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Division of Rheumatology, Department of Medicine, School of Medicine, Washington University, St. Louis, MO 63110
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Abstract |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionDisclosuresReferences |
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The AP was described first by Louis Pillemer and his collaborators (12) over 50 years ago. The centerpiece of their study was the serum protein properdin. In their report, Pillemer et al. (12) provided evidence that properdin:target complexes directed Ab-independent complement activation. At the time, most all of immunology dealt with Ab reactions of great specificity, while the properdin system was relatively nonspecific and one of the first examples of "natural immunity" (13). Thus, amid much publicity, the properdin system was received with great interest. Soon, however, the properdin work encountered formidable objections, notably those of Nelson (14), who showed that properdin’s "activity" could be due to Ab contaminants. With Pillemer’s tragic death (15), properdin lost its strongest advocate and was largely dismissed by the scientific community (16). The properdin system was reborn as the AP >20 years later (16), but the properdin protein itself was cast in a relatively nonspecific role (17).
The C3 convertases are serine proteases that cleave the serum protein C3 at a single site, generating C3b and C3a. C3a is an anaphylatoxin that stimulates local inflammatory processes, while nascent C3b covalently attaches nonselectively to surfaces via its highly reactive thioester moiety. Membrane-bound C3b promotes immune clearance and cell lysis. In the long-standing "textbook" model for the initiation of the AP, nascent C3b generated by the slow nonspecific fluid-phase activation of C3 covalently attaches to nearby targets (18, 19, 20). Target-bound C3b then associates with factor B and the resulting C3bB complex is cleaved by factor D. Two factor B fragments are generated, with the Ba fragment released and the Bb fragment remaining as part of the AP C3 convertase, C3bBb. The C3bBb complex is relatively unstable, with a t1/2 of 90 s under physiological conditions (21, 22). Properdin associates with C3bBb, resulting in a 5- to 10-fold more stable complex (17). By this model, target specificity is largely determined by the action of regulatory proteins that suppress complement activation on host tissues by inactivating bound C3b and resulting convertases. Although this mechanism can be demonstrated in vitro (23, 24), it does not provide a comprehensive accounting for in vivo target specificity (25). Recently, we observed that properdin bound to a biosensor surface serves as a platform for in situ convertase assembly (26). These new results raise again the possibility that properdin:target complexes initiate AP activation. In this report, we re-examine this issue.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionDisclosuresReferences |
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Properdin, C3b, factor B, and factor D were obtained from CompTech, formerly Advanced Research Technologies. The properdin was analyzed by SDS gel electrophoresis followed by Coomassie-staining and formed a single band of 
53 kDa; no other bands were visible. Mouse anti-human properdin mAb (IgG1 
catalog no. A235) and mouse anti-human Bb mAb (IgG1 binds factor B and the Bb fragment; catalog no. A227) were purchased from Quidel. The mouse anti-human C3d mAb (IgG1 binds C3, C3b, and C3d) used for the experiments described in Figs. 3–5 and 7 was obtained from Antibody Shop (catalog no. HAV 003-05). The mouse anti-human C3d mAb (IgG1 binds C3, C3b, and C3d) used for the experiments described in Fig. 6 was obtained from Quidel (catalog no. A207). FITC-labeled goat anti-mouse IgG was obtained from Sigma-Aldrich. FITC-labeled goat anti-human C3 was obtained from Cappel. C8-depleted human serum was obtained from Quidel. C7-depleted human serum was obtained from CompTech. Properdin-deficient and C7-deficient human sera were gifts from P. Densen (University of Iowa, Iowa City, IA). Normal human serum was obtained from healthy volunteers and prepared as described (27).
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Neisseria gonorrhoeae strain N401 (recA6, (kan); Ref. 28), a gift from M. Koomey (University of Oslo, Oslo, Norway) was used throughout. Rec A6 is an isopropyl 
-D-thiogalactoside-inducible allele of rec A. In this study, rec A was not expressed, minimizing antigenic variation. Plates made with GC agar supplemented with 1% IsoVitaleX (BD Biosciences) were inoculated with N. gonorrhoeae from frozen stock and incubated for 18–20 h at 37°C in 5% CO2 before use. In some cases, 24-h cultures were transferred to fresh plates and grown 18–20 h. Bacteria were washed in Mg2+ EGTA buffer (29), and resuspended to an OD595 of 0.5 (5 x 108/ml). They were treated in the same buffer with various human complement proteins including properdin (10 µg/ml), C3b (10 µg/ml), factor B (10 µg/ml), and factor D (1 µg/ml) for 30 min at 37°C and washed twice in this buffer between incubations. In some experiments, Neisseria were first incubated with properdin, washed twice, and then incubated in 5% properdin-deficient human serum in Mg2+ EGTA buffer for 10 min at 37°C. Under these buffer conditions, AP activation is permitted but CP and LP activation are excluded. In some experiments, the 5% properdin-deficient serum was supplemented with purified properdin (10 µg/ml). The data presented in each panel of Fig. 3 is representative of at least four independent experiments.
Escherichia coli K-12 strains (Table I) were obtained from the E. coli Genetic Stock Center (Yale University, New Haven, CT). E. coli cultures were grown from single colonies in Luria-Bertani medium overnight at 37°C, diluted 20-fold in fresh medium, and allowed to grow 1.5 h before use. Bacteria were suspended and washed in Mg2+ EGTA buffer, diluted to an OD595 of 0.5 (5 x 108/ml), and treated with a properdin and/or human normal or C7-deficient serum and analyzed by FACS as described for Neisseria. Strains were initially screened for properdin-binding capacity by incubating the bacteria with properdin at 10 µg/ml for 10 min at 37°C. Strains that bound properdin were later found to bind properdin within 1 min at 37°C. The FACS data presented in each panel of Fig. 7 and summarized in Table I is representative of at least three independent experiments.
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Rabbit erythrocytes (ER) and sheep erythrocytes (ES) were purchased from the Colorado Serum Company, washed in Mg2+ EGTA buffer and resuspended at 108/ml. They were treated in the same buffer with various human complement proteins including properdin (3 µg/ml), C3b (10 µg/ml), factor B (10 µg/ml), and factor D (1 µg/ml) for 30 min at 37°C and washed twice in buffer between incubations. The data presented in each panel of Fig. 5 is representative of at least four independent experiments.
Cells
Human 293T kidney cells (CRL-11268; American Type Culture Collection) were used for protein generation. They were maintained in DMEM (Mediatech) containing 10% FCS (Harlan Breeders). Media were supplemented with L-glutamine (2 mM final concentration; Sigma-Aldrich), nonessential amino acids (BioWhittaker), and penicillin and streptomycin (10 U/ml and 100 µg/ml; Cellgro, Mediatech). The hybridoma cell line BRIC 256 was maintained in RPMI 1640 containing all the supplements mentioned for the DMEM medium described above and additionally supplemented with 1 mM sodium pyruvate and 0.05 mM 2-ME (Sigma-Aldrich).
Construction of scFv-properdin that binds mouse erythrocytes
The human properdin coding sequence (minus the signal peptide region) was amplified by PCR from a human U937 cDNA library provided by J. Verbsky (Medical College of Wisconsin, Milwaukee, WI). Its 1356-bp sequence (flanked with the BsiWI and XbaI restriction enzyme cutting sites at the 5' and 3' termini, respectively) is identical to the published sequence that was derived from the same cell line (30). The 735-bp anti-mouse glycophorin A (GPA) (m)scFv sequence flanked by BsiWI restriction sites was derived directly from sT-DAF (31). The NH2-(m)scFv-properdin-COOH construct was prepared from the sT-DAF plasmid and the above properdin and (m)scFv-coding segments in the following steps: 1) the sT-DAF plasmid was digested with BsiWI and XbaI, releasing a 960-bp DAF fragment and a 735-bp scFv fragment but retaining the signal peptide sequence; 2) the two excised fragments were then replaced by the properdin sequence at the BsiWI and XbaI sites; 3) the intermediary plasmid (designated pNCIThProp) was then cut at the BsiWI site (between the signal peptide and properdin coding sequences) and the (m)scFv fragment (above) was introduced in the appropriate direction at that site. The mature plasmid was designated pNscThProp.
Construction of scFv-properdin that binds human erythrocytes
The scFv with specificity for human GPA was generated essentially as described (31) from the mouse hybridoma cell line BRIC 256 (32), provided by R. Mushens (International Blood Group Reference Laboratory, Bristol, U.K.). This line was chosen because it secretes an IgG1 mAb that recognizes a blood group-independent epitope on human GPA (33) and is therefore broadly applicable. Total RNA was isolated (RNeasy; Qiagen). Reverse transcription, followed by PCR (RT-PCR), was conducted using the SMART technology (BD Clontech) using primer combinations described previously (34). The resulting H and L chain variable cDNA fragments were subcloned into pCR2.1-TOPO (Invitrogen Life Technologies). After introducing suitable flanking restriction sites via PCR, the VH and VL chains were combined with a (G4S)3 linker resulting in the scFv Bric-256. Analysis of the amino acid primary sequence to determine the complementarity determining regions of the scFv Bric-256 was performed by applying the rules described at Ab Structure and Sequence Information version 2.0 (www.rubic.rdg.ac.uk). The 744-bp BsiWI fragment of scFv Bric-256 was inserted into pNCIThProp to complete the NH2-(hu)scFvP-COOH coding region. The mature plasmid was designated pN256hProp. All PCR-derived sequences were verified by DNA sequencing.
Production and characterization of recombinant proteins
Recombinant protein was prepared by transient expression in 293T cells using Invitrogen Life Technologies Opti-mem serum-free medium and TransIT-293 (MIR2700; Mirus) transfection reagent, as per the manufacturer’s instructions. To obtain concentrated protein stocks, the supernatants were applied to centrifugal filter devices with a 10-kDa molecular cut-off (Centricon Plus-20; Millipore). Concentration of the recombinant forms of human properdin, (m)scFv-P and (hu)scFv-P, was initially determined by ELISA using goat anti-human properdin polyclonal Ab (1/1000) for capture, anti-human properdin mAb (1/5000) and peroxidase-conjugated donkey anti-mouse IgG polyclonal Ab 1/3000) for detection. Native properdin was used to construct a standard concentration curve.
Recombinant proteins were analyzed by Western blot (35) using goat anti-properdin (1/10,000) followed by peroxidase-conjugated rabbit anti-goat IgG polyclonal Ab (1/10,000) for detection. Both scFvP forms yielded single polypeptide chains of length consistent with their sequence (Fig. 1).
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For these experiments, C8-depleted and normal human serum (NHS) were first absorbed 4 times with 4% (v/v) of washed, packed mouse erythrocytes (EM) for 15 min at 4°C to remove Abs directed against EM surface Ags (36). The serum from normal individuals exhibited only minimal background activity in CP-mediated lysis assays of mouse E. Absorbed sera were immediately frozen at –80°C.
C57BL/6 wild-type (WT) mice were used as an erythrocyte (EM) source. Blood was collected from the tail vein using heparinized glass capillaries. Procedures involving mice were approved by the local Animal Studies Committee and conducted in accordance with the guidelines for the care and use of laboratory research animals established by the National Institutes of Health.
In the experiment shown in Fig. 6, A and B, 4 µl of mouse whole blood (4 x 107 RBC) was incubated with 1.0 ml of the filtered, scFvP transfection supernatant for 2 h at room temperature. Control EM were treated for 2 h at room temperature with supernatants from nontransfected cells. EM samples were washed, resuspended in FACS buffer, and analyzed for properdin binding by flow cytometry. We estimated 1000 copies scFvP/E by comparing fluorescence values to those of human decay accelerating factor (DAF) on human E (31) and assuming 3300 DAF/human erythrocytes (EHU) (37). Some EM samples were also treated with 10% C8-depleted serum for 30 min at 37°C, washed, and analyzed for the deposition of complement fragments by flow cytometry. Lysis was observed when the NHS was used in place of the C8-depleted serum. The data presented in Fig. 6, A and B, are representative of at least four independent experiments.
Human erythrocytes
EHU were obtained from healthy volunteers according to the Washington University Medical Center Human Studies Committee guidelines. In Fig. 6, C and D, 10 µl of washed, packed EHU were incubated with 1.0 ml of the filtered, transfection supernatant for 2 h at room temperature. Control EHU were treated for 2 h at room temperature with supernatants from nontransfected cells. EHU samples were washed, resuspended in Mg2+ EGTA buffer, and analyzed for properdin binding by flow cytometry. Copy number per E (4500 scFvP/E) was determined as for EM. EHU were treated with 20% autologous human serum for 60 min at 37°C, washed, and analyzed for the deposition of complement activation products by flow cytometry. The data presented in Fig. 6, C and D, are representative of at least four independent experiments.
Zymosan
Zymosan (Sigma-Aldrich) was prepared as described (38). Particles were counted with a Coulter counter (6.55 x 109 particles/ml), and stored at 4°C until use. Zymosan was diluted in Mg2+ EGTA buffer to 1.7 x 107 particles/ml before treatment with various human complement proteins including properdin (10 µg/ml), C3b (10 µg/ml), factor B (10 µg/ml), and factor D (1 µg/ml) for 30 min at 37°C and washed twice in the same buffer between incubations. In some experiments, zymosan was first incubated with properdin, washed twice, and incubated in 5% properdin-deficient human serum for 10 min at 37°C. The data presented in Fig. 4 are representative of three independent experiments.
Flow cytometry
Erythrocytes, bacteria, or zymosan were washed and then incubated for 20–30 min at 4°C with a primary mAb (1/400). After washing, FITC-conjugated secondary Abs were added (1/100) and the incubation continued for an additional 15–20 min at 4°C. The Ab staining was conducted either in Mg2+ EGTA buffer (bacteria, zymosan, ER and ES) or in PBS supplemented with 1% heat inactivated FCS (EM and EHU). The E. coli experiments of Fig. 7E were performed with a single FITC-conjugated goat anti-human polyclonal IgG (Cappel Laboratories) incubated for 20 min at 4°C (1/1000). Cells were washed and analyzed by flow cytometry (FACScan or FACSCalibur; CellQuest software; BD Biosciences).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionDisclosuresReferences |
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The standard model for the initiation of the AP C3 convertase (C3bBbP) on a target surface (Fig. 2A) begins with nascent C3b generated nonspecifically in the fluid phase. Nascent C3b features a highly reactive thioester that can form a covalent bond with a nearby target surface. Once a C3b:target complex is established, it binds factor B, the resulting C3bB complex is cleaved by factor D yielding C3bBb, and then the C3bBb complex is stabilized by properdin (C3bBbP). Recently, we observed that properdin attached to a biosensor surface binds C3b and that treatment of the resulting C3bP complexes with factor B and factor D leads to the formation of C3bBbP (26). This process provided a new order for C3bBbP assembly (Fig. 2B) and raised the possibility that properdin:target complexes could initiate AP activation.
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Complement-deficient individuals are vulnerable to Neisseria infections (3, 4, 5), with meningococcal disease frequencies 6000-fold greater than in normal individuals (39). Those with properdin deficiencies suffer mortality rates of 34–63%. From our model of properdin function (Fig. 2B), we would predict that properdin binds to the Neisseria surface, initiating assembly of the AP C3 convertase, and promotes the opsonization and clearance or lysis of the pathogen (see also Ref. 40). To test this possibility, we examined the capacity of properdin to interact with Neisseria. As demonstrated in Fig. 3A, properdin binds WT Neisseria gonorrhoeae. C3b binds Neisseria, but only if the bacteria were pretreated with properdin (Fig. 3B). Surface-bound C3bBbP is generated when the bacteria-C3bP complexes are treated with factor B and factor D (Fig. 3C). These results demonstrate that properdin can initiate assembly of AP convertases on the Neisseria surface via bacterium-P and bacterium-C3bP intermediates. Importantly, Neisseria treated with properdin-deficient serum, under conditions that permit AP activation but exclude CP and LP activation, undergo low levels of C3 opsonization whereas Neisseria pretreated with properdin (forming bacterium-P complexes) become highly opsonized (Fig. 3D). C3 deposition achieved in the later case was comparable to that obtained with properdin-deficient serum reconstituted with purified properdin, and with serum bearing normal properdin levels (data not shown). These experiments demonstrate that Neisseria:properdin complexes promote the serum- and AP-dependent opsonization of the pathogen.
Properdin initiates complement activation on zymosan
In their original studies, Pillemer and his collaborators (12) incubated human serum with zymosan, an insoluble yeast cell wall preparation, and eluted the properdin protein from the resulting activation complexes. The remaining serum was depleted of complement activity that could be reconstituted by addition of the properdin protein or the zymosan activation complexes. In their straightforward interpretation, the authors proposed that properdin combined with zymosan to activate complement. Later, it was concluded that additional serum factors were required to form the zymosan:properdin complexes (19). We have re-examined this issue and show by FACS analysis that purified properdin does indeed bind zymosan (Fig. 4A) and that the zymosan:properdin complexes bind C3b more readily than do the untreated zymosan particles (Fig. 4B). Moreover, zymosan:properdin complexes promote complement activation in properdin-deficient human serum under conditions that permit AP activation but exclude CP and LP activation (Fig. 4C). These results support Pillemer’s original explanation of properdin function although they do not preclude activation of complement via the standard AP model.
Effects of properdin on erythrocyte target specificity
The ER is a target commonly used to study AP activation (19). Our observations with Neisseria and zymosan (above), suggested to us that ER:properdin complexes might also initiate AP activation. Thus, we treated ER with native properdin, analyzed the result by FACS, and observed that properdin bound ER surfaces (Fig. 5A). ES, the classic nonactivator of the AP (19), did not bind properdin (Fig. 5B). C3b bound ER pretreated with properdin (ERP), but did not bind ER alone (Fig. 5C). Treatment of ERP with C3b, factor B, and factor D led to the generation of ER-C3bBbP complexes (Fig. 5D). These results demonstrate that properdin can initiate assembly of AP convertases, in this case on the ER surface.
Directing complement activation with properdin
The above experiments establish that properdin:target complexes can direct the in situ assembly and function of C3 convertases (C3bBbP). In principle, the AP could be activated wherever properdin is bound (26, 41). To test this prediction, we constructed a bifunctional recombinant protein consisting of a human properdin domain fused to a single-chain Ab (scFv) domain that recognizes the abundant EM surface Ag GPA (31). The recombinant single-chain polypeptide, NH2-(mouse)scFv-(hu) properdin-COOH (or scFvP), bound EM, as shown by FACS analysis (Fig. 6A). Our rationale was to use this reagent to "tag" a target with properdin and to determine whether it would activate the alternative pathway. To that end, EM were pretreated with scFvP and incubated with C8-depleted human serum in Mg2+ EGTA buffer. As predicted, the EM-scFvP complex promoted deposition of C3 fragments on the EM surface (Fig. 6B). Deposition was dependent on the surface dosage of scFvP. Relatively low levels of complement-dependent C3 deposition occurred on mouse E that were not coated with scFvP. This activity could be attributed to the nonspecific AP initiation model. Additional experiments indicated mouse E bound little human properdin directly. No deposition was evident when reactions used heat-inactivated serum. In other experiments, if C8-depleted serum was replaced with absorbed normal human serum, properdin-tagged EM directed C-dependent cell lysis (data not shown).
A scFvP construct was also prepared to target EHU in a totally autologous system. In these experiments, in which EHU and serum were derived from the same individual, erythrocytes carried the human complement regulators CD59, DAF, and complement receptor 1 (CR1), which in this case would be expected to actively inhibit C3 deposition and hemolysis. An scFv domain was generated from a mAb that recognizes human GPA (32). The scFv-coding region was combined with human properdin-coding region to form a cDNA that encodes a NH2-(hu)scFv-(hu)properdin-COOH single-chain polypeptide, similar in design to the mouse-scFvP protein (above). The scFvP protein bound EHU (Fig. 6C) and the EHU-scFvP complex promoted C3b deposition in the presence of autologous human serum (Fig. 6D). EHU did not bind human properdin directly (data not shown). C3 opsonization was lower than in the case of EM, probably due to the presence of human complement regulators DAF and CR1 on the EHU surface. Similarly, the EHU did not lyse, likely due to the presence of CD59, which inhibits the membrane attack complex.
Properdin binds E. coli and Salmonella typhimurium LPS mutants
Experiments designed to identify the specific targets recognized by properdin were begun. We observed that properdin did not bind WT strains of E. coli K12 (Table I) or S. typhimurium (our unpublished observations). However, the major surface component of the enteric bacteria, LPS, differs significantly from its Neisseria counterpart lipo-oligosaccharide; although LPS is composed of three major regions: a lipid A anchor, a "core" oligosaccharide, and a highly repeated penta-saccharide, known as the O-Ag (42), lipo-oligosaccharide is made up of only the lipid A anchor and the core oligosaccharide. Thus, we also examined "rough" LPS mutants that lack the O-Ag and all or part of the core oligosaccharide. Interestingly, properdin bound all E. coli (Table I) and S. typhimurium (our unpublished results) rough mutants tested, suggesting that properdin recognizes a common bacterial surface component that is readily exposed in the case of Neisseria but masked by the O-Ag in the WT ("smooth") enteric bacteria.
Properdin-binding capacity of several bacterial strains correlates with their respective serum-dependent AP activation rate
If properdin initiates the AP in vivo, then we would expect that, in human serum, targets that bind properdin would activate the AP more readily than targets that do not bind properdin. We tested this hypothesis with several E. coli strains that differed markedly in their capacities to bind properdin (above and Table I). We included W1485, the strain was used by Schreiber et al. (43) in a detailed study of the bactericidal effects of the AP including the kinetics of serum-dependent C3 deposition. W1485 is a smooth strain which features the WT LPS structure and does not bind properdin (Fig. 7A). D31 is a rough strain which features reduced O-Ag (44) and binds properdin, D31m3 is a rough strain which features LPS that is truncated in the core oligosaccharide region (44). D31m3 binds properdin most readily of the three strains (Fig. 7A). We incubated the three bacteria strains for up to 10 min at 37°C in 5 or 20% human serum under buffer conditions that permit AP activation but exclude CP and LP activation, and then analyzed them by flow cytometry for C3 deposition. We found that at 5% serum levels (Fig. 7, B–D), W1485, which did not bind properdin, induced C3 deposition slowly with a lag of 5 min. These observations are consistent with those of Schreiber et al. (43). In contrast, D31m3, which bound purified properdin most readily, induced C3 deposition rapidly. In that case, activation did not appear to require the capacity of properdin to partially stabilize the AP C3 convertase, because C3bBb on its own has a half-life of 90 s, and the rapid reaction was evident within 1 min (data not shown). Finally, D31, which bound intermediate levels of properdin, induced C3 deposition with intermediate kinetics. These experiments were repeated with two additional WT (smooth) strains that did not bind properdin and two additional mutant (rough) strains that bound properdin readily and comparable results were obtained: the strains that bound properdin activated the AP more quickly than those that did not bind properdin (Table I). Similar observations were made when the strains were treated at 20% serum levels (Fig. 7E).
E. coli were also treated with 5% human serum as above, and C3 deposition was analyzed by SDS-PAGE under reducing conditions followed by Western blot (Fig. 8). In the case of D31m3, surface-bound C3b was seen by 2 min, evident by its 110-kDa 
' fragment and 75-kDa subunit. By 10 min, C3b was gone, having been converted to iC3b. This is confirmed by the loss of ' and the appearance of its 68-kDa 1 and 37-kDa 2 cleavage products. Thus, for the D31m3 strain, C3 convertase formation and function occurred rapidly. In contrast, in the case of W1485, C3 deposition occurred relatively slowly: C3b was not seen at 2 min but was evident at 10 min, along with iC3b. These results, obtained also with C7-deficient serum (data not shown), are consistent with the kinetics of C3 deposition observed by FACS analysis (above). Together, these experiments demonstrate that E. coli that bind properdin, activate the AP rapidly, while E. coli that do not bind properdin, activate the AP slowly.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionDisclosuresReferences |
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We propose that the AP can be initiated via two different mechanisms, one that is nonspecific and is described by the standard model, and a second that is directed to specific target surfaces by properdin and described here: 1) in the case of nonspecific initiation, nascent C3b* formed through fluid-phase C3 tickover becomes covalently attached to nonspecific target surfaces where it initiates the assembly of a C3 convertase (Fig. 2A). Once that convertase begins to function, activation can be propagated via the AP amplification loop. This model explains AP activation observed with targets that do not bind properdin. 2) In the case of properdin-directed initiation, properdin binds directly to a specific target surface where it provides a platform for the assembly of C3 convertases (Fig. 2B). Fluid-phase tickover provides C3b and iC3, a functional analog of C3b. In principle, target-bound properdin could also be a receptor for any available fluid-phase C3bB and C3bBb.
Properdin is a multivalent protein composed of identical subunits (45), which may enable it to bind strongly to ligands clustered on a microbial surface while interacting weakly with the same ligands in solution. By this reasoning, target recognition is based on multiple weak protein:ligand interactions. This model accounts for AP activation with targets that bind properdin such as Neisseria, the E. coli LPS mutants, and zymosan. It could also account for the sensitivity of properdin-deficient individuals to Neisseria. Results with the enteric bacteria suggest that properdin can recognize a common bacterial surface component that is readily exposed in the case of Neisseria but masked by the O-Ag in the WT enteric bacteria. We conclude that properdin-directed initiation is a major pathway to AP activation on targets that bind properdin.
Although most complement proteins are produced in the liver, properdin is synthesized instead by blood monocytes, blood T cells (both CD4+ and CD8+), and blood neutrophils (46). In the case of neutrophils, properdin is stored in secondary granules and released upon specific stimulation, including activation through LPS and chemokines/cytokines (47). This process would provide an influx of properdin, making the properdin-directed mechanism of AP activation particularly effective at sites of inflammation. It may also represent a major physiological instance of properdin-directed AP activation because the binding of bacteria to native properdin was not readily detected when bacteria were simply incubated in serum in the absence of complement activation (our unpublished results). Alternatively, in the presence of potentially inhibitory factors in serum, association of properdin directly to a target may be stabilized only if C3bBbP target is assembled. We are currently examining potential interactions among AP targets, neutrophils, properdin, and other serum components.
Pillemer and his collaborators (12) first described properdin and the alternative pathway over 50 years ago. Their primary observations were made with zymosan, an extract derived from yeast cell walls. The authors proposed that properdin combined with zymosan to activate complement, but later attempts to detect the binding of purified properdin to zymosan failed and the model was abandoned (19). We have re-examined this issue and obtained strong evidence in support of the Pillemer model. It is likely that part of the apparent discrepancy lies in the complexity of properdin:target interactions: initially, relatively few properdin proteins may bind to a target, but they are sufficient in number to initiate the AP. Once the AP is activated (as in the incubation of zymosan with serum), many more properdin-binding sites are generated through C3b opsonization. We have examined the initial steps in this process using purified proteins and highly sensitive methods including surface plasmon resonance technology (26), mAbs, flow cytometry, and recombinant DNA technology. The tools that were available during Pillemer’s time were sufficient to detect zymosan:properdin complexes achieved via the full activation process, but not sensitive enough to detect the initial zymosan:properdin interaction. Many of the AP studies conducted since than were performed with ES or with WT E. coli, which do not bind properdin (Figs. 5B and 7A, Table I).
Factor H is a serum protein that suppresses the assembly and function of the AP convertases. Previous work demonstrated that surface sialic acid promotes local factor H function and that some surfaces that feature sialic acid (ES) are less sensitive to the AP than others devoid of sialic acid (ER). It is not surprising that ER both binds properdin and is devoid of sialic acid because ER are among the most AP sensitive of erythrocyte species. Conversely, it is not surprising that ES both binds properdin poorly and harbors surface sialic acid (23, 24). Our new results do not preclude the importance of factor H, but instead indicate that a positive activation mechanism is also at work. In principle, we might expect that pathogens would evolve to avoid or control properdin affinity while hosts would evolve to maximize properdin:pathogen recognition. However, the interplay of different bacterial structures with properdin, factor H, Ab, and other immunity factors is complex so that avoidance of properdin-directed complement activation could result in sensitivity to another pathway. This may account for the particular vulnerability of properdin-deficient individuals to relatively rare N. meningitidis serogroups (48).
The alternative pathway has been implicated in lupus nephritis, rheumatoid arthritis, antiphospholipid syndrome, ischemia/reperfusion injury, asthma, atypical hemolytic-uremic syndrome, type II membranoproliferative glomerulonephritis, spontaneous fetal loss, and macular degeneration (49). The role of properdin in these instances now must be reconsidered. Properdin is stored in neutrophil granules that are released at sites of inflammation (46), and neutrophils are known to facilitate damage in mouse models of rheumatoid arthritis (50) and antiphospholipid syndrome (51). Thus, it is possible in these and other cases that properdin released locally by neutrophils initiates complement activation on self-tissue, disrupting the delicate balance between complement regulation and complement activation. In contrast, properdin discharged from neutrophils at sites of infection would direct complement activation appropriately to pathogen surfaces.
In summary, we present evidence that properdin:target interactions can initiate complement activation. Thus, properdin participates in two distinct activation pathways: one that occurs by the standard model and one that proceeds by a properdin-directed model. Our observations could account for the sensitivity of properdin-deficient individuals to Neisseria infections and we expect that other microbial targets also initiate complement activation via this mechanism. A reappraisal of properdin activity could shed new light on the evolution and function of the complement alternative pathway and its involvement in innate immunity and human disease.
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Acknowledgments |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionDisclosuresReferences |
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Footnotes |
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1 This work was supported by National Institutes of Health Grant R01 AI05143 (to D.E.H.). 
2 Address correspondence and reprint requests to Dr. Dennis E. Hourcade, Division of Rheumatology, Washington University School of Medicine, 660 South Euclid Avenue, Box 8045, St. Louis, MO 63110. E-mail address: dhourcad{at}im.wustl.edu
3 Abbreviations used in this paper: CP, classical pathway; LP, lectin pathway; AP, alternative pathway; GPA, glycophorin A; ER, rabbit erythrocyte; ES, sheep erythrocyte; EM, mouse erythrocyte; EHU, human erythrocyte; ERP, ER pretreated with properdin; NHS, normal human serum; DAF, decay accelerating factor; CR1, complement receptor 1; scFv, single-chain Ab; WT, wild type.
Received for publication March 16, 2007. Accepted for publication May 30, 2007.
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1H-dependent decay-dissociation of amplification C3 convertase of the alternative complement pathway. Proc. Natl. Acad. Sci. USA 75: 1971-1975. 1H control and generation of restriction on neuraminidase-treated cells. Proc. Natl. Acad. Sci. USA 75: 2416-2420. : evidence for heterogeneity in the molecule. Immunology 68: 283-289. [Medline]-galactosyl epitopes on nucleated cells. J. Biol. Chem. 263: 17755-17762. -glucosyltransferase provides a novel attachment site for O-polysaccharides. J. Biol. Chem. 273: 29497-29505. Related articles in The JI:
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