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D
uring acute inflammation, the complement system can be generated by spontaneous hydrolyses, the so-called “tick-over”
activated by at least three well-known pathways: the (2). All three pathways merge at the level of C3, and activation
classical pathway, the lectin or mannose-binding lectin of either pathway ultimately results in generation of the potent
(MBL) pathway, and the alternative pathway (1). The classical proinflammatory complement split products C3a and C5a as well
pathway is activated, for example, by Ag–Ab complexes that react as the terminal membrane attack complex (MAC) (3). Excessive
with activated C1q. The lectin pathway is initiated by either serum generation of C5a during the onset of sepsis causes various
MBL or ficolins that recognize certain oligosaccharide moieties on harmful effects mediated by C5aR (1, 4, 5), and blockade of either
microbial surfaces. The alternative pathway can be activated either C5a or C5aR leads to greatly improved survival of rodents in
by the presence of foreign surfaces such as LPSs or through C3b 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
*Department of Anesthesiology and Intensive Care Therapy, Jena University Hospi- suppression (8). We recently demonstrated that C5a alters in-
tal, Friedrich Schiller University Jena, 07747 Jena, Germany; †Institute for Func-
tional and Applied Anatomy, Hannover Medical School, 30625 Hannover, Germany; tracellular signaling pathways in neutrophils in vitro and during
‡
Department of Medical Microbiology, Jena University Hospital, Friedrich Schiller the onset of sepsis in vivo (9–12), offering an explanation for the
University Jena, 07747 Jena, Germany; xDepartment of Medical Microbiology, Hann- above-mentioned suppression of innate immune functions.
over Medical School, 30625 Hannover, Germany; {Center for Experimental Thera-
peutics and Reperfusions Injury, Anesthesiology, Perioperative Medicine and Pain Studies employing different complement knockout mice in a
Medicine, Brigham and Woman’s Hospital, Harvard Medical School, Boston, MA model of streptococcal pneumonia first suggested that the clas-
02115; and ‖Division of Clinical Immunology and Rheumatology, Department of
Medicine, University of Alabama, Birmingham, AL 35294
sical activation pathway appeared to be dominant for clearance
of these bacteria (13). However, little is known about the contri-
Received for publication August 13, 2010. Accepted for publication December 14,
2010. bution of the separate complement pathways during the onset
Address correspondence and reprint requests to Dr. Niels C. Riedemann, Department phase of sepsis. Initial experimental infection studies with knock-
of Anesthesiology and Intensive Care Therapy, Jena University Hospital, Friedrich out mice lacking all three complement pathways demonstrated
Schiller University Jena, Erlanger Allee 101, 07747 Jena, Germany. E-mail address: the importance of an intact complement activation system for
niels.riedemann@med.uni-jena.de
successful survival, and results with C1q knockout mice showed
Abbreviations used in this article: CBA, cytometric bead assay; CLP, cecal ligation
and puncture; GOT/AST, glutamate oxaloacetate transaminase/aspartate transami- involvement of the classical pathway (14, 15). Although
nase; LDH, lactate dehydrogenase; MAC, membrane attack complex; MBL, both reports suggested importance of the alternative and lectin
mannose-binding lectin; MPO, myeloperoxidase; PAMP, pathogen-associated molec- pathways, the individual contribution has not been dissected so
ular pattern.
far. We therefore conducted cecal ligation and puncture (CLP)
Copyright Ó 2011 by The American Association of Immunologists, Inc. 0022-1767/11/$16.00 studies with C1q2/2 mice, lacking the classical pathway, and
www.jimmunol.org/cgi/doi/10.4049/jimmunol.1002741
The Journal of Immunology 3067
fD2/2 mice, lacking the alternative pathway, and investigated the after CLP and analyzed for urea, bilirubin, glutamate oxaloacetate
effect of impaired activation of these complement pathways on transaminase/aspartate transaminase (GOT/AST), and lactate dehydroge-
nase (LDH) using an automated clinical chemistry analyzer (Fuji Dri-
outcome and innate immune responses in experimental sepsis. Chem 3500i; Sysmex).
After induction of CLP, animals were sacrificed at the indicated time points Quantitation of myeloperoxidase in the lung
and blood was drawn using direct needle puncture of the heart. Blood
samples were analyzed immediately after collection by automated veteri- Immediately after sacrificing the animals, organs were flushed with sterile
nary hematology analyzer (PocH-100iV Diff; Sysmex, Leipzig, Germany). physiological sodium chloride solution retrograde through the caudal caval
For plasma collection, the samples were stored at 4˚C, centrifuged at vein, and the organs were harvested and snap-frozen in liquid nitrogen.
4700 3 g for 10 min at 4˚C, and the plasma was collected and immediately Tissue homogenates were prepared according to the manufacturer’s in-
snap-frozen in liquid nitrogen and stored at 280˚C until used for further structions and the myeloperoxidase (MPO) concentration was analyzed by
analyses. For both plasma collection and whole blood analyses, EDTA or a commercially available ELISA (Hycult Biotechnology, Uden, The
heparin was used as anticoagulant as warranted for the particular test. Netherlands). The protein content of the homogenates was determined
with Bradford’s method.
C5a ELISA
Quantitation of MPO in plasma samples
The C5a concentration in various plasma samples was analyzed by the
commercially available ELISA mouse complement component C5a DuoSet Plasma samples were collected as outlined above. The MPO concentration
according to the manufacturer’s instructions (R&D Systems, Minneapolis, in various plasma samples was analyzed by a commercially available
MN). ELISA according to the manufacturer’s instructions (Hycult Biotech-
nology).
Immunohistochemical staining for C3/C3b/C3c deposition in Determination of histological organ damage during sepsis
kidney sections in vivo
Immediately after sacrificing an animal, the organs were flushed with sterile To visualize organ damage on the histological level, we conducted CLP
physiological sodium chloride solution retrograde through the caudal caval studies in the various mouse strains and isolated fresh organs at 0, 6, and
vein, and the organs were harvested and snap-frozen in liquid nitrogen. The
24 h after CLP immediately after sacrificing the animals. Organs were fixed
organs were embedded in Tissue-Tek OCT compound (Sakura Finetek,
for 24 h in 5% formaldehyde, embedded in paraffin (Leica TP 1020 tissue
Heppenheim, Germany), and 6-mm tissue sections were prepared, air-dried,
processor, Leica EG1160 embedding center) and sectioned at a thickness of
and then fixed in acetone for 10 min at 220˚C. Next, the tissue sections
3 mm, placed on glass slides, and stained with H&E according to the
were washed with 0.01% TBST (Sigma-Aldrich Chemie), incubated with following protocol: The tissue sections were deparaffinized and rehydrated
methanol containing 3% H2O2 for 10 min in the dark, and washed with with xylene and decreasing graded ethanol (100, 100, 96, 80, 60, 50%),
TBST again. After 30 min blocking with ChemMate Ab diluent (Dako,
dyed for 2 min with Mayer’s hemalaun, rinsed for 10 min in water, coun-
Hamburg, Germany), a polyclonal rabbit anti-human C3c Ab (Dako) in
terstained for 7 min with eosin, and rinsed with water again. All reagents
a dilution of 1:800 of the stock (0.5 mg/ml) in Tween 20 and TBS with
used for histology were purchased from Roth (Karlsruhe, Germany).
1% BSA (PAA Laboratories, Coelbe, Germany) was applied on the sec-
Staining was documented using light microscopy (AxioVision; Zeiss).
tions 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 Quantitation of IL-6, MCP-, IFN-g, and TNF-a
according to the manufacturer’s manual. Thereafter, the tissue sections
were washed for 2 min with TBST before incubation with undiluted Plasma samples were collected as outlined above. For quantification of
EnVision+/HRP, Rb (Dako) for 30 min at room temperature. After washing various mediators, a commercially available flow cytometric bead assay
with TBST the sections were stained with diaminobenzidine solution (CBA) was performed according to the manufacturer’s instructions (mouse
(0.05% diaminobenzidine/0.01% H2O2 in TBST) (Dako). Counterstaining inflammation kit; BD Biosciences, Heidelberg, Germany).
was achieved with Mayer’s hemalaun (Dako) for 10 min. Tissue sections
were fixed and cover slides were mounted with Vitro-Clud (Langenbrinck, Isolation, culture, and activation of peripheral murine
Emmendinge, Germany). Staining was documented using light microscopy leukocytes
(Axiophot; Zeiss, Jena, Germany) and digital imaging (Spot Advanced
software; VisitronSystems, Puchheim, Germany). Peripheral leukocytes from whole blood of all mouse strains were separated.
Whole blood was drawn from 24 untreated mice of each strain as described
Quantitation of organ dysfunction during sepsis in vivo above. In the first step platelet-rich plasma was demounted after centri-
fugation (15 min, 80 3 g, room temperature plus 12 min, 240 3 g, room
To determine organ dysfunction of various organs in vivo, plasma samples temperature). Cells were purified from erythrocytes using ammonium
were collected from the various groups of mice at different time points chloride lysis buffer (0.155 M NH4Cl, 0.01 M KHCO3, and 0.1233 M
3068 CONTRIBUTION OF COMPLEMENT PATHWAYS IN SEPSIS
EDTA [pH 7.27]), and the remaining leukocytes were cultured for 2 or
24 h (RPMI 1640 medium containing 10% mouse serum and 1% Gluta-
Max, 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 pro-
tein content of the homogenates was determined with Bradford’s method.
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 im-
paired survival capacity of C1q2/2 and fD2/2 mice when com-
pared with the wild-type controls, suggesting an important role for
the classical and the alternative pathway for successful host re-
sponse during sepsis. We therefore tried to investigate whether the
expected underlying lack of overall complement activation and FIGURE 2. C5a levels in plasma from wild-type, fD2/2, and C1q2/2
therefore a lack of bacterial clearance capacity would be a suffi- mice. C5a in plasma was measured by ELISA. Samples for ELISA were
analyzed in duplicates. A, There were no differences of C5a generation in
cient explanation for these findings.
plasma of untreated mice detected. B, C5a levels during the onset of sepsis
C5a generation in plasma from wild-type, fD2/2, and C1q2/2 in various mice strains as depicted. *p , 0.05 for statistical significance
mice 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
To underline the comparability of the used mouse strains with are representative of 5–12 animals per group and are depicted as mean 6
respect to their ability to generate one known key player, C5a, we SEM. wt, wild-type.
The Journal of Immunology 3069
(Fig. 3, right panel) and compared them to the corresponding nounced organ dysfunction in fD2/2 and C1q2/2 mice when
sham group (Fig. 3, left panel). In wild-type animals we observed compared with control mice, with these effects being most pro-
a low baseline of complement C3/C3b/C3c deposition in the renal minent in fD2/2 mice. No significant increases during the onset
tubuli at 0 h that was clearly increased 3 h after CLP (Fig. 3, top of sepsis were found in any of the three groups for alkaline
panel). In C1q2/2 mice as well as in fD2/2 mice we did not find phosphatase and plasma albumin (data not shown).
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). Bacterial load in blood, lung, liver, and kidney
Similar observations were made in the renal cortex section with Bacterial clearance represents one major endpoint for innate host
mild C3c deposition in the glomeruli in wild-type mice that was immune response to infection that is thought to be greatly influ-
strongly increased 3 h after CLP, whereas no C3/C3b/C3c de- enced by intact complement activation and formation of the ter-
position was detected in both complement knockout mice strains minal complement activation products. We therefore sought to
(data not shown). These data indicated a crucial contribution of determine the capacity of alternative and classical complement
both the classical and the alternative pathway to overall C3 cleavage pathway knockout mice to clear bacteria during the onset of sepsis
and therefore activation of the complement system in sepsis. in comparison with untreated control mice. Given the above-
depicted lowered survival and increased organ dysfunction in
Effects of fD and C1q knockout on organ dysfunction during
fD2/2 and C1q2/2 mice, we expected to find increased bacterial
CLP-induced sepsis
loads in both knockout strains. C1q2/2 mice demonstrated sig-
To establish whether the lack of factor D or C1q could be spe- nificantly elevated aerobic bacterial loads in lung, liver, and blood
cifically linked to organ dysfunction during experimental sepsis, we and a strong but not significant similar tendency in kidney 6 h after
analyzed organ-specific indicators in the plasma of septic mice at 6 CLP when compared with control mice (Fig. 5A–D). Surprisingly,
and 24 h after induction of CLP (Fig. 4A–D). All groups, C1q2/2, fD2/2 mice did not demonstrate such increases in bacterial organ
PAMP. We found that NF-kB activation, as detected in leukocytes function of factor D for NF-kB–dependent cytokine generation in
pooled from 24 mice per experiment upon stimulation with LPS, mouse leukocytes.
reached higher levels in fD2/2 mice when compared with wild-
type and C1q2/2 mice (Fig. 10A), which became even more ev- Discussion
ident after grayscale value density analysis (Fig. 10B). It is well known that sufficient activation of the complement system
To investigate the significance of this finding for mediator during infection is essential for a successful immune response and
generation in isolated mouse leukocytes ex vivo, supernatants of defense. However, the individual contributions of the separate
complement activation pathways for known changes in host de-
leukocytes pooled from 24 mice per strain were obtained 2 h after
fense and innate immune functions during sepsis have not yet been
stimulation with LPS and analyzed for cytokine generation using
investigated in detail. Knockout mice lacking all three complement
CBA assay. The results in Fig. 10C–F demonstrated that, cor- activation pathways demonstrated extreme susceptibility to in-
responding to the findings during sepsis in whole blood, leuko- fection caused by bacterial challenge, and results with C1q2/2
cytes from fD2/2 mice generated higher levels of IL-6, TNF-a, mice demonstrated involvement of the classical pathway (14).
MCP-1, and IFN-g when compared with neutrophils from C1q2/2 Similar results were previously found using these two knockout
and control mice. These results suggested a potential controlling strains in the CLP sepsis model (15). Both reports suggested
3072 CONTRIBUTION OF COMPLEMENT PATHWAYS IN SEPSIS
intact clearance of bacterial challenges early during sepsis. In- upon infectious stimuli (Fig. 10) and found this to be strongly
terestingly, we found that the bacterial clearance at 24 h after CLP enhanced. NF-kB activation is well known to promote cytokine
displayed a different situation when compared with the 6 h values: generation (25) as well as mechanisms leading to neutrophil re-
fD2/2 mice now presented with a significantly increased bacterial cruitment (26). We therefore suggest this mechanism to be at least
load in liver when compared with wild-type and C1q2/2 mice and partially responsible for the observed increases in inflammatory
a similar, but not statistically significant, tendency for kidney and mediator generation and increased MPO levels in lung and whole
whole blood (Fig. 5E–H). These findings add a new aspect since blood. Note that based on the knockout of a soluble serum factor
the importance of the separate pathways for bacterial clearance such as factor D, it may not directly be expected that cell-derived
appears to be time-dependent. The classical pathway is apparently effects can be observed. Based on the available data, we can
very important for clearing bacteria in the early development of currently only speculate that factor D may be involved in con-
sepsis, whereas the alternative pathway may play a more impor- trolling NF-kB activation in blood neutrophils and that knockout
tant role for the later phase of development. of this factor may lead to increased cell signaling via NF-kB.
A recent report suggested an important protective role of pro- Further experiments are warranted to define the underlying mech-
perdin, a cofactor of the alternative complement system, for out- anisms in more detail.
come during experimental sepsis (24). These findings are in line Interestingly, in models of chronic-type noninfectious in-
with our findings in fD2/2 mice with respect to the outcome flammatory diseases, such as asthma or autoimmune nephropathy,
results, demonstrating an important role of the alternative pathway inactivation of the alternative pathway showed beneficial effects,
for survival during experimental sepsis. A critical role for the partially by lowering the inflammatory response (2, 27, 28). In
alternative pathway has been also identified before for survival of these models, the activation of the alternative pathway was sug-
mice infected with Pseudomonas aeruginosa in a murine model of gested to act as an amplification loop triggering the hyperactive
pneumonia (22). In vitro studies with serum from fD2/2 mice
Acknowledgments 18. Ebanks, R. O., and D. E. Isenman. 1996. Mouse complement component C4 is
devoid of classical pathway C5 convertase subunit activity. Mol. Immunol. 33:
We thank Marina Botto for the use of C1q knockout mice for the experi- 297–309.
ments, and we are grateful to Iris Suckert and Ulrike Vetterling for excellent 19. Huber-Lang, M., J. V. Sarma, F. S. Zetoune, D. Rittirsch, T. A. Neff,
assistance. S. R. McGuire, J. D. Lambris, R. L. Warner, M. A. Flierl, L. M. Hoesel, et al.
2006. Generation of C5a in the absence of C3: a new complement activation
pathway. Nat. Med. 12: 682–687.
Disclosures 20. Ward, P. A. 2010. The harmful role of c5a on innate immunity in sepsis. J. Innate
Immun. 2: 439–445.
The authors have no financial conflicts of interest. 21. Takahashi, K., L. Shi, L. D. Gowda, and R. A. Ezekowitz. 2005. Relative roles of
complement factor 3 and mannose-binding lectin in host defense against in-
fection. Infect. Immun. 73: 8188–8193.
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