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Properdin Plays a Protective Role in Polymicrobial Septic Peritonitis¹

Cordula M. Stover^2,*, Jeni C. Luckett^*, Bernd Echtenacher, Aline Dupont^*, Sue E. Figgitt^*, Jane Brown, Daniela N. Männel and Wilhelm J. Schwaeble^*

^* Department of Infection, Immunity and Inflammation and Biomedical Services, University of Leicester, Leicester, United Kingdom; and Institute of Immunology, University of Regensburg, Regensburg, Germany

	Abstract

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Properdin is a positive regulator of complement activation so far known to be instrumental in the survival of infections with certain serotypes of Neisseria meningitidis. We have generated a fully backcrossed properdin-deficient mouse line by conventional gene-specific targeting. In vitro, properdin-deficient serum is impaired in alternative pathway-dependent generation of complement fragment C3b when activated by Escherichia coli DH5. Properdin-deficient mice and wild-type littermates compare in their levels of C3 and IgM. In an in vivo model of polymicrobial septic peritonitis induced by sublethal cecal ligation and puncture, properdin-deficient mice appear immunocompromised, because they are significantly impaired in their survival compared with wild-type littermates. We further show that properdin localizes to mast cells and that properdin has the ability to directly associate with E. coli DH5. We conclude that properdin plays a significant role in the outcome of polymicrobial sepsis.

	Introduction

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Cecal ligation and puncture (CLP)³ is a model for acute polymicrobial septic peritonitis following perforated appendicitis or diverticulitis. The severity of this model for the animal varies with length of the ligated cecum, needle size, and number of perforations (1). Complement activation, mast cells, and neutrophils are some of the outcome-determining factors of the host. Blockade of C5a or its receptor C5aR is beneficial for survival of the acute model of CLP in mice (2, 3) by suppression of C5a-mediated functions of peritoneal neutrophils (migration, oxidative burst, phagocytic response) (4). In the subacute model of CLP, however, neutrophil recruitment to the peritoneal cavity (5) and IL-12-mediated, IFN--driven neutrophil phagocytosis (6) are important for survival. During acute CLP, neutrophil migration is effected by TNF, which is produced locally by peritoneal mast cells, which, thereby, are significant players in disease outcome (7). Mast cell degranulation, TNF release, and neutrophil infiltration are significantly impaired in C3-deficient mice during acute CLP, accounting for their high mortality compared with wild-type (WT) mice (8). Properdin is an oligomeric serum protein that amplifies ongoing complement activation. By virtue of each of its monomers to bind to C3b, it acts to extend the half-lives of the C3-converting and C5-converting enzyme complexes (C3bBb and C3b_nBb), respectively (9, 10, 11). These are generated after activation of the alternative pathway of complement via deposition of C3b to target surfaces by so-called spontaneous tickover of fluid phase C3 or by activation of classical or lectin pathways (via recognition of immune complexes or carbohydrate moieties on microorganisms, respectively). C1q-deficient mice (deficient of the recognition molecule of the classical pathway) and mice with a dual deficiency of factor B and C2 (affecting activities for lectin, classical and alternative pathways) (all on Sv/129 background) have previously been studied in sublethal CLP (12). The study concluded that intact lectin and alternative pathways are important for survival of sublethal CLP; however, it did not allow quantification of the separate contribution of the alternative pathway amplification to this effect. Therefore, the sublethal CLP model seemed a highly suitable model to characterize the phenotype of a properdin-deficient mouse line, which we have developed and describe herewith for the first time. So far, properdin deficiency is known in humans and shows variable penetrance with predilection to succumb to fatal meningococcal disease when infected with uncommon serotypes Y and W-135 (13).

	Materials and Methods

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Generation of a properdin-deficient mouse line

The mouse properdin gene was isolated from a mouse (129Sv) genomic bacteriophage library (2001) using mouse properdin cDNA (European Molecular Biological Laboratory/GenBank accession number X12905) as a probe. DNA of three hybridizing clones was purified using a Nucleobond phage purification kit (Clontech) (14). BamH1 digest of two overlapping clones yielded 7.6-, 3.4-, and 0.5-kb fragments (corresponding to bp position 10,309–21,407 of the mouse BAC clone, European Molecular Biological Laboratory/GenBank accession number AL671853.7) that were subcloned in pBSKS+ and sequenced. A targeting construct was generated in pKO-NTKV 1901 (Stratagene) designed to contain the 5' part of the properdin gene (promoter region, exon for thrombospondin repeat (TSR) 1) and the 3' part of the gene (comprising exons for TSR 5, 6a, and 6b) flanking a positive selection marker gene (neo^r). Embryonic stem cells derived from male 129/Ola mice (E14.1a) were transfected with the linearized construct by electroporation. Clones were grown in G418 (positive selection for the presence of neo^r) and gancyclovir (negative selection against random integration, which produces thymidine kinase activity). Homologous recombination of this vector with the WT gene results in a replacement of exons 3–7 (1.6 kb) coding for the TSR 2–4 modules by the neo^r gene (1.6 kb) of the targeting vector. Positive clones were identified by Southern blotting through the presence of a 6-kb band in HindIII-digested genomic DNA (an additional HindIII site is introduced during targeting) using a probe external to the targeted region. One of the 170 embryonic stem cell clones screened contained the disrupted allele of the targeted gene and was microinjected into C57BL/6 blastocysts which were implanted in pseudopregnant mice. Two male chimeras were derived. Germline transmission of the targeted allele was obtained upon mating to C57BL/6 mice (agouti offspring). As expected, all male offspring were WT and all female offspring were heterozygous for the targeting event. Target-specific PCR with restriction mapping of this fragment with BstX1 showed correct replacement in these heterozygotes. Intercrossing resulted in hemizygous males, heterozygous females, and WT offspring, as identified by PCR, at the expected ratio. The properdin-deficient mice appear normal in weight and fertility and are maintained in a barrier unit according to institutional guidelines. The mice used in these experiments were backcrossed onto the C57BL/6 background for 9 and 10 generations. WT littermates were controls.

Rabbit RBC lysis

Target cells were prepared as follows. Rabbit blood (Biomedical Services, University of Leicester, Leicester, U.K.) was collected in Alsever’s solution (114 mM sodium citrate, 27 mM glucose, and 72 mM sodium chloride, pH 6.1), spun down at 200 x g, washed twice in PBS, and the pellet was resuspended in 1x VBS (727 mM sodium chloride, 16 mM 5,5-diethylbarbituric acid, and 9 mM 5,5-diethylbarbituric acid sodium salt)-20 mM Mg²⁺-8 mM EGTA (pH 7.4) to where lysis of 50 µl of RBC in 200 µl of water gave an absorption at 413 nm of 1.0–1.2 (positive control). The assay was set up in duplicate, diluting 100 µl of mouse serum (sample and heat-inactivated control) with 100 µl of VBS-Mg²⁺-EGTA, then adding 50 µl of adjusted rabbit RBC. The cells in 200 µl of VBS-Mg²⁺-EGTA were the negative control. After 2 h at 37°C, absorptions were read and hemolysis was expressed as a percentage against the controls (relative hemolysis = [(mean OD sample – mean OD heat inactivated mouse serum)/(mean OD water – mean OD buffer)] x 100%) (15).

C3 activation of WT and properdin-deficient sera

Properdin-deficient or WT mouse sera were diluted 1/20 in either PBS⁺⁺ (with Ca²⁺ and Mg²⁺), GVB⁺⁺, GVB/Mg²⁺-EGTA, or GVB⁺⁺-EDTA buffers and incubated at 37°C with 1 x 10⁶/ml formalin-fixed (10% formal saline) E. coli DH5. (washed in the respective buffers). After 30 min, the supernatant, that is, activated serum, was removed and separated under reducing conditions (2-ME, boiling) on 10% SDS-polyacrylamide gels. After electrophoretic transfer using cellulose membrane 2micron (Bio-Rad Laboratories), the membrane was incubated with monoclonal biotin-coupled rat anti-mouse C3 (Cedarlane Laboratories), detected with a streptavidin-HRP ABC kit (DakoCytomation), and developed using ECL reagent (Pierce).

IgM and C3 levels in mouse sera

Serum was prepared from mouse blood obtained via the tail vein and frozen in aliquots. Serum levels for IgM and C3 were determined by sandwich ELISAs according to the manufacturers’ manuals (Bethyl Laboratories, Universal Biologicals and Immunology Consultants Laboratory, Immune Systems) and analyzed using a five-parameter logistics curve.

Sublethal CLP

Mice were anesthetized by i.p. injection of 75 mg/kg Ketanest (Parke-Davis) and 16 mg/kg Rompun (Bayer). The cecum was exteriorized and the distal end (<30%) was ligated and punctured once with a needle (0.4-mm diameter, 27-gauge) to achieve a sublethal CLP as previously described (16). Mice were observed for 2 wk. Kaplan-Meier survival curves were compared using the log rank test. The experiments were performed in compliance with federal guidelines for animal experimentation (State of Bavaria, Germany).

Immunohistochemical and immunofluorescent analysis of mouse mast cells

Four-micrometer sections were cut from paraffin-embedded connective tissue obtained from the abdominal wall, incubated with proteinase K (2 mg/ml), 3% H₂O₂, and, after blocking with 5% BSA and application of SeroBlock FcR (BUF041A; Serotec) were incubated with goat anti-human properdin IgG (1/100; Nordic Immunology), or as control, goat anti-rabbit IgG (P0448; DakoCytomation), and rabbit anti-goat Igs, HRP-conjugated (1/400, A5420; Sigma-Aldrich). After development with diaminobenzidine tetrahydrochloride (D5637; Sigma-Aldrich), sections were counterstained with toluidine blue. For immunofluorescent analysis, sections were processed as above, incubated with FITC-coupled goat anti-human properdin IgG (1/200; Nordic Immunology) or rabbit anti-human mast cell tryptase Ab (1/400, sc-32889; Santa Cruz Biotechnology) and goat-anti-rabbit IgG, F(ab')₂-TRITC (1/400, sc-3841; Santa Cruz Biotechnology). Fluorescence signals were analyzed using a Nikon TE300 wide-field epifluorescence microscope.

Binding of properdin to E. coli DH5

Formalin-fixed E. coli DH5 (1 x 10⁶/ml) were incubated with human sera (diluted 1/20 in PBS/Mg²⁺/5% BSA) of known concentration of properdin (determined using a properdin ELISA kit obtained from Antibodyshop). After washing with PBS, monoclonal anti-human properdin Ab (HYB 039-06, diluted 1/1000; AntibodyShop) was added for 1 h and, after another washing step with PBS, FITC-coupled anti-mouse Fab Igs (Sigma-Aldrich) were added (1/4000). Fluorescence signals were analyzed as above. In parallel, an equal concentration of purified properdin (purity >98%; Europa Bioproducts) was added (diluted in PBS/Mg²⁺/5% BSA to the predetermined level for the respective sera) and slides were processed as above. Part of this latter incubation, along with a control (E. coli without properdin), was used for immunoprecipitation studies, in which the microbes are washed, then lysed in radioimmunoprecipitation assay buffer with protease inhibitors (P8340; Sigma-Aldrich), sheared, precleared with protein G-Sepharose (P3296; Sigma-Aldrich), and incubated with Sepharose-coupled monoclonal anti-human properdin Ab (HYB 039-06; AntibodyShop). The immunoprecipitate was run on a 10% SDS-polyacrylamide gel, blotted, and probed with rabbit anti-human properdin Ab.

	Results

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Generation of properdin-deficient mice

A properdin-deficient mouse line was generated by specific targeting and disruption of the murine properdin gene, which is located on chromosome X (17). The strategy was to delete those exons coding for thrombospondin repeats (TSR) with importance in binding to C3b and thereby stabilization of the C3bBb complex or for oligomerization of the protein (18) (Fig. 1A). Homologous recombination of mouse properdin DNA contained in the targeting vector and segments of the properdin gene in 129/Ola embryonic stem cells was confirmed in 1 of 170 clones (Fig. 1B). This clone was microinjected in C57BL/6 blastocysts and two chimeric mice were derived. Both showed germline transmission and produced heterozygous female and WT male offspring. These were intercrossed, obtaining genotypes at the expected Mendelian ratio and X chromosome linkage. Heterozygous mice were backcrossed to C57BL/6 to obtain male WT and properdin-deficient littermates (Fig. 1C). The hemizygous properdin gene-targeted mice lack properdin protein in their sera (Fig. 1D) and they are grossly impaired in the alternative pathway-dependent diagnostic test of lysing rabbit RBC (19) (Fig. 1E). Heterozygous mice showed an intermediate phenotype (data not shown).

View larger version (51K): [in this window] [in a new window]   FIGURE 1. Generation of a properdin-deficient mouse line. A, Representation of the strategy to target the murine properdin gene, to replace exons 3–7 with a selection marker, and to detect specificity of this event. B, Southern blot analysis of HindIII-restricted genomic DNA prepared from positively and negatively selected embryonic stem cell clones using the external probe of 781 bp (5' external probe) and the 3.4-kb fragment of cassette A (internal probe), see A, as probes. They both hybridize with a 6-kb band as indicated for lane 3. The size of the WT gene on HindIII restriction is 11 kb. The clones in lanes 1, 2, and 4 are either negative for any recombination event or nonhomologous recombinants. C, Typical agarose gel electrophoresis of PCR products obtained when genotyping offspring (see A for location of products). D, Verification of the absence of properdin in serum of hemizygous, gene-targeted mice using monoclonal anti-human properdin Ab with cross-reactivity to mouse protein (HYB 039-04, diluted 1/1000; AntibodyShop) by Western blot. (The secondary Ab (rabbit anti-mouse Ig, HRP conjugated) used for detection of the primary (anti-properdin) Ab) reacts with Igs contained in the sera.) E, Impairment of alternative pathway-mediated rabbit RBC lysis by properdin-deficient mice in comparison with WT littermate control. One of three representative experiments using two-sera set up in duplicate is shown.

Western blot analysis was performed for different WT and properdin-deficient sera activated by E. coli DH5 in four different buffer conditions. One of three representative experiments is shown (Fig. 2): incubation of serum diluted in PBS/Mg²⁺/Ca²⁺ with the microbe leads to complete conversion of C3 to C3b and iC3b for both WT and properdin-deficient serum. In buffer conditions that restrict activation of complement, either selectively (GVB⁺⁺, GVB⁺⁺EGTA) or globally (GVB⁺⁺EDTA), by contrast, the uncut 113-kDa C3 chain is seen (lanes 2–4 and 6–8, Fig 2). In conditions favoring activation of complement via the alternative pathway (lanes 3 and 7, Fig. 2), properdin-deficient serum shows a clear reduction of the 104-kDa activation product C3b. Further degradation of iC3b to the 40-kDa -chain product occurs in conditions that preclude C3 binding (EDTA) and in those inhibiting assembly of all recognition molecules to surfaces (GVB⁺⁺/GVB⁺⁺EGTA), but not in the most physiologic of these conditions (PBS⁺⁺).

View larger version (89K): [in this window] [in a new window]   FIGURE 2. C3 activation in properdin-deficient mice. Western blot for C3 activation products in WT and properdin-deficient sera after incubation with E. coli DH5 in different buffer conditions (lanes 1 and 5: PBS with Ca2+ and Mg2+; lanes 2 and 6: GVB++; lanes 3 and 7: GVB/Mg2+-EGTA; and lanes 4 and 8: GVB++-EDTA). One of three experiments is shown using WT and properdin-deficient littermate sera. The band indicated to represent C3dg is seen on longer exposure for the properdin-deficient sera.

Survival of WT and properdin-deficient mice in CLP

The severity of the CLP model was chosen to produce sublethal and prolonged inflammation. In the first 7 days of the observation period, 14 of 16 WT mice survived, whereas only 6 of 16 properdin-deficient mice survived (Fig. 3).

View larger version (14K): [in this window] [in a new window]   FIGURE 3. Properdin-deficient mice are impaired in their survival after sublethal CLP. Properdin-deficient mice and WT littermates (n = 16, each group) were subjected to sublethal CLP and mortality was recorded (p = 0.003; log rank statistic).

Sections of connective tissue mast cells of unchallenged WT mice were analyzed for reactivity of properdin by standard immunohistochemistry. Representative figures from two independent experiments clearly demonstrate properdin reactivity localizing to the granules of mast cells (Fig. 4A), which compares to the granule-specific metachromatic stain produced by toluidine blue on the control section (Fig. 4B). Subsequent immunofluorescence analysis of gut sections from WT and properdin-deficient mice using a mast cell marker, tryptase, confirmed that reactivities of properdin and tryptase overlap for WT, but not properdin-deficient mice (Fig. 4, C–F).

View larger version (53K): [in this window] [in a new window]   FIGURE 4. Properdin is produced by mast cells. Immunohistochemical analysis of connective tissue mast cells using goat anti-human properdin (A) or control Ab (B). Both sections were developed with diaminobenzidine tetrahydrochloride (brown stain) and counterstained with toluidine blue (purple stain). Immunofluorescent analysis showing reactivity of properdin (C), mast cell marker tryptase (D), and overlap (E) for gut mucosa (4',6-diamidino-2-phenylindole stained, F) of WT, but not properdin-deficient mice.

Properdin associates directly with E. coli DH5

Incubation of formalin-fixed E. coli DH5 with human serum of known properdin concentrations or with the same amount of purified properdin on its own leads to the same patchy reactivity with the monoclonal anti-properdin Ab (Fig. 5, A–C). This association is absent on chelation of serum with EDTA (data not shown). There is no detectable C3 in the properdin preparation (Fig. 5D). To ascertain the physical association between E. coli and properdin, immunoprecipitation was performed using the monoclonal anti-properdin Ab or a control Ab followed by Western blot analysis of immunoprecipitates using a polyclonal anti-properdin Ab. A 53- kDa band for monomeric properdin is detected for the specific immunoprecipitate (Fig. 5E).

View larger version (39K): [in this window] [in a new window]   FIGURE 5. Properdin associates with E. coli DH5. A, After incubation of E. coli DH5 with human serum (determined at 27 µg/ml properdin), properdin was detected using a monoclonal anti-human properdin Ab and FITC-coupled anti-mouse Fab Igs. B, After incubation of E. coli DH5 with purified properdin (25 µg/ml stock), bound properdin was detected as above. C, Negative control (addition of the secondary Ab only). Different sera were used and the amount of exogenous properdin was adjusted to the respective serum concentration. This is a representative figure of three such experiments. D, Aliquots of the purified properdin preparation were separated by SDS-PAGE and analyzed for possible C3 content using mouse anti-human C3 (ab11871; Abcam) (strip 1). Strip 2 of the same transfer was developed with mouse anti-human properdin Ab (HYB 039-06; AntibodyShop) and shows the expected 53-kDa band for monomeric properdin. E, Immunoprecipitation of properdin bound to E. coli DH5 (lane 2) and control (lane 1) using monoclonal anti-human properdin Ab. Transferred lysates were developed with polyclonal goat anti-human properdin Ab. The 53-kDa band for properdin is indicated. The secondary Ab (rabbit anti-goat Ig, HRP conjugated) reacts with Igs contained in both immunoprecipitates. The H chain of Ig is indicated.

	Discussion

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Properdin-deficient and WT littermates were subjected to sublethal CLP, a model of subacute peritoneal inflammation provoked by polymicrobial leakage from punctured distal cecum after ligation, which develops into sepsis. In this model, severity is measured by mortality (22). The present study finds that in the absence of properdin, survival after sublethal CLP-induced polymicrobial sepsis over an observation period of 14 days is significantly impaired. Natural Abs, germline-expressed Igs of the IgM type, bind to bacterial Ags and are protective in models of septic and endotoxin shock (23, 24). However, there was no difference between properdin-deficient (n = 4; 41.9 µg/ml ± 2.45 SD) and WT (n = 5; 43.14 µg/ml ± 9.89 SD) mice in their total serum IgM levels.

Mast cells are crucially important to survive CLP, relating to a significant degree to their ability to synthesize TNF- (7) and this study finds that mast cells express properdin. A direct association of properdin on the surface of fixed E. coli DH5 was investigated by incubation with human serum or with purified properdin. Properdin is found associated with E. coli DH5 and may thereby provide a scaffold for increased C3b or C3Bb binding as recently proposed (25). The properdin protein contained in the specific immunoprecipitate from E. coli after prior incubation with human properdin (Fig. 5E) compares to the protein identified as mouse properdin in WT serum (Fig. 1D). Our finding that properdin associates directly with E. coli DH5 (exhibiting so-called rough LPS) is strongly supported by recently published work showing that properdin binds to rough, not WT, so-called smooth, E. coli LPS mutants (26).

Based on the findings obtained for the in vitro and in vivo phenotype of the properdin-deficient mice, the immune response of the WT mice in this model of sublethal CLP is likely to be influenced by these properdin-dependent factors: optimal, i.e., properdin-stabilized, C3b generation is crucial to attract neutrophils to the peritoneal cavity (5, 6) through C3b-mediated TNF- release by mast cells (27). Neutrophils store, among other factors, properdin in their secondary granules (28) and so do mast cells. On release of properdin, it can associate with, and independent of, C3b to the surface of the microorganism (26). The molecular implications of these modes of properdin binding are currently under investigation.

	Acknowledgments

 
This publication contains a part of Lynda Dupont’s doctoral thesis. We are grateful to Prof. K. B. M. Reid (University of Oxford, Oxford, U.K.) for providing the mouse properdin cDNA clone and rabbit anti-human properdin antiserum and to Dr. C. Pritchard (University of Leicester) for advice and for providing the genomic library. We thank Mariya Hristova (University College London, London, U.K.) for helpful advice regarding the hemolysis test, Kathryn Staley (University of Leicester) for performing properdin ELISA measurements, Dr. Rana El-Rachkidy-Lonnen for useful discussions, and Claudia Dembek (Ludwig-Maximilians-University, Munich, Germany) and Irina Elliott (University of Leicester) for technical assistance.

	Disclosures

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The authors have no financial conflict of interest.

	Footnotes

 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ The work was funded by the Medical Research Council (Grant G0400300 to C.M.S.), the Wellcome Trust (Grant 060574 to W.J.S.), and Deutsche Forschungsgemeinschaft (Grant MA760/10-3 to D.N.M).

² Address correspondence and reprint requests to Dr. Cordula M. Stover, Department of Infection, Immunity and Inflammation, University of Leicester, University Road, Leicester LE1 9HN, U.K. E-mail address: cms13{at}le.ac.uk

³ Abbreviations used in this paper: CLP, cecal ligation and puncture; TSR, thrombospondin repeat; WT, wild type.

Received for publication August 10, 2007. Accepted for publication December 14, 2007.

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