a 6 hourly basis. Experiments are representative of 24
animals per group. wt, wild-type.
measured C5a concentrations in plasma by ELISA. There were no
detectable differences in baseline C5a generation in plasma of
untreated knockout or control mice (Fig. 2A). Control mice as well
as C1q2/2 mice displayed an expected increase of C5a levels at
3 h after CLP, which was most pronounced in C1q2/2 mice. The
latter also displayed a statistically significant initial drop at 1.5 h
after CLP when compared with control mice (Fig. 2B). Even
though fD2/2 mice showed some decrease in C5a levels at 6 h,
these changes were not statistically different from control levels.
These results demonstrated that fD2/2 mice were capable of
generating equal baseline C5a levels when compared with control
and C1q2/2 mice, but they displayed a lowered and timely
delayed increase in C5a generation after the onset of sepsis.
Immunohistochemical staining for C3/C3b/C3c deposition
during sepsis in kidney sections from wild-type, C1q2/2, and
fD2/2 mice
To determine the contribution of the classical and the alternative
pathway for C3 activation, we detected C3/C3b/C3c deposition
in the kidney of wild-type, C1q2/2, and fD2/2 mice 3 h after CLP
Results
Outcome in experimental sepsis CLP study in wild-type, fD2/2,
and C1q2/2 mice
To investigate the effect of impaired activation of the classical
complement activation pathway in C1q2/2 mice and the impaired
activation in fD2/2 mice on outcome in experimental sepsis, we
conducted CLP experiments in these mice. Mice were followed up
for 7 d and monitored every 6 h for various signs of sickness.
C1q2/2 mice started to die very rapidly after 24 h and after 55 h
none of the animals survived (Fig. 1). Although fD2/2 mice
survived slightly longer, the mortality also reached 100% after
approximately 4 d. Wild-type mice displayed a significantly
higher survival rate at ∼30%. These results demonstrated an impaired survival capacity of C1q2/2 and fD2/2 mice when compared with the wild-type controls, suggesting an important role for
the classical and the alternative pathway for successful host response during sepsis. We therefore tried to investigate whether the
expected underlying lack of overall complement activation and
therefore a lack of bacterial clearance capacity would be a sufficient explanation for these findings.
C5a generation in plasma from wild-type, fD2/2, and C1q2/2
mice
To underline the comparability of the used mouse strains with
respect to their ability to generate one known key player, C5a, we
FIGURE 2. C5a levels in plasma from wild-type, fD2/2, and C1q2/2
mice. C5a in plasma was measured by ELISA. Samples for ELISA were
analyzed in duplicates. A, There were no differences of C5a generation in
plasma of untreated mice detected. B, C5a levels during the onset of sepsis
in various mice strains as depicted. *p , 0.05 for statistical significance
to 0 h control groups. #p , 0.05, ##p , 0.01, ###p = 0.0002 for statistical
significance among different groups of corresponding time points. Data
are representative of 5–12 animals per group and are depicted as mean 6
SEM. wt, wild-type.
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The avidin–biotin complex DNA assay is based on immobilization of
protein–DNA complexes via binding of a biotinylated oligonucleotide to
a streptavidin matrix. Biotinylated oligos were used from Biomers.net
(Ulm, Germany) and were composed of the following sequences: NF-kB
consensus oligonucleotide, sense, 59-AGT TGA GGG GAC TTT CCC
AGG C-39 and antisense, 59-GCC TGG GAA AGT CCC CTC AAC T-39.
A total of 200 ml leukocyte whole-cell extract (same protein content) was
incubated with 200 ml buffer H (50 mM KCl, 20 mM HEPES [pH 7.8],
20% glycerol, 1 mM DTT, 0.1% Nonidet P-40), 2 ml biotinylated oligo,
10 ml herring sperm DNA, and 5 ml 2 M KCl were boiled for 5 min at 37˚C
and stored on ice for 1 h. After addition of 41 ml equilibrated streptavidin
agarose beads (Novagen, Madison, WI), incubation was continued for
30 min at 4˚C on a rotator. Beads were washed repeatedly with buffer H
and boiled in Laemmli sample buffer, and proteins were separated via
SDS-PAGE under nonreducing conditions. NF-kB (65 kDa) was detected
by Western blot (anti–NF-kB p65 Ab C22B4; Cell Signaling Technology,
Danvers, MA) via chemiluminescence detection (Immobilon Western
chemiluminescent HRP substrate; Millipore, Billerica, MA). Gray scale
value density of detected results was analyzed by Aida Image Analyzer
v.3.52 (Raytest, Straubenhardt, Germany). To demonstrate the equal protein load in SDS-PAGE, b-actin (45 kDa) was detected in Western blot, too
(b-actin [13E5]; Cell Signaling Technology, Danvers, MA).
Cell supernatants were also obtained after 2 h incubation and analyzed
for cytokine generation using CBA assays as described above.
4
CONTRIBUTION OF COMPLEMENT PATHWAYS IN SEPSIS
(Fig. 3, right panel) and compared them to the corresponding
sham group (Fig. 3, left panel). In wild-type animals we observed
a low baseline of complement C3/C3b/C3c deposition in the renal
tubuli at 0 h that was clearly increased 3 h after CLP (Fig. 3, top
panel). In C1q2/2 mice as well as in fD2/2 mice we did not find
such deposits at 0 h, and induction of sepsis did not lead to marked
depositions 3 h after CLP (Fig. 3, middle and lower panels).
Similar observations were made in the renal cortex section with
mild C3c deposition in the glomeruli in wild-type mice that was
strongly increased 3 h after CLP, whereas no C3/C3b/C3c deposition was detected in both complement knockout mice strains
(data not shown). These data indicated a crucial contribution of
both the classical and the alternative pathway to overall C3 cleavage
and therefore activation of the complement system in sepsis.
nounced organ dysfunction in fD2/2 and C1q2/2 mice when
compared with control mice, with these effects being most prominent in fD2/2 mice. No significant increases during the onset
of sepsis were found in any of the three groups for alkaline
phosphatase and plasma albumin (data not shown).
Effects of fD and C1q knockout on organ dysfunction during
CLP-induced sepsis
Bacterial clearance represents one major endpoint for innate host
immune response to infection that is thought to be greatly influenced by intact complement activation and formation of the terminal complement activation products. We therefore sought to
determine the capacity of alternative and classical complement
pathway knockout mice to clear bacteria during the onset of sepsis
in comparison with untreated control mice. Given the abovedepicted lowered survival and increased organ dysfunction in
fD2/2 and C1q2/2 mice, we expected to find increased bacterial
loads in both knockout strains. C1q2/2 mice demonstrated significantly elevated aerobic bacterial loads in lung, liver, and blood
and a strong but not significant similar tendency in kidney 6 h after
CLP when compared with control mice (Fig. 5A–D). Surprisingly,
fD2/2 mice did not demonstrate such increases in bacterial organ
loads and demonstrated an equal capability of clearing bacteria
when compared with control mice. However, 24 h after CLP this
situation changed and aerobic bacterial loads showed an increase
compared with the other strains, which was statistically significant
for liver samples and present as a strong tendency in blood and
kidney samples (Fig. 5E–H). Anaerobic bacterial loads displayed
a higher level of variance between different study animals after 6
and 24 h, but showed similar tendencies when compared with
aerobic results (data not shown).
Effects of C1q and fD knockout on phagocytosis activity in
granulocytes and monocytes during sepsis
In light of the above detailed findings of increased bacterial loads in
C1q2/2 mice, we sought to investigate whether the phagocytosis
activity in granulocytes and monocytes would also be affected
by impaired activation of the alternative or classical complement
cascade. Whole blood samples were collected at 0 and 6 h after
CLP from wild-type, fD2/2, and C1q2/2 mice and then stimulated
with opsonized FITC-conjugated E. coli bacteria (1.7 3 109/ml)
for 20 min. Phagocytosis activity was then measured using
a flow cytometer. Granulocytes from wild-type mice demonstrated
a comparatively high baseline ability to phagocytose bacteria,
which significantly decreased 6 h after CLP (Fig. 6A). Both fD2/2
and C1q2/2 mice showed only marginal ability to phagocytose
bacteria under untreated conditions. However, there was a somewhat inducible potential for phagocytosis in C1q2/2 mice 6 h after
CLP, which was absent in fD2/2 mice (Fig. 6A). Similar results
were obtained in monocytes (Fig. 6B). These findings could explain in part the reduced bacterial clearance capacity during sepsis
in C1q2/2 mice. However, despite the detected low phagocytosis
capacity in fD2/2 mice, the latter group was capable of clearing
bacteria similarly well when compared with control mice.
MPO content in the lung and plasma during sepsis in
wild-type, fD2/2, and C1q2/2 mice
FIGURE 3. Immunohistochemical staining for C3c deposition in kidney
sections during sepsis from wild-type, C1q2/2, and fD2/2 mice. Compared are C3c staining results from kidney sections from wild-type (top
panel), C1q2/2 (middle panel), and fD2/2 mice at 0 h (left panel) and 3 h
after CLP (right panel). Results are representative of three to six staining
experiments from two independent experiments per condition. Original
magnification 3200. wt, wild-type.
To better understand and interpret the findings in our phagocytosis
and bacterial clearance experiments, we evaluated the invasion of
neutrophils into the lung during the onset of sepsis by measuring
the amount of MPO with ELISA technique in homogenized lung
tissue samples from wild-type, fD2/2, and C1q2/2 mice before
(CLP 0 h) and 6 h after CLP. The concentration of MPO was
normalized to 100 mg protein. In all three groups (wild-type, fD2/2,
and C1q2/2 mice) the MPO concentration was significantly
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To establish whether the lack of factor D or C1q could be specifically linked to organ dysfunction during experimental sepsis, we
analyzed organ-specific indicators in the plasma of septic mice at 6
and 24 h after induction of CLP (Fig. 4A–D). All groups, C1q2/2,
fD2/2, and wild-type control mice, demonstrated elevated levels
of the following parameters when compared with their corresponding control values at 0 h CLP: urea, indicating a reduced
filtrating function of the kidneys (Fig. 4A); GOT/AST, indicating
liver cell damage (Fig. 4C); bilirubin, indicating a reduced liver
capacity to convert hemoglobin and/or excrete bilirubin (Fig. 4B);
and LDH as an indicator for tissue and organ damage (Fig. 4D).
Wild-type, fD2/2, and C1q2/2 mice displayed significantly increased values of urea, GOT/AST, and LDH at 24 h after CLP
when compared with control mice (Fig. 4A, 4C, 4D), indicating
significantly worsened organ dysfunction in these mice. LDH was
significantly elevated in C1q2/2 mice 24 h after CLP when
compared with wild-type and fD2/2 mice. In case of bilirubin,
only fD2/2 and C1q2/2 mice showed significantly increased
levels 24 h after CLP, which could not be observed in wild-type
mice. In summary, we observed a tendency toward more pro-
Bacterial load in blood, lung, liver, and kidney
The Journal of Immunology
5
FIGURE 4. A–D, Analysis of various blood and
plasma parameters indicative of organ dysfunction
during sepsis in wild-type, fD2/2, and C1q2/2 mice.
Automated plasma parameter analysis for urea, bilirubin, GOT/AST, and LDH in plasma samples from
septic mice at different time points after CLP. *p ,
0.05, **p , 0.01, ***p , 0.0001 for statistical significance to 0 h value of the groups. #p , 0.05, ##p ,
0.01, ###p , 0.0001 for statistical significance between
different groups. Data are representative of 10–12
animals per group and are depicted as mean 6 SEM.
wt, wild-type.
Effects of C1q and fD knockout on structural organ changes
during CLP-induced sepsis
In light of the above findings, demonstrating rapid mortality in
fD2/2 and C1q2/2 mice during CLP-induced sepsis, we were able
to demonstrate significantly increased organ dysfunction in both
knockout strains when compared with control mice 24 h after
CLP. We therefore conducted additional staining experiments
for organ sections from septic animals of the above-mentioned
groups of mice to visualize the potential extent of structural organ changes in livers (not shown) and lungs from septic animals
(Fig. 8). In all groups, signs of inflammation were observed 6 and
24 h post CLP. However, lungs from fD2/2 and C1q2/2 mice
demonstrated more signs of severe structural organ changes, such
as alveolar swelling and infiltration of mononuclear cells, when
compared with organ sections from wild-type mice, which displayed such changes to a lesser extent. Structural organ changes
appeared to be most pronounced in fD2/2 mice. These findings
were in line with the results of the organ dysfunction parameters
and suggested an overall deleterious effect of fD2/2 and C1q2/2
knockout during the onset of sepsis.
Cytokine generation during sepsis in wild-type, fD2/2, and
C1q2/2 mice
To gain more information about the cause of death in fD2/2mice,
which apparently were capable of undisturbed bacterial clearance,
we investigated differences in cytokine patterns in whole blood
during CLP-induced sepsis 6 and 24 h after CLP in wild-type,
fD2/2 , and C1q2/2 mice (Fig. 9). Because production of inflammatory mediators reflects one arm of the innate host response, which is thought to be directly dependent on the
concentration of pathogen-associated molecular patterns (PAMPs)
and therefore also on the bacterial load, we predicted that we
would find the highest levels of such mediators in C1q2/2 mice.
As depicted in Fig. 9, in all three groups the serum levels for TNFa (Fig. 9B), IL-6 (Fig. 9A), MCP-1 (Fig. 9D), and IFN-g (Fig. 9C)
were significantly increased 24 h after CLP when compared with
corresponding 0 h values. It is known that wild-type mice typically
already demonstrate very high cytokine levels in this sepsis model.
However, in both knockout strains, IL-6, TNF-a, and IFN-g
generation was significantly higher 24 h after CLP when compared with wild-type controls (Fig. 9A–C). In fD2/2 mice, levels
of IL-6 and TNF-a even demonstrated a tendency toward higher
levels at 24 h after CLP when compared with C1q2/2 mice, which
did not reach statistical significance (Fig. 9A, 9B). With respect to
the earlier described normal bacterial clearance capacity of fD2/2
mice, these results suggested that these mice potentially suffered
from a loss of control over the host cytokine response to bacterial
challenge or that these mice were capable of producing significantly increased cytokine amounts through the established increased neutrophil recruitment alone.
NF-kB generation and cytokine generation in isolated mouse
leukocytes
Based on the above-depicted findings, we hypothesized that factor
D may exert a controlling function for cytokine generation and
investigated the ability of fD2/2 mice to activate NF-kB in isolated
leukocytes upon stimulation with bacterial LPS as a prototype
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elevated at 6 h after CPL when compared with the corresponding
0 h control groups (Fig. 7A). In fD2/2 mice the total MPO concentrations as well as the increase in MPO content were significantly higher when compared with C1q2/2 or wild-type mice at
6 h after CLP (Fig. 7A, 7C). To assess the amount of MPO secreted or released from neutrophils in the plasma during the onset
of sepsis, we measured the MPO concentration with an ELISA in
plasma samples from wild-type, fD2/2, and C1q2/2 mice at 0 and
3 h after CLP. In all three groups (wild-type, fD2/2, and C1q2/2
mice), significantly higher MPO concentration were measured 3 h
after CLP when compared with the corresponding 0 h control
groups (Fig. 7B). Again, the total MPO concentrations in plasma,
as well as the increase in MPO in fD2/2 mice at 3 h after CLP,
were significantly higher when compared with wild-type or C1q2/2
mice (Fig. 7D). These results indicated that fD2/2 mice could
potentially compensate their low phagocytosis capacity by a significantly increased neutrophil recruitment to reach an overall
normal bacterial clearance capacity when compared with healthy
control animals.
6
CONTRIBUTION OF COMPLEMENT PATHWAYS IN SEPSIS
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FIGURE 5. Bacterial load in blood, lung, liver, and
kidney during sepsis in wild-type, fD2/2, and C1q2/2
mice. A–H, CFU aerobic bacteria in blood and various
organ samples from wild-type, fD2/2, and C1q2/2
mice. Samples were collected 6 (A–D) and 24 h (E–H)
after CLP-induced sepsis in mice. *p , 0.05, **p ,
0.01 compared with wild-type group. #p , 0.05 compared with fD2/2 group. Data are representative of four
to six animals per group and are depicted as mean 6
SEM. wt, wild-type.
PAMP. We found that NF-kB activation, as detected in leukocytes
pooled from 24 mice per experiment upon stimulation with LPS,
reached higher levels in fD2/2 mice when compared with wildtype and C1q2/2 mice (Fig. 10A), which became even more evident after grayscale value density analysis (Fig. 10B).
To investigate the significance of this finding for mediator
generation in isolated mouse leukocytes ex vivo, supernatants of
leukocytes pooled from 24 mice per strain were obtained 2 h after
stimulation with LPS and analyzed for cytokine generation using
CBA assay. The results in Fig. 10C–F demonstrated that, corresponding to the findings during sepsis in whole blood, leukocytes from fD2/2 mice generated higher levels of IL-6, TNF-a,
MCP-1, and IFN-g when compared with neutrophils from C1q2/2
and control mice. These results suggested a potential controlling
function of factor D for NF-kB–dependent cytokine generation in
mouse leukocytes.
Discussion
It is well known that sufficient activation of the complement system
during infection is essential for a successful immune response and
defense. However, the individual contributions of the separate
complement activation pathways for known changes in host defense and innate immune functions during sepsis have not yet been
investigated in detail. Knockout mice lacking all three complement
activation pathways demonstrated extreme susceptibility to infection caused by bacterial challenge, and results with C1q2/2
mice demonstrated involvement of the classical pathway (14).
Similar results were previously found using these two knockout
strains in the CLP sepsis model (15). Both reports suggested
The Journal of Immunology
importance of the alternative pathway, but the separate contribution could not be determined at that time. To investigate the effects
of impaired alternative complement activation in comparison with
defective classical complement activation on outcome and impaired innate immune responses during experimental sepsis, we
conducted CLP studies using C1q2/2 and fD2/2 mice, which have
been shown to lack specifically the alternative pathway (17). In
line with previous studies (15), we found C1q2/2 mice to be
highly susceptibile, with 100% mortality very early during CLPinduced sepsis. Factor D2/2 mice also displayed 100% mortality
but survived 2 d longer in the mean values (Fig. 1). Interestingly,
fD2/2 mice displayed normal baseline C5a levels when compared
with control mice (Fig. 2A). The same was true for C1q2/2 mice.
FIGURE 7. MPO levels in lung and plasma during sepsis in wild-type,
fD2/2, and C1q2/2 mice. MPO ELISA of lung homogenates (A) and
plasma samples (B) at 0, 3, and 6 h after induction of CLP also depicted as
corresponding increases in percentages (C, D). MPO concentrations were
normalized to the protein content measured with Bradford’s assay. *p ,
0.05, **p , 0.01, ***p , 0.0001 for statistically significant difference
from control groups (CLP 0 h) for A and B and from corresponding groups
of wild-type and C1q2/2 mice in C and D, respectively. #p , 0.05, ###p ,
0.0001 for statistically significant difference between indicated observational groups. Data are representative of four animals per group and are
depicted as mean 6 SEM. wt, wild-type.
FIGURE 8. Histological staining of lung sections from wild-type, fD2/2,
and C1q2/2 mice at different time points after CLP. H&E staining results
for lung sections of wild-type, fD2/2, and C1q2/2 mice 0, 6, and 24 h after
CLP. Data are representative of two to three staining sets from three animals per group. Original magnification 3200. wt, wild-type.
It has been previously reported that C57BL/6 mice, which served
at least partially as background also for both knockouts, appear to
have some defect in the classical C5 convertase, leading to compromised classical C5 activation (18). Our results in this study
suggest that both knockout strains employed in our studies did
not suffer from an inability to generate C5a. However, it is known
now that C5a can be generated in the absence of the classical or
alternative convertase (19). Surprisingly, fD2/2 mice displayed
a different characteristic in C5a levels in the time course of sepsis compared with wild-type and C1q2/2 mice (Fig. 2B), and
they demonstrated no significant changes over time. Additionally,
C1q2/2 mice exhibited a decrease after 1.5 h after CLP, which
was followed by a significantly higher increase after 3 h compared
with fD2/2 and wild-type mice, which could be a possible cause
for the rapid onset of mortality. Besides the possibility of local
additional generation independent of upstream complement activation, there are various other factors influencing C5a levels in
plasma. One of them is degradation, which is dependent on
availability and activation status of the degrading C5 processing
enzymes. Another, even more important factor is the existing large
C5a sink in form of an abundant number of C5a receptors such as
C5aR and C5L2 (20) on almost all tissues and especially on blood
neutrophils. C5aR is dramatically upregulated during sepsis in
various tissues (7). Because all of these factors display strong
dynamics during sepsis, we must conclude that measured C5a
levels in plasma may not accurately reflect the extent of the activation of the complement system.
Experiments with C32/2 mice have demonstrated before that
these animals are highly susceptible to a broad range of infectious
insults (3, 21–23), with these mice being incapable of generating
the important opsonin C3b and initiating the formation of the
MAC. On the other hand, there is accumulating evidence that
excessive production of the potent proinflammatory complement
split product C5a exerts harmful effects on innate immune functions and worsens outcome during experimental sepsis (1, 4–7).
These findings are in line with our findings in factor D knockout
mice with respect to the outcome studies. The increased susceptibility in both C1q and factor D knockout mice could be
explained by the strongly reduced activation of C3 and the MAC,
as suggested by our staining experiments for C3c deposition in the
kidneys (Fig. 3).
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FIGURE 6. E. coli-induced phagocytosis of granulocytes and monocytes during sepsis in wild-type, fD2/2, and C1q2/2 mice. Flow cytometric analysis of phagocytosis in whole blood samples gated for granulocytes (A) and monocytic cells (B). Whole blood samples were
drawn at 0 and 6 h after CLP and then stimulated with opsonized FITCconjugated E. coli bacteria (1.7 3 109/ml) for 20 min. Phagocytosis activity per single cell is depicted as mean fluorescence intensity (MFI; A, B).
**p , 0.01 statistical significance to 0 h wild-type group. #p , 0.05, ##p ,
0.01 indicates statistical significance to 6 h wild-type group. Data are
representative of four to six animals per group and are depicted as mean 6
SEM. wt, wild-type.
7
8
CONTRIBUTION OF COMPLEMENT PATHWAYS IN SEPSIS
FIGURE 9. A–D, Cytokine generation during sepsis in wild-type, fD2/2, and C1q2/2 mice. Timedependent changes in cytokine levels (IL-6, TNF-a,
IFN-g, and MCP-1) at 0, 6, and 24 h after CLP (n = 5–
7 animals/time point). Analysis was performed using
a flow cytometric bead assay of plasma samples from
septic animals. *p , 0.05, ** p , 0.01, ***p , 0.001
for statistically significant difference to 0 h control
groups. #p , 0.05, ##p , 0.01, ###p , 0.001 for statistically significant difference between indicated observational groups. Data are representative of five to
seven animals per group and are depicted as mean 6
SEM. wt, wild-type.
when compared with C1q2/2 and wild-type mice (Fig. 5A–D).
Our findings therefore confirm the important role of the classical
pathway for survival during sepsis by being mainly responsible for
FIGURE 10. NF-kB generation in isolated mouse
leukocytes. A, Western blot analysis for activated NFkB (65 kDa) generation in isolated leukocytes. Cells
were cultured overnight, left untreated or stimulated
for 30 min with 200 ng/ml LPS, lysed, and NF-kB
was isolated by an avidin–biotin complex DNA assay.
Results were interlocked by grayscale value density
analysis and are depicted in arbitrary units (B). C–F,
CBA assay analysis for various cytokines as depicted
in supernatants from wild-type, fD2/2, and C1q2/2
mice after 2 h culture and 6 h stimulation with LPS.
Each group was generated with neutrophils pooled
from 24 mice. wt, wild-type.
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We showed that C1q2/2 mice are not able to clear bacteria
effectively during the early onset of sepsis, whereas fD2/2 mice
were fully capable of clearing bacteria 6 h after induction of sepsis
The Journal of Immunology
upon infectious stimuli (Fig. 10) and found this to be strongly
enhanced. NF-kB activation is well known to promote cytokine
generation (25) as well as mechanisms leading to neutrophil recruitment (26). We therefore suggest this mechanism to be at least
partially responsible for the observed increases in inflammatory
mediator generation and increased MPO levels in lung and whole
blood. Note that based on the knockout of a soluble serum factor
such as factor D, it may not directly be expected that cell-derived
effects can be observed. Based on the available data, we can
currently only speculate that factor D may be involved in controlling NF-kB activation in blood neutrophils and that knockout
of this factor may lead to increased cell signaling via NF-kB.
Further experiments are warranted to define the underlying mechanisms in more detail.
Interestingly, in models of chronic-type noninfectious inflammatory diseases, such as asthma or autoimmune nephropathy,
inactivation of the alternative pathway showed beneficial effects,
partially by lowering the inflammatory response (2, 27, 28). In
these models, the activation of the alternative pathway was suggested to act as an amplification loop triggering the hyperactive
inflammatory response and thereby contributing negatively to the
progression of the disease, whereas in models of bacterial infection an intact activation of the complement system seemed to
be essential for successful and early control of the infection as
discussed above. It is therefore likely that the alternative pathway
plays a different role in aseptic inflammatory responses when
compared with immune responses during septic inflammation. In
a model of gastrointestinal ischemia and reperfusion injury, the
third pathway, the MBL-activated pathway, has been demonstrated
to be mainly responsible for gastrointestinal injury but not for codevelopment of lung injury (29). In this model, the alternative
pathway has also been suggested to act as an amplifier for complement activation with critical initial activation of the lectin
and/or classical complement pathway (30). Similarly, myocardial
ischemia/reperfusion injury was dependent on MBL-dependent
pathway activation, but not on the activation of the classical
pathway (31, 32) as suggested by earlier reports (33). Evidence
exists also for a key role of the lectin pathway in infection, as
MBL A/C2/2 mice have been shown to be highly susceptible to
Staphylococcus aureus and to postburn infection with P. aeruginosa (34, 35). Taken together, throughout the literature, one proposed main function of the alternative pathway has been the
amplification of complement activation after initial activation of
the classical or MBL pathway (30, 36, 37). However, our recent
study suggests a potentially new controlling and time-dependent
function of this pathway during experimental sepsis in mice. In
a recent study from our group, we were able to demonstrate
a similar direct regulatory function on the innate immune response
of the host for the PI3K pathway (9). In that study, PI3K-deficient
animals also displayed strongly increased inflammatory responses
and reduced phagocytosis function when compared with control
animals during sepsis, leading to worsened outcome.
We conclude from our findings an important and previously
unrecognized controlling function of the alternative complement
activation pathway, and particularly of complement factor D, for
various innate immune responses during experimental sepsis. We
further provide evidence for our hypothesis that the amplifying
function of the alternative complement pathway for overall complement activation as suggested in other models of inflammation
appears not to be a major mechanism in experimental sepsis. Our
report therefore suggests distinct different functions of these two
complement activation pathways for host innate immune responses
in experimental sepsis.
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intact clearance of bacterial challenges early during sepsis. Interestingly, we found that the bacterial clearance at 24 h after CLP
displayed a different situation when compared with the 6 h values:
fD2/2 mice now presented with a significantly increased bacterial
load in liver when compared with wild-type and C1q2/2 mice and
a similar, but not statistically significant, tendency for kidney and
whole blood (Fig. 5E–H). These findings add a new aspect since
the importance of the separate pathways for bacterial clearance
appears to be time-dependent. The classical pathway is apparently
very important for clearing bacteria in the early development of
sepsis, whereas the alternative pathway may play a more important role for the later phase of development.
A recent report suggested an important protective role of properdin, a cofactor of the alternative complement system, for outcome during experimental sepsis (24). These findings are in line
with our findings in fD2/2 mice with respect to the outcome
results, demonstrating an important role of the alternative pathway
for survival during experimental sepsis. A critical role for the
alternative pathway has been also identified before for survival of
mice infected with Pseudomonas aeruginosa in a murine model of
pneumonia (22). In vitro studies with serum from fD2/2 mice
demonstrated that the alternative pathway strongly contributed to
the deposition of C3 on the phosphocholine-rich surface in kidneys. The authors therefore suggested that this pathway critically
contributed to overall complement activation and was important
for opsonization and clearance of bacteria (17). Even though our
observations for C3b staining in kidneys emphasize that fD2/2
mice also demonstrate a compromised ability for C3 cleavage
during sepsis, our new findings demonstrating a normal bacterial
clearance of these knockout mice in early CLP-induced sepsis
when compared with control mice suggest an additional role of
this pathway and of factor D during sepsis other than only contributing to overall complement activation.
In our studies, both knockout strains showed a strongly reduced
baseline phagocytosis capacity when compared with control mice
(Fig. 6), which could also be explained by functional defects
in neutrophils of these knockout strains, but fD2/2 mice depicted
significantly higher MPO levels in lung and whole blood (Fig. 7).
We therefore hypothesize that an altered and somewhat less
controlled neutrophil recruitment in fD2/2 mice may compensate
for their reduced phagocytosis capability, leading to an overall
normal bacterial clearance in comparison with control mice at
least early during the onset phase of sepsis. Additional histological
experiments showed pronounced mononuclear cell infiltrations in
lungs and liver of fD2/2 mice in contrast to wild-type and C1q2/2
mice (Fig. 8), supporting this hypothesis.
We investigated cytokine generation in these mice (Fig. 9) and
found that fD2/2 mice displayed extremely high levels of IL-6,
TNF-a, MCP-1, and IFN-g when compared with the cytokine
storm-like values already present in control mice during sepsis.
These findings further suggested some kind of loss of control over
PAMP-induced inflammatory mediator generation during sepsis
and could explain a worsened outcome in these animals, as it is
well known that inflammatory mediators such as IL-6 and TNF-a
exert harmful effects per se, leading also to death in mice when
administered in higher doses. Because cytokine changes are usually delayed several hours from maximum stimulus to maximum
observed phenotype (stimulation of isolated cells), it may well be
possible that, depending on the cytokine, the depicted 24 h levels
in whole blood might reflect the bacterial load differences present
earlier, such as 6 h after CLP. To further elucidate the hypothesis
that fD2/2 mice generate higher levels of inflammatory mediators
even though similar stimuli may be present, we investigated the
ability of neutrophils from fD2/2 mice to induce NF-kB activation
9
CONTRIBUTION OF COMPLEMENT PATHWAYS IN SEPSIS
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We thank Marina Botto for the use of C1q knockout mice for the experiments, and we are grateful to Iris Suckert and Ulrike Vetterling for excellent
assistance.
Disclosures
The authors have no financial conflicts of interest.
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