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The down-stream effects of mannan-induced lectin complement pathway activation depend quantitatively on alternative pathway amplification
Molecular Immunology 47 (2009) 373–380
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The down-stream effects of mannan-induced lectin complement pathway activation depend quantitatively on alternative pathway amplification Morten Harboe a , Peter Garred b , Ellen Karlstrøm a , Julie K. Lindstad a , Gregory L. Stahl c , Tom Eirik Mollnes a,∗ a b c
Institute of Immunology, University of Oslo and Rikshospitalet University Hospital, NO-0027 Oslo, Norway Department of Clinical Immunology, Section 7631 Rigshospitalet, Copenhagen University Hospital, DK-2100 Copenhagen Ø, Denmark Center for Experimental Therapeutics and Reperfusion Injury, Harward Medical School, Boston, MA 02115, USA
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Article history: Received 10 June 2009 Received in revised form 2 September 2009 Accepted 3 September 2009 Available online 1 October 2009 Keywords: Complement Lectin pathway Mannose-binding lectin Activation
a b s t r a c t Complement activation plays an important role in human pathophysiology. The effect of classical pathway activation is largely dependent on alternative pathway (AP) amplification, whereas the role of AP for the down-stream effect of mannan-induced lectin pathway (LP) activation is poorly understood. In normal human serum specific activation of LP was obtained after exposure to a wide concentration range of mannan on the solid phase. Reaction mechanisms in this system were delineated in inhibition experiments with monoclonal antibodies. Direct mannose-binding lectin (MBL) independent activation of AP was not observed even at high mannan concentrations since addition of the inhibiting anti-MBL mAb 3F8 completely abolished generation of the terminal C5b-9 complex (TCC). However, selective blockade of AP by anti-factor D inhibited more than 80% of TCC release into the fluid phase after LP activation showing that AP amplification is quantitatively responsible for the final effect of initial specific LP activation. TCC generation on the solid phase was distinctly but less inhibited by anti-fD. C2 bypass of the LP pathway could be demonstrated, and AP amplification was also essential during C2 bypass in LP as shown by complete inhibition of TCC generation in C2-deficient serum by anti-fD and anti-properdin antibodies. In conclusion, the down-stream effect of LP activation depends strongly on AP amplification in normal human serum and in the C2 bypass pathway. © 2009 Elsevier Ltd. All rights reserved.
1. Introduction The classical pathway (CP) and the lectin pathway (LP) of the complement system are initially activated by distinct recognition mechanisms. CP is activated by C1q interacting with antibodies or other agents like C-reactive protein, whereas LP typically is activated by mannose-binding lectin (MBL) or ficolins interacting with carbohydrate structures on microbial surfaces. The further initial activation stages in these two pathways show extensive similarities. In addition to MBL, ficolin-1 (M), ficolin-2 (L) and ficolin-3 (H) are recognition units in LP. Like MBL, the ficolins react with Nacetylglucosamine but the fine carbohydrate binding specificity differs from MBL and they do not react with mannan (Matsushita and Fujita, 2001; Roos et al., 2001; Krarup et al., 2004). Among the MBL-associated serine proteases, MASP-2 cleaves C4 and C2 to form the C3 convertase, while the physiological role of
∗ Corresponding author. Tel.: +47 90630015; fax: +47 23073510. E-mail address: [email protected] (T.E. Mollnes). 0161-5890/$ – see front matter © 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.molimm.2009.09.005
MASP-1 and MASP-3 has been uncertain (Sorensen et al., 2005). MASP-1 has been shown to activate C3 directly in vitro (Takahashi et al., 2007), while MASP-3 has a distinct substrate specificity in cleaving synthetic peptides and insulin-like growth factor-binding protein 5 (Cortesio and Jiang, 2006), but no known function in complement activation. Recent observations indicate that a truncated form of MASP-2, sMAP, plays a regulatory role in the activation of LP (Iwaki et al., 2006). Specific activation of CP by heat aggregated human IgG (HAIGG) has been extensively used, while development of systems for specific activation of LP in close to physiologic conditions has encountered considerable difficulties. However, we have recently developed a system for specific activation of LP without involvement of CP or direct AP activation using mannan coating on the solid phase of ELISA plates and normal human serum (NHS) at high concentration (diluted 1:2) (Harboe et al., 2006). Three main observations indicated LP specificity of this system. (i) Mannan on the solid phase induced activation of NHS but not of MBL deficient serum, showing that MBL is required for the activation. (ii) After reconstitution of MBL deficient serum with purified MBL, activation was obtained, showing that MBL is responsible for activation.
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(iii) Monoclonal anti-MBL antibody 3F8, documented to inhibit MBL function (Zhao et al., 2002), abolished the activation of NHS, showing that activation depends exclusively on MBL. A role of AP in amplification of CP has been known for decades. Only recently, however, this effect was quantified (Harboe et al., 2004). The quantitative role of AP amplification in LP activation is unknown. Thus, the aim of the present work was to delineate reaction mechanisms of LP with emphasis on the quantitative role of AP amplification on the down-stream effect of initial LP activation of normal human serum, and the influence of AP amplification in LP activation in the absence of C2 (“C2 bypass pathway”) (Harboe et al., 2006; Selander et al., 2006). 2. Materials and methods 2.1. Monoclonal antibodies Mouse anti-MBL mAb HYB131-01 (IgG1) was obtained from AntibodyShop, Gentofte, Denmark. Mouse anti-MBL mAbs 3F8 (IgG1) and 1C10 (IgG2b) were raised by immunization with purified human MBL, reacting with MBL with high affinity. 3F8 reacts with a conformation dependent epitope in the hinge within the carbohydrate recognition domain (CRD) of MBL and inhibits MBL-dependent C3 deposition on mannan-coated plates completely at 10 g/mL NHS (Zhao et al., 2002), while 1C10 reacts with another epitope being non-inhibiting (Collard et al., 2000). Mouse anti-human fD (clone 166-32, IgG1) (Fung et al., 2001), mouse anti-human C2 (clone 175-62, IgG1) and an isotypematched control mAb (clone G3-519, anti-HIV gp120, IgG1) were kindly provided by Michael Fung. Inhibiting mouse antihuman properdin, factor P (fP), from clone A233 (IgG) raised by immunization with purified human fP was obtained from Quidel Corporation, San Diego, CA. mAb aE11 reacting with a neoepitope exposed when C9 is incorporated in the terminal complement complex (TCC) (Mollnes et al., 1985) was raised in our own laboratory.
al., 2003). Reconstituting the serum with recombinant wild type MBL had previously shown that the capability of MASP-1/3 and MASP-2 to interact with MBL is normal as well as the capacity to deposit C4 (Larsen et al., 2004). C2-deficient (C2d) serum was obtained from an 18-year-old male who was referred to hospital at the age of 9 due to recurrent infections, primarily of the airways, but with no current tendency to infections. The serum contained no C2 protein reacting with polyclonal goat anti-human C2 (Quidel) in Western blotting. Further molecular studies including PCR and sequencing revealed a 28 base pair deletion in exon 6 characteristic of Type I C2 deficiency (Johnson et al., 1992). In the Wielisa assay there was no CP- or LP-activity and normal AP activity. The MBL concentration in this serum was 3696 g/L. 2.4. Specific lectin pathway activation Mannan from Saccharomyces cerevisiae (Sigma–Aldrich, St. Louis, MO) in a 100 L volume was coated on Costar 3590 flatbottomed polystyrene 96-well plates (Corning Inc., Corning, NY) in 50 mM Na-carbonate buffer pH 9.6 overnight at room temperature and the fluid was removed. After washing, the remaining binding sites in the wells were saturated with a blocking buffer, PBS pH 7.4 containing 1% BSA and 0.1% Tween 20 (Sigma–Aldrich), for 1 h at 37 ◦ C. After three times washing with PBS containing 0.1% Tween 20, 50 L NHS diluted 1:2 in veronal buffer pH 7.5 containing 0.5 mM MgCl2 , 2 mM CaCl2 , 0.05% Tween 20 and 0.1% gelatin (GVB + buffer) was added to each well for complement activation for 30 min at 37 ◦ C. To stop LP activation and to ensure complete inhibition of continuing background AP amplification the microtiter plate was incubated on ice and 10 L EDTA was added immediately to each well to a final concentration of 20 mM before assay for activation products on the solid phase and in the supernatant (fluid phase). 2.5. Readouts for activation products on the solid phase
2.2. Normal human serum and purified MBL Normal human serum (NHS) was collected from nine healthy volunteers, all showing normal CP, LP and AP activity in the Wielisa assay (Seelen et al., 2005). Prior to pooling, the sera were tested for LP activation after exposure to mannan on the solid phase (0.5 g/well) using solid phase TCC deposition as readout. Activation was obtained and TCC deposition was inhibited completely by 3F8 mAb in all nine sera, excluding interference by complement fixing anti-mannan antibodies occurring in occasional sera (Harboe et al., 1981, 2006). Subsequently the sera were pooled and stored as aliquots at −70 ◦ C. The MBL concentration of the pool was 740 g/L. To approach physiological conditions in activation assays and to ensure a fully active AP NHS was used at a final dilution of 1:2, which was the lowest possible dilution to obtain a constant final concentration of serum after addition of buffer and mAbs. Purified plasma-derived MBL was obtained from Statens seruminstitut, Copenhagen, Denmark (Laursen et al., 2007). 2.3. Complement deficiency sera MBL deficient (MBLd) serum was selected from an MBL D/D (codon 52 variant in the MBL2 gene) homozygous 35-year-old healthy male with no anti-mannan antibodies. The serum had normal CP and AP activity in the Wielisa assay (Seelen et al., 2005) and standard hemolytic assays. It contains dysfunctional low molecular weight MBL which cannot bind ligands or activate the complement system efficiently under physiological conditions (Garred et
Deposition of MBL on the solid phase following activation was demonstrated in ELISA with mouse anti-MBL HYB131-01 mAb 1:10,000 followed by HRP conjugated goat anti-mouse IgG 1:4000 (Southern Biotech, Birmingham, UK) using ABTS (Sigma–Aldrich) as substrate. Between each step the plates were washed 3× in PBS pH 7.4 with 0.1% Tween 20. In inhibition experiments, deposition of MBL was also assayed using polyclonal rabbit anti-human MBL2 antibody 1:50 (Atlas Antibodies AB, Stockholm, Sweden) obtained by immunization with PrEST technology (Larsson et al., 2006; Persson et al., 2006) followed by HRP conjugated donkey anti-rabbit Ig 1:2000 (Amersham Biosciences, Little Chalfont, UK) with ABTS as substrate. Deposition of C4b and C3b on the solid phase was demonstrated with polyclonal rabbit anti-human C4 (OSAO 194 1:10,000) and anti-human C3 (OSAP 192 1:40,000) antibodies (Dade Behring, Marburg, Germany), HRP conjugated donkey anti-rabbit Ig 1:2000 and ABTS as substrate. Deposition of fP was demonstrated with polyclonal goat anti-human fP (A239 45 mg/mL 1:1000) (Complement Technology, Tyler, TX), HRP conjugated mouse anti-goat Ig 1:1000 (Jackson ImmunoResearch Europe Ltd., Newmarket, Suffolk, UK) with ABTS as substrate. Deposition of the terminal complement complex (TCC) was based on reaction with purified mAb aE11 (1 mg/mL 1:6000 in PBS with 0.2% Tween 20) followed by biotinconjugated rat anti-mouse IgG2a mAb 1:1000 (BD Biosciences Pharmingen, Erembodegem, Belgium) and HRP conjugated streptavidin 1:2000 (Amersham) with ABTS as substrate, using OD values at 405 nm to represent the relative amount of deposited TCC in the wells.
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2.6. Readout for TCC in the fluid phase TCC in the fluid phase was measured by a double-antibody assay described in detail previously (Mollnes et al., 1985, 1993). Briefly, mAb aE11 was used as catching antibody, biotinylated anti-C6 mAb 9C4-RQ-B (Mollnes et al., 1993) as detection antibody, HRP-labelled streptavidin (Amersham), and ABTS as substrate. 2.7. Inhibition assays mAbs were added to serum followed by incubation for 5 min at room temperature prior to activation and assay. Optimal concentration was determined in pilot experiments, control antibody being used in the same amount. The amount of purified mAb/well is recorded as transformed to amount/mL of undiluted serum, antiMBL 3F8 at 20 g/mL, anti-fD 166-32 at 10 g/mL, anti-fP A233 at 20 g/mL, and anti-C2 175-62 at 50 g/mL. The fraction remaining after inhibition was calculated as follows: (TCC U/mL after inhibition in mannan-coated wells minus TCC U/mL after inhibition in well without mannan) divided by (TCC U/mL without inhibition in mannan-coated well Minus TCC U/mL without inhibition in well without mannan). Percentage inhibition is then: (100 − fraction remaining) × 100. 2.8. Statistics The Mann–Whitney test was used to calculate two-tailed pvalues for differences between groups. 2.9. Ethics The study was approved by the Norwegian Government Regional Committee for Medical Research Ethics. 3. Results 3.1. TCC generation after lectin pathway activation A dose-dependent deposition of TCC on the solid phase was observed after activation of NHS by different amounts of mannan on the solid phase (0–8 g/well) (Fig. 1A). Addition of the inhibiting anti-MBL mAb 3F8 abolished TCC deposition at all mannan concentrations, showing OD values similar to the negative buffer control without mannan, indicating that no direct, MBL-independent AP activation leading to TCC generation occurred in this system. Addition of the non-inhibiting mAb 1C10 gave OD values very similar to those obtained with NHS alone. Generation of TCC in the fluid phase also occurred dosedependently with increasing mannan concentrations on the solid phase (Fig. 1B). At lower concentrations of mannan, activation was limited, but it continued to increase at mannan concentration higher than required to get a maximum deposition of TCC on the solid phase (Fig. 1A and B). 3.2. Reaction pattern and reconstitution of MBL deficient serum Fig. 2 shows the reaction pattern of NHS compared with MBL deficient serum after activation with a single dose of mannan on the solid phase, 8 g/well. Evidence of activation was observed in all of four readouts, deposition of C4b, C3b, and TCC on the solid phase, as well as release of TCC into the fluid phase. With the MBL deficient serum, distinctly lower values were obtained in all of the four readouts, similar to the controls without mannan on the solid phase during activation.
Fig. 1. Deposition of TCC on the solid phase (A) and generation of TCC in the fluid phase (B) after activation of normal human serum (NHS) by different amounts of mannan on the solid phase without and in the presence of 3F8 and 1C10 = anti-MBL inhibiting and non-inhibiting monoclonal antibodies, respectively. The standard for quantification of TCC in the fluid phase was NHS activated with zymosan and defined to contain 1000 arbitrary units (AU)/mL. The data are presented as mean values and range from two independent, identical experiments.
Reconstitution with MBL showed distinct activation with increasing values in all four readouts after addition of 10 and 20 g MBL/mL MBL deficient serum. 3.3. Deposition of MBL after lectin pathway activation Deposition of MBL and inhibition with anti-MBL mAb 3F8 was first studied using mAb HYB131-01 for readout in analogy with previous experiments (Harboe et al., 2006). Without addition with mAb 3F8 the maximal OD value (1.22) was observed at 1 g mannan/well followed by a decrease to OD 0.06 in the well without mannan on the solid phase. Wells with inhibiting anti-MBL 3F8 showed consistent low OD values (0.07 or lower). Interpretation of these low OD values as inhibition of MBL deposition by mAb 3F8 should be made with caution since interference in reactivity may be caused by the corresponding two epitopes lying close to each other in the carbohydrate recognition domain of MBL. Therefore, a rabbit polyclonal anti-MBL antibody generated by PrEST immunization (Larsson et al., 2006; Persson et al., 2006) was used for additional readout. The recombinant peptide used for immunization covered amino acids 102–240 of the MBL sequence (www.atlasantibodies.com), and antigenicity plot analysis (Hopp and Woods, 1981) indicated a series of antibody-reactive epitopes in this area. The readout with polyclonal anti-human MBL2 is shown in Fig. 3. No deposition of MBL was observed in wells without mannan, whereas a dose-dependent increase was observed up to 1 g mannan/well. Addition of mAb 3F8 inhibited MBL deposition completely with consistent low OD values comparable to the buffer control, whereas addition of the non-inhibiting mAb 1C10 gave OD values very similar to those obtained with NHS alone.
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Fig. 2. Reaction pattern of NHS compared with MBL deficient (D/D) serum after activation with a single dose of mannan on the solid phase, 8 g/well. Compared with NHS, the MBL deficient serum gave low values in four different readouts. Reconstitution with purified MBL showed distinct activation with increasing values in all four readouts after addition of 10 and 20 g MBL/mL MBL deficient serum. The bar heights show mean values and range from two independent, identical experiments.
3.4. Anti-fD and anti-fP inhibit TCC generation after lectin pathway activation Addition of anti-fD inhibited deposition of TCC on the solid phase consistently but not completely after activation with different amounts of mannan on the solid phase (Fig. 4A). At 4 g mannan/well inhibition with anti-fD the OD value for deposition of TCC was reduced approximately 50%. Inhibition was also obtained with anti-fP, but to a distinctly lower degree than with anti-fD. Again, mAb 3F8 inhibited completely, whereas mAb 1C10 and the IgG isotype control mAb did not inhibit. Addition of anti-fD inhibited generation of fluid phase TCC profoundly (Fig. 4B). This was also the case for anti-fP, while the results
Fig. 3. Deposition of MBL on the solid phase after activation of NHS by different amounts of mannan on the solid phase. The data are presented as mean values and range from two independent, identical experiments.
Fig. 4. Inhibition of deposition of TCC on the solid phase (A) and generation of TCC in the fluid phase (B) by anti-fD (a-fD) and anti-fP (a-fP) monoclonal antibodies after activation of NHS by different amounts of mannan on the solid phase. The data are presented as mean values and range from two independent, identical experiments.
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3.6. C2 bypass in the lectin pathway The reaction of C2d serum with mannan was also investigated. TCC deposition on the solid phase increased dose-dependently to modest OD values with increasing mannan concentration from 4 to 8 g/well (Fig. 7A). In all experiments with C2d serum NHS without mAb was included as a positive control giving distinctly higher values, similar to those shown in Figs. 1, 4 and 6. These are not included in Fig. 7 to adjust the ordinate to facilitate illustration of the particular features of C2d serum. Similar to NHS, the deposition of TCC from C2d serum was completely inhibited by anti-MBL mAb 3F8; consistent with the previous observations regarding deposition of C3b (Harboe et al., 2006), then interpreted as C2 bypass in LP. The
Fig. 5. Inhibition of TCC release into the fluid phase by anti-fD after activation of NHS by 8 or 16 g mannan/well on the solid phase in 20 identical setups. Bar heights indicate mean values and error bars 95% confidence intervals.
for the other antibodies were as described for the solid phase (Fig. 4 A). For quantification of degree of inhibition in the fluid phase NHSp was activated in 20 identical setups on two ELISA plates without and 8 or 16 g mannan/well on the solid phase with release of TCC in the fluid phase as readout. The inhibition was profound as illustrated in Fig. 5. At 8 g mannan/well the mean inhibition was 81.2% (95% CI 78.6–83.8). At 16 g mannan/well the mean inhibition was 85.4% (95% CI 83.4–87.3). The difference between TCC values in the control without mAb and anti-fD was highly significant at both mannan concentrations (p < 0.001).
3.5. Deposition of fP after lectin pathway activation After LP activation, deposition of fP on the solid phase increased dose-dependently reaching a plateau of high OD values at 2 g mannan/well (Fig. 6). No fP deposition was observed in the wells without mannan. Deposition of fP was inhibited by mAb 3F8, although a very modest increase was observed at 8 g mannan/well. Addition of the non-inhibiting mAb 1C10 did not attenuate fP deposition (Fig. 6). Deposition of fP was completely inhibited by anti-fP, but anti-fD had no effect.
Fig. 6. Inhibition of deposition of fP on the solid phase by various monoclonal antibodies after activation of NHS by different amounts of mannan on the solid phase. The data are presented as mean values and range from two independent, identical experiments.
Fig. 7. Deposition of TCC on the solid phase (A), generation of TCC in the fluid phase (B), and deposition of fP on the solid phase (C) after LP activation of NHS compared with C2-deficient serum (C2m). The data are presented as mean values and range from two independent, identical experiments.
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Fig. 8. Effect of anti-fD mAb, anti-C2 mAb, and combination of these two antibodies on deposition of C4b on the solid phase after LP activation. The data are presented as mean values and range from two independent, identical experiments.
finding with 3F8 again confirmed that no direct, MBL-independent activation of AP was induced leading to generation of TCC at these mannan concentrations on the solid phase. TCC deposition in C2d serum was completely inhibited by anti-fD and anti-fP (Fig. 7 A). This extends our previous observations, showing that the C2 LP bypass requires AP involvement. With C2d serum, distinct generation of TCC in the fluid phase was also observed (Fig. 7 B), albeit in lower amounts than with NHS. Generation of TCC in the fluid phase was also completely inhibited by anti-fD or anti-fP. Distinct deposition of fP on the solid phase was obtained during LP activation of C2d serum being markedly and similarly inhibited by 3F8, anti-fD or anti-fP mAbs, whereas the 1C10 mAb had no effect (Fig. 7C). 3.7. Deposition of C4b on the solid phase after lectin pathway activation To explore the initial LP activation in more detail we measured deposition of C4b, the first component to be activated by MBLinduced MASP-2 activation, in the presence and absence of the antibodies used in this study (Fig. 8). After LP activation of NHS with increasing doses of mannan coated to the wells, solid phase deposition of C4b increased dose-dependently and similarly in the absence of mAb as well as in the presence of mAbs 1C10, antifD, anti-C2, and anti-fD combined with anti-C2 (Fig. 8). Notably, mAb 3F8 completely inhibited C4b deposition, consistent with MBL being essential for C4b deposition and further down-stream activation. No deposition of C4b was observed in wells without mannan, further indicating that no CP or LP activation occurred in serum in contact with the plastic. In control plates with different amounts of mannan on the solid phase processed in parallel, the findings after addition of anti-C2 alone were as in the experiments with C2-deficient serum (not shown). 4. Discussion It is well established that AP amplification is a main determinant for the final effect of initial HAIGG induced specific CP activation, while only limited information is available on quantification of this effect (Harboe et al., 2004). In LP activation similar AP amplification has been described (Suankratay et al., 1998; Brouwer et al., 2006, 2008) but this effect has previously not been studied quantitatively. Here we show that AP amplification is crucial for the down-stream effect of initial LP activation, being responsible for more than 80% of TCC release into the fluid phase in a system with highly specific LP activation of human whole serum. fD was essential for this effect, observed both in normal serum and in C2d serum (“MBL-dependent C2 bypass pathway”).
Experiments comparing activation of NHS and MBL deficient serum by a single concentration of mannan on the solid phase followed by reconstitution with purified MBL (Fig. 2) show conclusively that MBL is required for LP activation. The inhibition experiments with anti-fD (Figs. 4 and 5) further show that AP amplification is essential for the final effect of the initial LP activation. CP and LP share common features in their recognition and activation mechanisms. In our previous studies leading to development of a system for specific LP activation (Harboe et al., 2006), the use of two anti-MBL antibodies reacting with different epitopes on the MBL molecule was essential, 3F8 being inhibiting (Zhao et al., 2002) and 1C10 non-inhibiting (Collard et al., 2000). Similar experimental setups provide essential information regarding CP activation, mAbs reacting with the globular heads of the C1q molecule being inhibiting and others reacting with the collagen-like region of C1q being non-inhibiting (McGrath et al., 2006). The dependency of CP and LP on AP amplification is similar. Thus, in our earlier study, anti-fD mAb inhibited more than 80% of C5a and TCC release into the fluid phase after HAIGG induced activation of CP (Harboe et al., 2004) which is in accordance with the data observed for LP in the present study and recent detailed structural studies showing analogous interactions in the initiating complexes of CP and LP (Phillips et al., 2009). To distinguish between direct activation of AP and initial activation of CP or LP where the final effect to a large extent is determined by AP amplification, is often difficult. Here, inhibition experiments with mAbs are very informative regarding demonstration of initial activation of CP or LP versus direct activation of AP as further outlined in a recent review (Harboe and Mollnes, 2008). Direct activation of AP may also be initiated by fP acting as a recognition molecule (Hourcade, 2006; Spitzer et al., 2007; Kimura et al., 2008; Stover et al., 2008). Our data indicate that fP might to some extent might bind to mannan, but this binding was very modest and only seen at the highest mannan concentrations. Furthermore, down-stream activation with generation of TCC was not seen at this concentration. Thus, activation of AP induced by fP as recognition molecule does not seem to occur in this model, but would require other conditions. Demonstration of activation products after specific LP activation in close to physiologic conditions has met with considerable difficulties. This is partly due to the common reaction sequence of C4 and C2 after initial reaction of C1q or MBL with their respective targets, both pathways then leading to AP involvement and amplification. Initial specific LP activation by mannan on a solid phase was associated with deposition of TCC on the solid phase. TCC was also generated in the fluid phase, in particular at high mannan concentrations on the solid phase, probably to a great extent explaining why attempts to detect LP activation products in the fluid phase have been so difficult. MBL deposition on the solid phase was observed after LP activation in vitro (Fig. 3). In analogy, MBL deposition at lesional sites has been taken as evidence of LP activation in vivo. This has been demonstrated in renal biopsy material as indicator of LP activation in IgA nephropathy (Matsuda et al., 1998; Hisano et al., 2001; Oortwijn et al., 2006) and Henoch–Schönlein purpura nephritis (Endo et al., 2000). In IgA nephropathy MBL deposition was demonstrated in glomeruli in 11 patients (Endo et al., 1998). In five of these no simultaneous deposition of IgG, IgM or C1q were demonstrable, indicating selective activation of LP and no involvement of CP. Corresponding in vitro studies have shown that polymeric human IgA activates the complement system via the LP (Roos et al., 2001). Glomerular LP activation in IgA nephropathy is associated with more severe renal disease (Roos et al., 2006), and the role of LP activation in IgA nephropathy has recently been reviewed (Oortwijn et al., 2008).
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Deposition of C4b on the solid phase after LP activation in the presence of inhibitory antibodies to fD and C2, but not when the MBL inhibitory antibody 3F8 was used is consistent with a “traditional” MBL-dependent MASP-2 mediated activation of C4 taking place in this system. The complement system is a typical cascade system with a strict order of activation and amplification at specific sites. In genetic deficiencies of individual components “bypass” mechanisms have been identified, as outlined in a recent review (Degn et al., 2007). CP C2 bypass in humans has been demonstrated in vitro (Matsushita and Okada, 1986; Deguchi et al., 1987; Knutzen Steuer et al., 1989; Farries et al., 1990) while basic observations on CP C2 bypass in vivo were made in guinea pigs (Wagner et al., 1999). High concentrations of anti-Forssman antibody induced efficient lysis of sheep erythrocytes in vitro in C2-deficient serum. Inhibition experiments showed that a fully functional AP was required for hemolysis. A Forssman shock model was used to test the relevance of C2 bypass in vivo: intravenous injection of anti-Forssman antibody in normal guinea pigs resulted in rapid death from pulmonary shock whereas C2-deficient guinea pigs died in a delayed fashion or had a sublethal reaction. Recently, LP C2 bypass in humans has been demonstrated independently in two different model systems. In C2-deficient serum, specific LP activation by mannan on the solid phase in ELISA would be expected to stop at the C4 stage, while direct activation of AP might occur at high mannan concentrations (Roos et al., 2001; Degn et al., 2007). We observed a markedly increased deposition of C3b on the solid phase at 10 g mannan/well compared with 0.5 g/well (Harboe et al., 2006). This deposition was not inhibited by neutralizing anti-C1q clone 85 mAb, but completely by anti-MBL mAb 3F8 demonstrating LP C2 bypass (Harboe et al., 2006). In the present investigation, similar findings were made concerning deposition of TCC on the solid phase and generation of TCC in the fluid phase after activation of C2d serum by high concentrations of mannan on the solid phase. This LP C2 bypass depends on intact AP function and was completely inhibited by anti-fD and anti-fP showing that AP is essential for LP C2 bypass as originally observed at high antibody concentrations in CP (Wagner et al., 1999). LP C2 bypass was also demonstrated by quantification of C3 deposition onto wells coated with Salmonella O antigen-specific oligosaccharides after incubation with normal human serum compared with C2-deficient serum (Selander et al., 2006). Deposition in C2-deficient serum was completely dependent on MBL and intact AP function (Selander et al., 2006) analogous with our observations. Incubation of Aspergillus conidia in normal human serum has recently also been reported to activate AP involving an MBL C2 bypass mechanism (Dumestre-Perard et al., 2008). The quantitative role of AP amplification in LP activation was more pronounced in the fluid than the solid phase, consistent with the fluid phase convertase being more dependent on AP amplification. This may explain the efficient specific detection of CP and LP activity using the final TCC as readout, e.g. in the Wielisa test (Seelen et al., 2005). Here, CP and LP activation is demonstrated by exposure of serum to IgM and mannan, respectively, on a solid phase, at a serum dilution of 1:101, where there is no remaining AP activity. This would appear surprising in relation to the profound down-stream effect of AP amplification on the result in the fluid phase of initial CP and LP activation. Our data on the solid phase, however, support a sufficient deposition of TCC in the absence of AP to be locally detected. This might even have clinical implications in relation to treatment by complement inhibitors in cases where a systemic activation is regarded as undesirable, since inhibition of AP would efficiently inhibit the fluid-phase convertase triggered either by CP or LP.
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Molecular Immunology journal homepage: www.elsevier.com/locate/molimm
The down-stream effects of mannan-induced lectin complement pathway activation depend quantitatively on alternative pathway amplification Morten Harboe a , Peter Garred b , Ellen Karlstrøm a , Julie K. Lindstad a , Gregory L. Stahl c , Tom Eirik Mollnes a,∗ a b c
Institute of Immunology, University of Oslo and Rikshospitalet University Hospital, NO-0027 Oslo, Norway Department of Clinical Immunology, Section 7631 Rigshospitalet, Copenhagen University Hospital, DK-2100 Copenhagen Ø, Denmark Center for Experimental Therapeutics and Reperfusion Injury, Harward Medical School, Boston, MA 02115, USA
a r t i c l e
i n f o
Article history: Received 10 June 2009 Received in revised form 2 September 2009 Accepted 3 September 2009 Available online 1 October 2009 Keywords: Complement Lectin pathway Mannose-binding lectin Activation
a b s t r a c t Complement activation plays an important role in human pathophysiology. The effect of classical pathway activation is largely dependent on alternative pathway (AP) amplification, whereas the role of AP for the down-stream effect of mannan-induced lectin pathway (LP) activation is poorly understood. In normal human serum specific activation of LP was obtained after exposure to a wide concentration range of mannan on the solid phase. Reaction mechanisms in this system were delineated in inhibition experiments with monoclonal antibodies. Direct mannose-binding lectin (MBL) independent activation of AP was not observed even at high mannan concentrations since addition of the inhibiting anti-MBL mAb 3F8 completely abolished generation of the terminal C5b-9 complex (TCC). However, selective blockade of AP by anti-factor D inhibited more than 80% of TCC release into the fluid phase after LP activation showing that AP amplification is quantitatively responsible for the final effect of initial specific LP activation. TCC generation on the solid phase was distinctly but less inhibited by anti-fD. C2 bypass of the LP pathway could be demonstrated, and AP amplification was also essential during C2 bypass in LP as shown by complete inhibition of TCC generation in C2-deficient serum by anti-fD and anti-properdin antibodies. In conclusion, the down-stream effect of LP activation depends strongly on AP amplification in normal human serum and in the C2 bypass pathway. © 2009 Elsevier Ltd. All rights reserved.
1. Introduction The classical pathway (CP) and the lectin pathway (LP) of the complement system are initially activated by distinct recognition mechanisms. CP is activated by C1q interacting with antibodies or other agents like C-reactive protein, whereas LP typically is activated by mannose-binding lectin (MBL) or ficolins interacting with carbohydrate structures on microbial surfaces. The further initial activation stages in these two pathways show extensive similarities. In addition to MBL, ficolin-1 (M), ficolin-2 (L) and ficolin-3 (H) are recognition units in LP. Like MBL, the ficolins react with Nacetylglucosamine but the fine carbohydrate binding specificity differs from MBL and they do not react with mannan (Matsushita and Fujita, 2001; Roos et al., 2001; Krarup et al., 2004). Among the MBL-associated serine proteases, MASP-2 cleaves C4 and C2 to form the C3 convertase, while the physiological role of
∗ Corresponding author. Tel.: +47 90630015; fax: +47 23073510. E-mail address: [email protected] (T.E. Mollnes). 0161-5890/$ – see front matter © 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.molimm.2009.09.005
MASP-1 and MASP-3 has been uncertain (Sorensen et al., 2005). MASP-1 has been shown to activate C3 directly in vitro (Takahashi et al., 2007), while MASP-3 has a distinct substrate specificity in cleaving synthetic peptides and insulin-like growth factor-binding protein 5 (Cortesio and Jiang, 2006), but no known function in complement activation. Recent observations indicate that a truncated form of MASP-2, sMAP, plays a regulatory role in the activation of LP (Iwaki et al., 2006). Specific activation of CP by heat aggregated human IgG (HAIGG) has been extensively used, while development of systems for specific activation of LP in close to physiologic conditions has encountered considerable difficulties. However, we have recently developed a system for specific activation of LP without involvement of CP or direct AP activation using mannan coating on the solid phase of ELISA plates and normal human serum (NHS) at high concentration (diluted 1:2) (Harboe et al., 2006). Three main observations indicated LP specificity of this system. (i) Mannan on the solid phase induced activation of NHS but not of MBL deficient serum, showing that MBL is required for the activation. (ii) After reconstitution of MBL deficient serum with purified MBL, activation was obtained, showing that MBL is responsible for activation.
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(iii) Monoclonal anti-MBL antibody 3F8, documented to inhibit MBL function (Zhao et al., 2002), abolished the activation of NHS, showing that activation depends exclusively on MBL. A role of AP in amplification of CP has been known for decades. Only recently, however, this effect was quantified (Harboe et al., 2004). The quantitative role of AP amplification in LP activation is unknown. Thus, the aim of the present work was to delineate reaction mechanisms of LP with emphasis on the quantitative role of AP amplification on the down-stream effect of initial LP activation of normal human serum, and the influence of AP amplification in LP activation in the absence of C2 (“C2 bypass pathway”) (Harboe et al., 2006; Selander et al., 2006). 2. Materials and methods 2.1. Monoclonal antibodies Mouse anti-MBL mAb HYB131-01 (IgG1) was obtained from AntibodyShop, Gentofte, Denmark. Mouse anti-MBL mAbs 3F8 (IgG1) and 1C10 (IgG2b) were raised by immunization with purified human MBL, reacting with MBL with high affinity. 3F8 reacts with a conformation dependent epitope in the hinge within the carbohydrate recognition domain (CRD) of MBL and inhibits MBL-dependent C3 deposition on mannan-coated plates completely at 10 g/mL NHS (Zhao et al., 2002), while 1C10 reacts with another epitope being non-inhibiting (Collard et al., 2000). Mouse anti-human fD (clone 166-32, IgG1) (Fung et al., 2001), mouse anti-human C2 (clone 175-62, IgG1) and an isotypematched control mAb (clone G3-519, anti-HIV gp120, IgG1) were kindly provided by Michael Fung. Inhibiting mouse antihuman properdin, factor P (fP), from clone A233 (IgG) raised by immunization with purified human fP was obtained from Quidel Corporation, San Diego, CA. mAb aE11 reacting with a neoepitope exposed when C9 is incorporated in the terminal complement complex (TCC) (Mollnes et al., 1985) was raised in our own laboratory.
al., 2003). Reconstituting the serum with recombinant wild type MBL had previously shown that the capability of MASP-1/3 and MASP-2 to interact with MBL is normal as well as the capacity to deposit C4 (Larsen et al., 2004). C2-deficient (C2d) serum was obtained from an 18-year-old male who was referred to hospital at the age of 9 due to recurrent infections, primarily of the airways, but with no current tendency to infections. The serum contained no C2 protein reacting with polyclonal goat anti-human C2 (Quidel) in Western blotting. Further molecular studies including PCR and sequencing revealed a 28 base pair deletion in exon 6 characteristic of Type I C2 deficiency (Johnson et al., 1992). In the Wielisa assay there was no CP- or LP-activity and normal AP activity. The MBL concentration in this serum was 3696 g/L. 2.4. Specific lectin pathway activation Mannan from Saccharomyces cerevisiae (Sigma–Aldrich, St. Louis, MO) in a 100 L volume was coated on Costar 3590 flatbottomed polystyrene 96-well plates (Corning Inc., Corning, NY) in 50 mM Na-carbonate buffer pH 9.6 overnight at room temperature and the fluid was removed. After washing, the remaining binding sites in the wells were saturated with a blocking buffer, PBS pH 7.4 containing 1% BSA and 0.1% Tween 20 (Sigma–Aldrich), for 1 h at 37 ◦ C. After three times washing with PBS containing 0.1% Tween 20, 50 L NHS diluted 1:2 in veronal buffer pH 7.5 containing 0.5 mM MgCl2 , 2 mM CaCl2 , 0.05% Tween 20 and 0.1% gelatin (GVB + buffer) was added to each well for complement activation for 30 min at 37 ◦ C. To stop LP activation and to ensure complete inhibition of continuing background AP amplification the microtiter plate was incubated on ice and 10 L EDTA was added immediately to each well to a final concentration of 20 mM before assay for activation products on the solid phase and in the supernatant (fluid phase). 2.5. Readouts for activation products on the solid phase
2.2. Normal human serum and purified MBL Normal human serum (NHS) was collected from nine healthy volunteers, all showing normal CP, LP and AP activity in the Wielisa assay (Seelen et al., 2005). Prior to pooling, the sera were tested for LP activation after exposure to mannan on the solid phase (0.5 g/well) using solid phase TCC deposition as readout. Activation was obtained and TCC deposition was inhibited completely by 3F8 mAb in all nine sera, excluding interference by complement fixing anti-mannan antibodies occurring in occasional sera (Harboe et al., 1981, 2006). Subsequently the sera were pooled and stored as aliquots at −70 ◦ C. The MBL concentration of the pool was 740 g/L. To approach physiological conditions in activation assays and to ensure a fully active AP NHS was used at a final dilution of 1:2, which was the lowest possible dilution to obtain a constant final concentration of serum after addition of buffer and mAbs. Purified plasma-derived MBL was obtained from Statens seruminstitut, Copenhagen, Denmark (Laursen et al., 2007). 2.3. Complement deficiency sera MBL deficient (MBLd) serum was selected from an MBL D/D (codon 52 variant in the MBL2 gene) homozygous 35-year-old healthy male with no anti-mannan antibodies. The serum had normal CP and AP activity in the Wielisa assay (Seelen et al., 2005) and standard hemolytic assays. It contains dysfunctional low molecular weight MBL which cannot bind ligands or activate the complement system efficiently under physiological conditions (Garred et
Deposition of MBL on the solid phase following activation was demonstrated in ELISA with mouse anti-MBL HYB131-01 mAb 1:10,000 followed by HRP conjugated goat anti-mouse IgG 1:4000 (Southern Biotech, Birmingham, UK) using ABTS (Sigma–Aldrich) as substrate. Between each step the plates were washed 3× in PBS pH 7.4 with 0.1% Tween 20. In inhibition experiments, deposition of MBL was also assayed using polyclonal rabbit anti-human MBL2 antibody 1:50 (Atlas Antibodies AB, Stockholm, Sweden) obtained by immunization with PrEST technology (Larsson et al., 2006; Persson et al., 2006) followed by HRP conjugated donkey anti-rabbit Ig 1:2000 (Amersham Biosciences, Little Chalfont, UK) with ABTS as substrate. Deposition of C4b and C3b on the solid phase was demonstrated with polyclonal rabbit anti-human C4 (OSAO 194 1:10,000) and anti-human C3 (OSAP 192 1:40,000) antibodies (Dade Behring, Marburg, Germany), HRP conjugated donkey anti-rabbit Ig 1:2000 and ABTS as substrate. Deposition of fP was demonstrated with polyclonal goat anti-human fP (A239 45 mg/mL 1:1000) (Complement Technology, Tyler, TX), HRP conjugated mouse anti-goat Ig 1:1000 (Jackson ImmunoResearch Europe Ltd., Newmarket, Suffolk, UK) with ABTS as substrate. Deposition of the terminal complement complex (TCC) was based on reaction with purified mAb aE11 (1 mg/mL 1:6000 in PBS with 0.2% Tween 20) followed by biotinconjugated rat anti-mouse IgG2a mAb 1:1000 (BD Biosciences Pharmingen, Erembodegem, Belgium) and HRP conjugated streptavidin 1:2000 (Amersham) with ABTS as substrate, using OD values at 405 nm to represent the relative amount of deposited TCC in the wells.
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2.6. Readout for TCC in the fluid phase TCC in the fluid phase was measured by a double-antibody assay described in detail previously (Mollnes et al., 1985, 1993). Briefly, mAb aE11 was used as catching antibody, biotinylated anti-C6 mAb 9C4-RQ-B (Mollnes et al., 1993) as detection antibody, HRP-labelled streptavidin (Amersham), and ABTS as substrate. 2.7. Inhibition assays mAbs were added to serum followed by incubation for 5 min at room temperature prior to activation and assay. Optimal concentration was determined in pilot experiments, control antibody being used in the same amount. The amount of purified mAb/well is recorded as transformed to amount/mL of undiluted serum, antiMBL 3F8 at 20 g/mL, anti-fD 166-32 at 10 g/mL, anti-fP A233 at 20 g/mL, and anti-C2 175-62 at 50 g/mL. The fraction remaining after inhibition was calculated as follows: (TCC U/mL after inhibition in mannan-coated wells minus TCC U/mL after inhibition in well without mannan) divided by (TCC U/mL without inhibition in mannan-coated well Minus TCC U/mL without inhibition in well without mannan). Percentage inhibition is then: (100 − fraction remaining) × 100. 2.8. Statistics The Mann–Whitney test was used to calculate two-tailed pvalues for differences between groups. 2.9. Ethics The study was approved by the Norwegian Government Regional Committee for Medical Research Ethics. 3. Results 3.1. TCC generation after lectin pathway activation A dose-dependent deposition of TCC on the solid phase was observed after activation of NHS by different amounts of mannan on the solid phase (0–8 g/well) (Fig. 1A). Addition of the inhibiting anti-MBL mAb 3F8 abolished TCC deposition at all mannan concentrations, showing OD values similar to the negative buffer control without mannan, indicating that no direct, MBL-independent AP activation leading to TCC generation occurred in this system. Addition of the non-inhibiting mAb 1C10 gave OD values very similar to those obtained with NHS alone. Generation of TCC in the fluid phase also occurred dosedependently with increasing mannan concentrations on the solid phase (Fig. 1B). At lower concentrations of mannan, activation was limited, but it continued to increase at mannan concentration higher than required to get a maximum deposition of TCC on the solid phase (Fig. 1A and B). 3.2. Reaction pattern and reconstitution of MBL deficient serum Fig. 2 shows the reaction pattern of NHS compared with MBL deficient serum after activation with a single dose of mannan on the solid phase, 8 g/well. Evidence of activation was observed in all of four readouts, deposition of C4b, C3b, and TCC on the solid phase, as well as release of TCC into the fluid phase. With the MBL deficient serum, distinctly lower values were obtained in all of the four readouts, similar to the controls without mannan on the solid phase during activation.
Fig. 1. Deposition of TCC on the solid phase (A) and generation of TCC in the fluid phase (B) after activation of normal human serum (NHS) by different amounts of mannan on the solid phase without and in the presence of 3F8 and 1C10 = anti-MBL inhibiting and non-inhibiting monoclonal antibodies, respectively. The standard for quantification of TCC in the fluid phase was NHS activated with zymosan and defined to contain 1000 arbitrary units (AU)/mL. The data are presented as mean values and range from two independent, identical experiments.
Reconstitution with MBL showed distinct activation with increasing values in all four readouts after addition of 10 and 20 g MBL/mL MBL deficient serum. 3.3. Deposition of MBL after lectin pathway activation Deposition of MBL and inhibition with anti-MBL mAb 3F8 was first studied using mAb HYB131-01 for readout in analogy with previous experiments (Harboe et al., 2006). Without addition with mAb 3F8 the maximal OD value (1.22) was observed at 1 g mannan/well followed by a decrease to OD 0.06 in the well without mannan on the solid phase. Wells with inhibiting anti-MBL 3F8 showed consistent low OD values (0.07 or lower). Interpretation of these low OD values as inhibition of MBL deposition by mAb 3F8 should be made with caution since interference in reactivity may be caused by the corresponding two epitopes lying close to each other in the carbohydrate recognition domain of MBL. Therefore, a rabbit polyclonal anti-MBL antibody generated by PrEST immunization (Larsson et al., 2006; Persson et al., 2006) was used for additional readout. The recombinant peptide used for immunization covered amino acids 102–240 of the MBL sequence (www.atlasantibodies.com), and antigenicity plot analysis (Hopp and Woods, 1981) indicated a series of antibody-reactive epitopes in this area. The readout with polyclonal anti-human MBL2 is shown in Fig. 3. No deposition of MBL was observed in wells without mannan, whereas a dose-dependent increase was observed up to 1 g mannan/well. Addition of mAb 3F8 inhibited MBL deposition completely with consistent low OD values comparable to the buffer control, whereas addition of the non-inhibiting mAb 1C10 gave OD values very similar to those obtained with NHS alone.
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Fig. 2. Reaction pattern of NHS compared with MBL deficient (D/D) serum after activation with a single dose of mannan on the solid phase, 8 g/well. Compared with NHS, the MBL deficient serum gave low values in four different readouts. Reconstitution with purified MBL showed distinct activation with increasing values in all four readouts after addition of 10 and 20 g MBL/mL MBL deficient serum. The bar heights show mean values and range from two independent, identical experiments.
3.4. Anti-fD and anti-fP inhibit TCC generation after lectin pathway activation Addition of anti-fD inhibited deposition of TCC on the solid phase consistently but not completely after activation with different amounts of mannan on the solid phase (Fig. 4A). At 4 g mannan/well inhibition with anti-fD the OD value for deposition of TCC was reduced approximately 50%. Inhibition was also obtained with anti-fP, but to a distinctly lower degree than with anti-fD. Again, mAb 3F8 inhibited completely, whereas mAb 1C10 and the IgG isotype control mAb did not inhibit. Addition of anti-fD inhibited generation of fluid phase TCC profoundly (Fig. 4B). This was also the case for anti-fP, while the results
Fig. 3. Deposition of MBL on the solid phase after activation of NHS by different amounts of mannan on the solid phase. The data are presented as mean values and range from two independent, identical experiments.
Fig. 4. Inhibition of deposition of TCC on the solid phase (A) and generation of TCC in the fluid phase (B) by anti-fD (a-fD) and anti-fP (a-fP) monoclonal antibodies after activation of NHS by different amounts of mannan on the solid phase. The data are presented as mean values and range from two independent, identical experiments.
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3.6. C2 bypass in the lectin pathway The reaction of C2d serum with mannan was also investigated. TCC deposition on the solid phase increased dose-dependently to modest OD values with increasing mannan concentration from 4 to 8 g/well (Fig. 7A). In all experiments with C2d serum NHS without mAb was included as a positive control giving distinctly higher values, similar to those shown in Figs. 1, 4 and 6. These are not included in Fig. 7 to adjust the ordinate to facilitate illustration of the particular features of C2d serum. Similar to NHS, the deposition of TCC from C2d serum was completely inhibited by anti-MBL mAb 3F8; consistent with the previous observations regarding deposition of C3b (Harboe et al., 2006), then interpreted as C2 bypass in LP. The
Fig. 5. Inhibition of TCC release into the fluid phase by anti-fD after activation of NHS by 8 or 16 g mannan/well on the solid phase in 20 identical setups. Bar heights indicate mean values and error bars 95% confidence intervals.
for the other antibodies were as described for the solid phase (Fig. 4 A). For quantification of degree of inhibition in the fluid phase NHSp was activated in 20 identical setups on two ELISA plates without and 8 or 16 g mannan/well on the solid phase with release of TCC in the fluid phase as readout. The inhibition was profound as illustrated in Fig. 5. At 8 g mannan/well the mean inhibition was 81.2% (95% CI 78.6–83.8). At 16 g mannan/well the mean inhibition was 85.4% (95% CI 83.4–87.3). The difference between TCC values in the control without mAb and anti-fD was highly significant at both mannan concentrations (p < 0.001).
3.5. Deposition of fP after lectin pathway activation After LP activation, deposition of fP on the solid phase increased dose-dependently reaching a plateau of high OD values at 2 g mannan/well (Fig. 6). No fP deposition was observed in the wells without mannan. Deposition of fP was inhibited by mAb 3F8, although a very modest increase was observed at 8 g mannan/well. Addition of the non-inhibiting mAb 1C10 did not attenuate fP deposition (Fig. 6). Deposition of fP was completely inhibited by anti-fP, but anti-fD had no effect.
Fig. 6. Inhibition of deposition of fP on the solid phase by various monoclonal antibodies after activation of NHS by different amounts of mannan on the solid phase. The data are presented as mean values and range from two independent, identical experiments.
Fig. 7. Deposition of TCC on the solid phase (A), generation of TCC in the fluid phase (B), and deposition of fP on the solid phase (C) after LP activation of NHS compared with C2-deficient serum (C2m). The data are presented as mean values and range from two independent, identical experiments.
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Fig. 8. Effect of anti-fD mAb, anti-C2 mAb, and combination of these two antibodies on deposition of C4b on the solid phase after LP activation. The data are presented as mean values and range from two independent, identical experiments.
finding with 3F8 again confirmed that no direct, MBL-independent activation of AP was induced leading to generation of TCC at these mannan concentrations on the solid phase. TCC deposition in C2d serum was completely inhibited by anti-fD and anti-fP (Fig. 7 A). This extends our previous observations, showing that the C2 LP bypass requires AP involvement. With C2d serum, distinct generation of TCC in the fluid phase was also observed (Fig. 7 B), albeit in lower amounts than with NHS. Generation of TCC in the fluid phase was also completely inhibited by anti-fD or anti-fP. Distinct deposition of fP on the solid phase was obtained during LP activation of C2d serum being markedly and similarly inhibited by 3F8, anti-fD or anti-fP mAbs, whereas the 1C10 mAb had no effect (Fig. 7C). 3.7. Deposition of C4b on the solid phase after lectin pathway activation To explore the initial LP activation in more detail we measured deposition of C4b, the first component to be activated by MBLinduced MASP-2 activation, in the presence and absence of the antibodies used in this study (Fig. 8). After LP activation of NHS with increasing doses of mannan coated to the wells, solid phase deposition of C4b increased dose-dependently and similarly in the absence of mAb as well as in the presence of mAbs 1C10, antifD, anti-C2, and anti-fD combined with anti-C2 (Fig. 8). Notably, mAb 3F8 completely inhibited C4b deposition, consistent with MBL being essential for C4b deposition and further down-stream activation. No deposition of C4b was observed in wells without mannan, further indicating that no CP or LP activation occurred in serum in contact with the plastic. In control plates with different amounts of mannan on the solid phase processed in parallel, the findings after addition of anti-C2 alone were as in the experiments with C2-deficient serum (not shown). 4. Discussion It is well established that AP amplification is a main determinant for the final effect of initial HAIGG induced specific CP activation, while only limited information is available on quantification of this effect (Harboe et al., 2004). In LP activation similar AP amplification has been described (Suankratay et al., 1998; Brouwer et al., 2006, 2008) but this effect has previously not been studied quantitatively. Here we show that AP amplification is crucial for the down-stream effect of initial LP activation, being responsible for more than 80% of TCC release into the fluid phase in a system with highly specific LP activation of human whole serum. fD was essential for this effect, observed both in normal serum and in C2d serum (“MBL-dependent C2 bypass pathway”).
Experiments comparing activation of NHS and MBL deficient serum by a single concentration of mannan on the solid phase followed by reconstitution with purified MBL (Fig. 2) show conclusively that MBL is required for LP activation. The inhibition experiments with anti-fD (Figs. 4 and 5) further show that AP amplification is essential for the final effect of the initial LP activation. CP and LP share common features in their recognition and activation mechanisms. In our previous studies leading to development of a system for specific LP activation (Harboe et al., 2006), the use of two anti-MBL antibodies reacting with different epitopes on the MBL molecule was essential, 3F8 being inhibiting (Zhao et al., 2002) and 1C10 non-inhibiting (Collard et al., 2000). Similar experimental setups provide essential information regarding CP activation, mAbs reacting with the globular heads of the C1q molecule being inhibiting and others reacting with the collagen-like region of C1q being non-inhibiting (McGrath et al., 2006). The dependency of CP and LP on AP amplification is similar. Thus, in our earlier study, anti-fD mAb inhibited more than 80% of C5a and TCC release into the fluid phase after HAIGG induced activation of CP (Harboe et al., 2004) which is in accordance with the data observed for LP in the present study and recent detailed structural studies showing analogous interactions in the initiating complexes of CP and LP (Phillips et al., 2009). To distinguish between direct activation of AP and initial activation of CP or LP where the final effect to a large extent is determined by AP amplification, is often difficult. Here, inhibition experiments with mAbs are very informative regarding demonstration of initial activation of CP or LP versus direct activation of AP as further outlined in a recent review (Harboe and Mollnes, 2008). Direct activation of AP may also be initiated by fP acting as a recognition molecule (Hourcade, 2006; Spitzer et al., 2007; Kimura et al., 2008; Stover et al., 2008). Our data indicate that fP might to some extent might bind to mannan, but this binding was very modest and only seen at the highest mannan concentrations. Furthermore, down-stream activation with generation of TCC was not seen at this concentration. Thus, activation of AP induced by fP as recognition molecule does not seem to occur in this model, but would require other conditions. Demonstration of activation products after specific LP activation in close to physiologic conditions has met with considerable difficulties. This is partly due to the common reaction sequence of C4 and C2 after initial reaction of C1q or MBL with their respective targets, both pathways then leading to AP involvement and amplification. Initial specific LP activation by mannan on a solid phase was associated with deposition of TCC on the solid phase. TCC was also generated in the fluid phase, in particular at high mannan concentrations on the solid phase, probably to a great extent explaining why attempts to detect LP activation products in the fluid phase have been so difficult. MBL deposition on the solid phase was observed after LP activation in vitro (Fig. 3). In analogy, MBL deposition at lesional sites has been taken as evidence of LP activation in vivo. This has been demonstrated in renal biopsy material as indicator of LP activation in IgA nephropathy (Matsuda et al., 1998; Hisano et al., 2001; Oortwijn et al., 2006) and Henoch–Schönlein purpura nephritis (Endo et al., 2000). In IgA nephropathy MBL deposition was demonstrated in glomeruli in 11 patients (Endo et al., 1998). In five of these no simultaneous deposition of IgG, IgM or C1q were demonstrable, indicating selective activation of LP and no involvement of CP. Corresponding in vitro studies have shown that polymeric human IgA activates the complement system via the LP (Roos et al., 2001). Glomerular LP activation in IgA nephropathy is associated with more severe renal disease (Roos et al., 2006), and the role of LP activation in IgA nephropathy has recently been reviewed (Oortwijn et al., 2008).
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Deposition of C4b on the solid phase after LP activation in the presence of inhibitory antibodies to fD and C2, but not when the MBL inhibitory antibody 3F8 was used is consistent with a “traditional” MBL-dependent MASP-2 mediated activation of C4 taking place in this system. The complement system is a typical cascade system with a strict order of activation and amplification at specific sites. In genetic deficiencies of individual components “bypass” mechanisms have been identified, as outlined in a recent review (Degn et al., 2007). CP C2 bypass in humans has been demonstrated in vitro (Matsushita and Okada, 1986; Deguchi et al., 1987; Knutzen Steuer et al., 1989; Farries et al., 1990) while basic observations on CP C2 bypass in vivo were made in guinea pigs (Wagner et al., 1999). High concentrations of anti-Forssman antibody induced efficient lysis of sheep erythrocytes in vitro in C2-deficient serum. Inhibition experiments showed that a fully functional AP was required for hemolysis. A Forssman shock model was used to test the relevance of C2 bypass in vivo: intravenous injection of anti-Forssman antibody in normal guinea pigs resulted in rapid death from pulmonary shock whereas C2-deficient guinea pigs died in a delayed fashion or had a sublethal reaction. Recently, LP C2 bypass in humans has been demonstrated independently in two different model systems. In C2-deficient serum, specific LP activation by mannan on the solid phase in ELISA would be expected to stop at the C4 stage, while direct activation of AP might occur at high mannan concentrations (Roos et al., 2001; Degn et al., 2007). We observed a markedly increased deposition of C3b on the solid phase at 10 g mannan/well compared with 0.5 g/well (Harboe et al., 2006). This deposition was not inhibited by neutralizing anti-C1q clone 85 mAb, but completely by anti-MBL mAb 3F8 demonstrating LP C2 bypass (Harboe et al., 2006). In the present investigation, similar findings were made concerning deposition of TCC on the solid phase and generation of TCC in the fluid phase after activation of C2d serum by high concentrations of mannan on the solid phase. This LP C2 bypass depends on intact AP function and was completely inhibited by anti-fD and anti-fP showing that AP is essential for LP C2 bypass as originally observed at high antibody concentrations in CP (Wagner et al., 1999). LP C2 bypass was also demonstrated by quantification of C3 deposition onto wells coated with Salmonella O antigen-specific oligosaccharides after incubation with normal human serum compared with C2-deficient serum (Selander et al., 2006). Deposition in C2-deficient serum was completely dependent on MBL and intact AP function (Selander et al., 2006) analogous with our observations. Incubation of Aspergillus conidia in normal human serum has recently also been reported to activate AP involving an MBL C2 bypass mechanism (Dumestre-Perard et al., 2008). The quantitative role of AP amplification in LP activation was more pronounced in the fluid than the solid phase, consistent with the fluid phase convertase being more dependent on AP amplification. This may explain the efficient specific detection of CP and LP activity using the final TCC as readout, e.g. in the Wielisa test (Seelen et al., 2005). Here, CP and LP activation is demonstrated by exposure of serum to IgM and mannan, respectively, on a solid phase, at a serum dilution of 1:101, where there is no remaining AP activity. This would appear surprising in relation to the profound down-stream effect of AP amplification on the result in the fluid phase of initial CP and LP activation. Our data on the solid phase, however, support a sufficient deposition of TCC in the absence of AP to be locally detected. This might even have clinical implications in relation to treatment by complement inhibitors in cases where a systemic activation is regarded as undesirable, since inhibition of AP would efficiently inhibit the fluid-phase convertase triggered either by CP or LP.
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