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Candidate inhibitors of porcine complement
Molecular Immunology 44 (2007) 1827–1834
Candidate inhibitors of porcine complement Ebbe B. Thorgersen a,∗ , Yohannes T. Ghebremariam b , Joshua M. Thurman c , Michael Fung d , Erik Waage Nielsen e , V. Michael Holers f , Girish J. Kotwal b , Tom Eirik Mollnes a a
Institute of Immunology, Rikshospitalet-Radiumhospitalet Medical Center and University of Oslo, N-0027 Oslo, Norway b Division of Medical Virology, IIDMM, University of Cape Town, HSC, Cape Town 7925, South Africa c Divisions of Nephrology and Hypertension, Department of Medicine, University of Colorado Health Sciences Center, Denver, CO 80262, USA d Tanox Inc., Houston, TX, USA e Department of Anaesthesiology, Nordland Hospital and University of Tromsø, Norway f Division of Rheumatology, Department of Medicine, University of Colorado Health Sciences Center, Denver, CO 80262, USA Received 18 September 2006; received in revised form 11 October 2006; accepted 13 October 2006 Available online 15 November 2006
Abstract Therapeutic complement inhibition is a promising strategy for treatment of a number of diseases as judged from rodent studies. The species distance from rodents to humans may limit the clinical relevance of these studies. The pig is an alternative animal for studies of human diseases like sepsis and ischemia/reperfusion injury. However, available complement inhibitors for use in pigs are scarce. The aim of the present study was to investigate and compare the efficacy of selected candidate inhibitors of porcine complement in vitro for possible future application in vivo. Sera from three different pigs were each incubated with three different activators of the complement system (zymosan, heat-aggregated immunoglobulin G (HAIGG) and Escherichia coli). Three groups of complement inhibitor candidates were tested: serine protease inhibitors (FUT-175 and C1-inhibitor), monoclonal antibodies (anti-factor B (fB) and anti-factor D (fD)) and a recombinant regulatory protein (vaccinia virus complement control protein (VCP)). Read-out was the terminal C5b-9 complement complex (TCC). The serine protease inhibitors FUT-175 and C1-inhibitor dose-dependently inhibited TCC formation in zymosan-, HAIGG- and E. coli-activated porcine sera, but with different efficacy. Complete inhibition of TCC was obtained using 0.2 mg/mL FUT-175, but required 16 mg/mL of C1inhibitor. The monoclonal anti-fB and -fD antibodies both inhibited TCC formation dose-dependently, but in different ways. Anti-fB at high dose (1 mg/mL) completely inhibited TCC formation in sera activated with zymosan and virtually completely in sera activated with HAIGG, but not in sera activated with E. coli. Anti-fD inhibited all three activators at low dose (0.05 mg/mL), and approximately 50% TCC reduction was obtained. The recombinant complement regulatory protein VCP efficiently and dose-dependently inhibited TCC formation with a complete inhibition found at 0.05 mg/mL for all three activators. All candidates tested inhibited porcine complement activation, but in different ways and to different degrees. Of the complement-specific candidates, VCP inhibited all activators completely at low doses. © 2006 Elsevier Ltd. All rights reserved. Keywords: Anti-factor B; Anti-factor D; C1-inhibitor; Complement; Complement inhibitors; Escherichia coli; FUT-175; Porcine; Vaccinia virus complement control protein (VCP)
1. Introduction In response to invading pathogens, the innate immune system plays an important role in protecting the host. The complement system is part of the innate immune defence. The system arose early in the evolution of species and is both preserved and
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0161-5890/$ – see front matter © 2006 Elsevier Ltd. All rights reserved. doi:10.1016/j.molimm.2006.10.004
developed later, reflecting the importance in protection against non-self (Zarkadis et al., 2001). Complement is a cascade system in blood. It consists of three known initiating pathways; the classical (CP), the lectin (LP) and the alternative pathway (AP) and one common final pathway. Recognition of opsonised microorganisms by the classical pathway, via antibodies that bind to C1q, and the lectin pathway, via MBL-MASP complexes, leads to activation of serine proteases that cleave complement components C4 and C2. This leads to the formation of the protease complex C4b2a, which then cleaves
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C3 into C3a and C3b and forms the C5 convertase C4b2a3b. The alternative pathway forms C3b by slow spontaneous hydrolysis of C3 to C3(H2 O). C3(H2 O) forms complex with factor B, which is then cleaved by factor D to yield C3(H2 O)Bb. This complex is able to cleave C3 to C3a and C3b. C3b binds factor B and the second alternative pathway C3 convertase, C3bBb, is formed and upon stabilization with properdin generates the C5 convertase C3b3bBb. After the cleavage of C5 by either C5 convertase, C5b binds to C6 and the terminal sequence then forms the terminal complement complex (TCC), either as the membrane-inserted C5b-9 (membrane attack complex) or as the soluble sC5b-9. A number of soluble and cell-bound regulatory proteins act to inhibit activation at different levels, keeping the system under tight control (Mollnes et al., 2002). The complement system plays an essential role in host defence, in particular by opsonising microbes (Goldfarb and Parrillo, 2005). However, increasing evidence has underscored the pathogenic side of excessive complement activation leading to tissue damage in several inflammatory conditions, including sepsis (Ward, 2004), trauma and shock (Yao et al., 1998), nephritis (Welch, 2002), arthritis (Linton and Morgan, 1999; Wouters et al., 2006), ischemia/reperfusion injury (Arumugam et al., 2004) and respiratory distress syndrome (Robbins et al., 1987). Thus, inhibitors of the complement system may be useful in the treatment of these conditions (Mollnes and Kirschfink, 2006). Inhibitors that target broadly several of the cascade proteins (such as serine proteases), complement specific monoclonal antibodies, and recombinant regulatory proteins targeting specific sites in the complement cascade are all candidate inhibitors. The pig is widely used as a model animal for human diseases. Only occasionally though, complement inhibitors have been used in pig studies, e.g. with a soluble complement receptor type 1 (sCR1) (Pierre et al., 1998; Szebeni et al., 1999) and an anti-C5a antibody (Amsterdam et al., 1995; Mohr et al., 1998; Tofukuji et al., 1998). However, a detailed evaluation and comparison of the effects of various candidate inhibitors on the three different complement activation pathways in pigs is missing. The current study was therefore undertaken to address the need.
2. Material and methods 2.1. Sera and reagents Sera from pigs (Norsvin, Norwegian farm pigs (landrace), out-bred stock) were prepared and frozen in aliquots at −70 ◦ C. Zymosan A (Z-4250) was purchased from Sigma (St. Louis, MO). Gamma globulin (human, 165 mg/mL, Vno. 027490) was purchased from Biovitrum (Stockholm, Sweden) and prepared to heat-aggregated immunoglobulin G (HAIGG) by heating a dilution of 10 mg/mL of gamma globulin to 63 ◦ C for 10 min. Escherichia coli was purchased from American Type Culture Collection (ATCC; Manassas, VA). Human serum albumin (HSA; 200 mg/mL, Vno. 478172) was purchased from Octapharma AG (Lachen, Switzerland). Sterile phosphatebuffered saline (PBS; cat no. 14040-083) was purchased from Gibco, Invitrogen Corporation (Paisley, Scotland).
2.2. Serine protease inhibitors FUT-175 (nafamostat mesilate, Futhan) was purchased from Torii Pharmaceutical Co. (Tokyo, Japan). Human C1-inhibitor was purchased from ZLB Behring AB (Danderyd, Sweden). 2.3. Monoclonal antibodies Mouse monoclonal antibody (mAb) to mouse factor B (clone 1379 5a IgG1), shown to cross-react with pig (Thurman et al., 2005), and pig factor D (clone 230A IgG1) (Fung unpublished) were produced in the co-authors laboratories and described in detail previously. A mouse-anti-human CD22 (clone IS7 IgG1) mAb was purchased from Diatec (Oslo, Norway). Anti-CD22 was tested in flow cytometry and showed no cross-reactivity with porcine antigens and could therefore be used as an isotype control. 2.4. Recombinant complement regulatory protein Vaccinia complement control protein (VCP) described previously (Kotwal and Moss, 1988; Kotwal et al., 1990) was produced using the Pichia pastoris yeast expression system as described before (Murthy et al., 2001; Ghebremariam et al., 2005). 2.5. Enzyme immunoassays The soluble terminal C5b-9 complement complex (TCC), was measured in an enzyme immunoassay (EIA), as described previously (Mollnes et al., 1985) and later modified (Mollnes et al., 1993b). Briefly, the monoclonal antibody aE11 reacting with a neoepitope exposed in C9 after incorporation in the C5b9 complex was used as capture antibody, the final concentration in the wells was 3 g/mL. A biotinylated monoclonal anti-C6 (Quidel Corporation, San Diego, CA) was used as detection antibody in a final concentration of 4 g/mL. Both antibodies cross-react with specific pig epitopes and the assay can be used to detect porcine TCC (Mollnes et al., 1993a). The standard was normal human serum activated with zymosan and defined to contain 1000 AU/mL. Zymosan-activated porcine serum was used as a positive control and the buffer used as a diluent for the standards and samples, containing PBS, ethylenediaminetetraacetic acid (EDTA) and a detergent (Tween), was used as a negative control. 3. In vitro experimental model 3.1. Test of complement pathways in serum Porcine serum from three different pigs were tested using a commercial available EIA (Wielisa, Wieslab, Lund, Sweden), for assessment of complement functional activity in classical, lectin, and alternative pathway (Seelen et al., 2005). The kit is developed for human use but because it contains the mAb aE11 as detection antibody, which cross-react with the porcine epitope (Mollnes et al., 1993a), it was here found to work with
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pig serum as well, provided that higher serum concentration was used. Porcine sera diluted 1/10 for CP and LP and 1/2 for AP gave similar results as for human serum diluted according to the recommendation of the kit instruction (1/101 for CP and LP and 1/18 for AP). 3.2. Serum experiments Porcine sera were incubated with three complement activators: zymosan, a polysaccharide from the yeast Saccaharomyces cerevisiae cell membrane, known to be a potent activator of the lectin pathway (Brouwer et al., 2006) and the alternative pathway (Fearon and Austen, 1977) to a final concentration of 1 mg/mL serum, heat-aggregated immunoglobulin G (HAIGG), known to activate the classical pathway of complement (Fust et al., 1978) to a final concentration of 1 mg/mL serum, and E. coli to a final concentration of 1 × 108 /mL serum. PBS was used as background control. Inhibition experiments were performed by pre-incubation for 5 min with the different candidate inhibitors and their controls. An isotype-matched control mAb and HSA were used as controls. The complement activators were added and incubated for 30 min at 37 ◦ C. Complement activation was stopped by adding EDTA to a final concentration of 20 mM. The samples were kept on ice and then centrifuged for 15 min at 3000 × g (4 ◦ C), for removal of E. coli and zymosan from the samples. The T0 baseline sample was processed immediately. The sera were stored at −70 ◦ C until analyzed. 3.3. Data presentation Data are presented as ratios (indicated as “normalized values” in the figure legends) between the measured value at each concentration of inhibitor and the baseline uninhibited value. Median and range of these values are presented. The quantities are presented as mass throughout the text and in the figures. In order to compare the efficacy on a molar basis, the lowest molar concentration of each agent giving maximum inhibition of complement activation is presented (Table 1). 3.4. Ethics The study was approved by the The Norwegian Animal Experimental Board.
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4. Results 4.1. Test of the complement pathways in serum The Wielisa kit, measuring the functional activity of the classical, lectin and alternative complement pathway, cross-reacted with porcine complement and all three pathways of the complement system were found to be intact in the sera used. 4.2. Effect of candidate inhibitors on complement activation in serum 4.2.1. Serine protease inhibitors FUT-175 dose-dependently inhibited complement activation, as measured by TCC, for all three activators, whereas HSA had no effect (Fig. 1). Inhibition was complete at 0.2 mg FUT175/mL serum. C1-inhibitor dose-dependently inhibited TCC formation for all three activators, whereas HSA had no effect (Fig. 2). Inhibition was complete at 16 mg (64 Units) C1-inhibitor/mL serum. HAIGG and E. coli were inhibited more efficiently at lower doses than zymosan (Fig. 2). 4.2.2. Monoclonal antibodies Anti-factor B dose-dependently and completely inhibited zymosan- and almost completely inhibited HAIGG-induced TCC formation, at a concentration of 1 mg/mL (Fig. 3). In serum incubated with E. coli, the antibody had virtually no effect. A marginal inhibition at low doses was observed, which was not seen at higher doses (Fig. 3). The isotype-matched control mAb showed no inhibitory effect (Fig. 3). Anti-factor D dose-dependently inhibited TCC formation for all three activators (Fig. 4). Maximal inhibition was observed using low doses (0.05 mg/mL), but was limited to a reduction of TCC formation of approximately 50% (Fig. 4). The isotypematched control mAb showed no inhibitory effect (Fig. 4). 4.2.3. Recombinant complement regulatory protein Vaccinia virus complement control protein (VCP) dosedependently inhibited TCC formation for all three activators, whereas HSA had no effect (Fig. 5). Inhibition was complete at 0.05 mg VCP/mL serum, but inhibited markedly at
Table 1 Molarity of the lowest concentration of each porcine complement candidate inhibitor giving maximum inhibitiona Candidate inhibitor
FUT-175 C1-inhibitor Anti-factor B Anti-factor Dd VCP a b c d
Molecular weight (Da)
540 105,000 150,000b 150,000b 28,800
Molarity (M) Zymosan
HAIGG
Escherichia coli
2.2 × 10−3
2.2 × 10−3
2.2 × 10−3 9.2 × 10−4 –c 2.0 × 10−6 1.1 × 10−5
9.2 × 10−4 4.0 × 10−5 2.0 × 10−6 1.1 × 10−5
9.2 × 10−4 4.0 × 10−5 5.1 × 10−7 1.1 × 10−5
Maximum inhibition varied considerably between the different inhibitors (see Section 4). Approximate molecular weight for anti-factors B and D. Anti-factor B had almost no inhibitory effect on E. coli-induced complement activation. The highest obtainable anti-factor D concentration used did not permit determination of maximum inhibition (see Section 5).
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Fig. 1. Effect of FUT-175 on complement activation in porcine serum. Sera from three different pigs were pre-incubated with FUT-175 or HSA for 5 min, and then complement activators (zymosan, HAIGG, Escherichia coli) were added and incubated for 30 min at 37 ◦ C. Read-out was TCC measured using ELISA. Median and range of normalized values of three separate experiments are shown.
Fig. 2. Effect of C1-inhibitor on complement activation in porcine serum. Sera from three different pigs were pre-incubated with C1-inhibitor or HSA for 5 min, and then complement activators (zymosan, HAIGG, E. coli) were added and incubated for 30 min at 37 ◦ C. Read-out was TCC measured using ELISA. Median and range of normalized values of three separate experiments are shown. One Unit of C1-inhibitor corresponds to 0.25 mg.
lower concentrations. Thus, at 10-fold lower concentration (0.00625 mg/mL) VCP inhibited TCC formation by approximately 40% in zymosan-activated sera, 70% in HAIGGactivated sera and 60% in E. coli-activated sera (Fig. 5). 4.2.4. Comparison of the inhibitors on a molar basis Due to the different molecular weights of the inhibitors, quantities are expressed as molarity as well in order to compare the efficacy on a molar basis. The lowest molar concentration which gave a maximum inhibition for each of the activators is therefore shown (Table 1).
5. Discussion Three groups of candidate complement inhibitors (serine protease inhibitors, monoclonal antibodies, and a recombinant complement regulatory protein) were examined in vitro in porcine serum. Sera were tested using the Wielisa functional kit for human complement, which was found to cross-react with porcine serum, and all three initiating pathways of complement were found to be intact. The synthetic serine protease inhibitor FUT-175 (nafamostat mesilate, Futhan) is known to inhibit several proteins in
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Fig. 3. Effect of anti-factor B on complement activation in porcine serum. Sera from three different pigs were pre-incubated with anti-factor B or an isotype matched control for 5 min, and then complement activators (zymosan, HAIGG, E. coli) were added and incubated for 30 min at 37 ◦ C. Read-out was TCC measured using ELISA. Median and range of normalized values of two separate experiments are shown.
the human complement cascade (C1r, C1s, C3, MASPs, factor B, factor D) and other serine proteases in the contact system and the coagulation system (Fujii and Hitomi, 1981; Ikari et al., 1983; Pfeifer et al., 1999). FUT-175 is a commercial available substance and is used in Japan for the treatment of pancreatitis (Imaizumi et al., 2004). In the present study it showed a dose-dependent inhibition to baseline TCC values at 0.2 mg/mL in porcine serum incubated with zymosan, HAIGG or E. coli. FUT-175 is easily available at a low price and thus a promising
candidate for inhibition of porcine complement. These advantages are counteracted by the fact that it is not a specific complement inhibitor. Furthermore, the molecular weight is low (approximately 540 Da (Miyata et al., 2006)) and therefore the bioavailability is probably limited due to its rapid elimination from circulation. C1-inhibitor is a serine protease inhibitor and naturally occurring regulator of the complement and the contact systems and, thus, like FUT-175 it is not a complement specific inhibitor. It is
Fig. 4. Effect of anti-factor D on complement activation in porcine serum. Sera from three different pigs were pre-incubated with anti-factor D or an isotype matched control for 5 min, and then complement activators (zymosan, HAIGG, E. coli) were added and incubated for 30 min at 37 ◦ C. Read-out was TCC measured using ELISA. Median and range of normalized values of three separate experiments are shown.
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Fig. 5. Effect of VCP on complement activation in porcine serum. Sera from three different pigs were pre-incubated with VCP or HSA for 5 min, and then complement activators (zymosan, HAIGG, E. coli) were added and incubated for 30 min at 37 ◦ C. Read-out was TCC measured using ELISA. Median and range of normalized values of three separate experiments are shown.
a glycosylated peptide of 478 amino acid residues with a molecular weight of approximately 105 kDa, consistent with a probable good bioavailability and prolonged retention. Physiological plasma concentration in man is 0.24 mg/mL corresponding to 1 U/mL (Caliezi et al., 2000). We used human C1-inhibitor in our experiments. According to a recent study the reactive site of porcine and human C1-inhibitor are highly homologous with similar complement inhibitory function in pig and human sera (Kobayashi et al., 2006). In porcine serum, the HAIGG-induced complement activation was inhibited in a dose-dependent manner, as expected since C1-inhibitor is a regulator of the classical pathway (Caliezi et al., 2000). In E. coli-activated serum C1inhibitor showed a similar dose-dependent inhibition of complement, whereas the inhibition of complement activation by zymosan was less profound. Jiang and co-authors have reported an inhibitory effect of C1-inhibitor on the alternative complement pathway by binding to C3b (Jiang et al., 2001). Therefore the effect observed in our study may be explained concurrently. Nevertheless, inhibition of TCC induced by all three activators required substantial amounts of the protein, which hardly would be attainable in vivo. Anti-factor B has previously been shown to be a promising inhibitor of the complement system, protecting against antiphospholipid antibody-induced pregnancy loss (Thurman et al., 2005) and renal ischemia/reperfusion injury in mice (Thurman et al., 2006). Recently it has been shown to be effective in reducing airway hyperresponsiveness in a murine model of allergic airway disease (Taube et al., 2006). Consistently, the present study documented a dose-response inhibition of TCC induced by zymosan and HAIGG, showing that mouse anti-factor B cross-reacts with pig factor B. The zymosan-induced complement activation was completely inhibited, whilst the HAIGG-induced activation was reduced by approximately 80%. This effectiveness of antifactor B on classical pathway induced TCC formation probably reflects the importance of the alternative pathway as an amplifi-
cation loop for this pathway (Harboe et al., 2004). A complete inhibition required a relatively high concentration, though corresponding well with theoretical dose needed for neutralization of factor B in serum. However, the effect on E. coli induced complement activation was surprising. A small inhibitory effect was seen at the lower doses of the mAb, which was not seen at higher doses. An unspecific activating effect of the antibody on complement in high doses is ruled out by the fact that the same dose efficiently reduced HAIGG- and zymosan-induced TCC formation. It is therefore tempting to speculate that E. coli has properties, on the surface or by released products to the fluid-phase, which abolish the effect of anti-factor B. Anti-factor B might therefore not be a suitable candidate for treatment of sepsis caused by E. coli, although it is promising in the treatment of the conditions mentioned above, as well as for inhibition of meconium-induced complement activation (own unpublished data). Factor D is the rate limiting protease of the alternative complement pathway because of its low concentration in blood (Volanakis and Narayana, 1996). It is therefore not surprising that a lower concentration was required for complement inhibition than that of anti-factor B. The alternative pathway is an amplification loop for the other two pathways, making it very important in the cascade for response to invading microorganisms (Harboe et al., 2004). This and other studies have shown that a very efficient inhibition of the human alternative complement pathway is obtained at concentrations below 0.01 mg anti-human factor D/mL serum. In the present study the anti-porcine factor D tested inhibited all three activators, but with a maximal inhibition limited to approximately 50% at 0.05 mg/mL. The anti-porcine factor D used in this study was produced by E. coli, and is therefore not glycosylated in contrast to the anti-human factor D mAb 166-32 (Fung et al., 2001). This may explain that the maximum inhibitory effect obtained in porcine serum was restricted. We cannot, however, fully exclude that an additional
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effect could have been obtained using higher concentration of the antibody since no plateau was reached. The amount of protein in the stock solution did not allow higher final concentration in the experiment. VCP is a 28.8 kDa 243 amino acid protein secreted by vaccinia virus infected mammalian cells. It is structurally related to C4b-binding protein, and functionally closely related to CR1. It binds to C3b and C4b in the rodent and human complement cascade and thereby arrests the C3 convertases (Kotwal and Moss, 1988; Jha and Kotwal, 2003). In a study of VCP’s ability to protect xenoendothelial cells from damage by the complement system, Al-Mohanna and colleagues found that VCP might bind to ␣-galactose residues on pig aortic endothelial cells thereby competitively blocking binding of xenoreactive natural antibodies, showing additional properties of this protein (Al-Mohanna et al., 2001). Tests under various and harsh conditions show that VCP is a robust protein and therefore easy to ship, store and administer (Smith et al., 2002). Several studies have demonstrated the beneficial effects of VCP as complement inhibitor in rodent disease models: collagen-induced arthritis (Jha et al., 2005), spinal cord injury (Reynolds et al., 2004), traumatic brain injury (Hicks et al., 2002; Pillay et al., 2005), experimental peritonitis (Scott et al., 2003), atherosclerosis (Thorbjornsdottir et al., 2005) and mouse-to-sensitized rat and guinea pig-to-rat heart xenotransplantations (Anderson et al., 2002, 2003). The effect of VCP on porcine complement was however not known prior to this study, which clearly document a potent complement inhibitory effect by all activators tested. A marked reduction in TCC formation was observed at 0.006 mg/mL and a complete inhibition was obtained at 0.05 mg/mL with all three activators. Provided that VCP can be retained in circulation after intravenous administration in pigs, it will probably be an excellent candidate for future in vivo studies. In summary, the serine protease inhibitors FUT-175 and C1inhibitor both inhibited porcine complement, FUT-175 being more efficient in inhibiting complement activation. Monoclonal antibodies to factor B and porcine factor D inhibited porcine complement differentially with respect to activation mechanism and dose required. VCP inhibited all activators of complement at low doses and emerges to be a promising candidate for future porcine studies.
Acknowledgements We thank Anne Pharo and Merethe Sanna Borgen for excellent laboratory technical assistance and Dorthe Christiansen for growing and preparing the bacteria. Financial support was kindly provided from The Norwegian Research Council, The Norwegian Council on Cardiovascular Disease, The Norwegian Foundation for Health and Rehabilitation, The Odd Fellow Foundation, The Research Council of Rikshospitalet, The Sonneborn Charitable Trust, Family Blix Foundation and Sigvald Bergesen d.y. and wife Nanki’s Foundation. GJK is senior International Wellcome Trust fellow for biomedical sciences in South Africa. YTG is a recipient of the poliomyelitis research foundation (PRF) and UCT fellowships and scholarships.
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Jiang, H., Wagner, E., Zhang, H., Frank, M.M., 2001. Complement 1 inhibitor is a regulator of the alternative complement pathway. J. Exp. Med. 194, 1609–1616. Kobayashi, C., Matsunami, K., Omori, T., Nakatsu, S., Nakahata, K., Xu, H., Fukuzawa, M., Shirakura, R., Miyagawa, S., 2006. Features of a newly cloned pig C1 esterase inhibitor1. J. Biochem. (Tokyo) 140, 421–427. Kotwal, G.J., Isaacs, S.N., McKenzie, R., Frank, M.M., Moss, B., 1990. Inhibition of the complement cascade by the major secretory protein of vaccinia virus. Science 250, 827–830. Kotwal, G.J., Moss, B., 1988. Vaccinia virus encodes a secretory polypeptide structurally related to complement control proteins. Nature 335, 176–178. Linton, S.M., Morgan, B.P., 1999. Complement activation and inhibition in experimental models of arthritis. Mol. Immunol. 36, 905–914. Miyata, M., Shirakawa, T., Acharya, B., Terao, S., Gotoh, A., 2006. Effects of nafamostat mesilate on ADP-induced platelet aggregation and disaggregation in hemodialysis patients. ASAIO J. 52, 272–275. Mohr, M., Hopken, U., Oppermann, M., Mathes, C., Goldmann, K., Siever, S., Gotze, O., Burchardi, H., 1998. Effects of anti-C5a monoclonal antibodies on oxygen use in a porcine model of severe sepsis. Eur. J. Clin. Invest. 28, 227–234. Mollnes, T.E., Kirschfink, M., 2006. Strategies of therapeutic complement inhibition. Mol. Immunol. 43, 107–121. Mollnes, T.E., Lea, T., Frøland, S.S., Harboe, M., 1985. Quantification of the terminal complement complex in human plasma by an enzyme-linked immunosorbent assay based on monoclonal antibodies against a neoantigen of the complex. Scand. J. Immunol. 22, 197–202. Mollnes, T.E., Redl, H., Hogasen, K., Bengtsson, A., Garred, P., Speilberg, L., Lea, T., Oppermann, M., Gotze, O., Schlag, G., 1993a. Complement activation in septic baboons detected by neoepitope-specific assays for C3b/iC3b/C3c, C5a and the terminal C5b-9 complement complex (TCC). Clin. Exp. Immunol. 91, 295–300. Mollnes, T.E., Redl, H., Hogasen, K., Bengtsson, A., Garred, P., Speilberg, L., Lea, T., Oppermann, M., Gotze, O., Schlag, G., 1993b. Complement activation in septic baboons detected by neoepitope-specific assays for C3b/iC3b/C3c, C5a and the terminal C5b-9 complement complex (TCC). Clin. Exp. Immunol. 91, 295–300. Mollnes, T.E., Song, W.C., Lambris, J.D., 2002. Complement in inflammatory tissue damage and disease. Trends Immunol. 23, 61–64. Murthy, K.H., Smith, S.A., Ganesh, V.K., Judge, K.W., Mullin, N., Barlow, P.N., Ogata, C.M., Kotwal, G.J., 2001. Crystal structure of a complement control protein that regulates both pathways of complement activation and binds heparan sulfate proteoglycans. Cell 104, 301–311. Pfeifer, P.H., Kawahara, M.S., Hugli, T.E., 1999. Possible mechanism for in vitro complement activation in blood and plasma samples: futhan/EDTA controls in vitro complement activation. Clin. Chem. 45, 1190–1199. Pierre, A.F., Xavier, A.M., Liu, M., Cassivi, S.D., Lindsay, T.F., Marsh, H.C., Slutsky, A.S., Keshavjee, S.H., 1998. Effect of complement inhibition with soluble complement receptor 1 on pig allotransplant lung function. Transplantation 66, 723–732. Pillay, N.S., Kellaway, L.A., Kotwal, G.J., 2005. Administration of vaccinia virus complement control protein shows significant cognitive improvement in a mild injury model. Ann. N. Y. Acad. Sci. 1056, 450–461. Reynolds, D.N., Smith, S.A., Zhang, Y.P., Mengsheng, Q., Lahiri, D.K., Morassutti, D.J., Shields, C.B., Kotwal, G.J., 2004. Vaccinia virus complement control protein reduces inflammation and improves spinal cord integrity following spinal cord injury. Ann. N. Y. Acad. Sci. 1035, 165–178.
Robbins, R.A., Russ, W.D., Rasmussen, J.K., Clayton, M.M., 1987. Activation of the complement system in the adult respiratory distress syndrome. Am. Rev. Respir. Dis. 135, 651–658. Scott, M.J., Burch, P.T., Jha, P., Peyton, J.C., Kotwal, G.J., Cheadle, W.G., 2003. Vaccinia virus complement control protein increases early bacterial clearance during experimental peritonitis. Surg. Infect. (Larchmt.) 4, 317– 326. Seelen, M.A., Roos, A., Wieslander, J., Mollnes, T.E., Sjoholm, A.G., Wurzner, R., Loos, M., Tedesco, F., Sim, R.B., Garred, P., Alexopoulos, E., Turner, M.W., Daha, M.R., 2005. Functional analysis of the classical, alternative, and MBL pathways of the complement system: standardization and validation of a simple ELISA. J. Immunol. Meth. 296, 187–198. Smith, S.A., Krishnasamy, G., Murthy, K.H., Cooper, A., Bromek, K., Barlow, P.N., Kotwal, G.J., 2002. Vaccinia virus complement control protein is monomeric, and retains structural and functional integrity after exposure to adverse conditions. Biochim. Biophys. Acta 1598, 55–64. Szebeni, J., Fontana, J.L., Wassef, N.M., Mongan, P.D., Morse, D.S., Dobbins, D.E., Stahl, G.L., Bunger, R., Alving, C.R., 1999. Hemodynamic changes induced by liposomes and liposome-encapsulated hemoglobin in pigs: a model for pseudoallergic cardiopulmonary reactions to liposomes. Role of complement and inhibition by soluble CR1 and anti-C5a antibody. Circulation 99, 2302–2309. Taube, C., Thurman, J.M., Takeda, K., Joetham, A., Miyahara, N., Carroll, M.C., Dakhama, A., Giclas, P.C., Holers, V.M., Gelfand, E.W., 2006. Factor B of the alternative complement pathway regulates development of airway hyperresponsiveness and inflammation. Proc. Natl. Acad. Sci. U.S.A. 103, 8084–8089. Thorbjornsdottir, P., Kolka, R., Gunnarsson, E., Bambir, S.H., Thorgeirsson, G., Kotwal, G.J., Arason, G.J., 2005. Vaccinia virus complement control protein diminishes formation of atherosclerotic lesions: complement is centrally involved in atherosclerotic disease. Ann. N. Y. Acad. Sci. 1056, 1–15. Thurman, J.M., Kraus, D.M., Girardi, G., Hourcade, D., Kang, H.J., Royer, P.A., Mitchell, L.M., Giclas, P.C., Salmon, J., Gilkeson, G., Holers, V.M., 2005. A novel inhibitor of the alternative complement pathway prevents antiphospholipid antibody-induced pregnancy loss in mice. Mol. Immunol. 42, 87–97. Thurman, J.M., Royer, P.A., Ljubanovic, D., Dursun, B., Lenderink, A.M., Edelstein, C.L., Holers, V.M., 2006. Treatment with an inhibitory monoclonal antibody to mouse factor B protects mice from induction of apoptosis and renal ischemia/reperfusion injury. J. Am. Soc. Nephrol. 17, 707–715. Tofukuji, M., Stahl, G.L., Agah, A., Metais, C., Simons, M., Sellke, F.W., 1998. Anti-C5a monoclonal antibody reduces cardiopulmonary bypass and cardioplegia-induced coronary endothelial dysfunction. J. Thorac. Cardiovasc. Surg. 116, 1060–1068. Volanakis, J.E., Narayana, S.V., 1996. Complement factor D, a novel serine protease. Protein Sci. 5, 553–564. Ward, P.A., 2004. The dark side of C5a in sepsis. Nat. Rev. Immunol. 4, 133–142. Welch, T.R., 2002. Complement in glomerulonephritis. Nat. Genet. 31, 333–334. Wouters, D., Voskuyl, A.E., Molenaar, E.T., Dijkmans, B.A., Hack, C.E., 2006. Evaluation of classical complement pathway activation in rheumatoid arthritis: measurement of C1q-C4 complexes as novel activation products. Arthritis Rheum. 54, 1143–1150. Yao, Y.M., Redl, H., Bahrami, S., Schlag, G., 1998. The inflammatory basis of trauma/shock-associated multiple organ failure. Inflamm. Res. 47, 201–210. Zarkadis, I.K., Mastellos, D., Lambris, J.D., 2001. Phylogenetic aspects of the complement system. Dev. Comp. Immunol. 25, 745–762.
Candidate inhibitors of porcine complement Ebbe B. Thorgersen a,∗ , Yohannes T. Ghebremariam b , Joshua M. Thurman c , Michael Fung d , Erik Waage Nielsen e , V. Michael Holers f , Girish J. Kotwal b , Tom Eirik Mollnes a a
Institute of Immunology, Rikshospitalet-Radiumhospitalet Medical Center and University of Oslo, N-0027 Oslo, Norway b Division of Medical Virology, IIDMM, University of Cape Town, HSC, Cape Town 7925, South Africa c Divisions of Nephrology and Hypertension, Department of Medicine, University of Colorado Health Sciences Center, Denver, CO 80262, USA d Tanox Inc., Houston, TX, USA e Department of Anaesthesiology, Nordland Hospital and University of Tromsø, Norway f Division of Rheumatology, Department of Medicine, University of Colorado Health Sciences Center, Denver, CO 80262, USA Received 18 September 2006; received in revised form 11 October 2006; accepted 13 October 2006 Available online 15 November 2006
Abstract Therapeutic complement inhibition is a promising strategy for treatment of a number of diseases as judged from rodent studies. The species distance from rodents to humans may limit the clinical relevance of these studies. The pig is an alternative animal for studies of human diseases like sepsis and ischemia/reperfusion injury. However, available complement inhibitors for use in pigs are scarce. The aim of the present study was to investigate and compare the efficacy of selected candidate inhibitors of porcine complement in vitro for possible future application in vivo. Sera from three different pigs were each incubated with three different activators of the complement system (zymosan, heat-aggregated immunoglobulin G (HAIGG) and Escherichia coli). Three groups of complement inhibitor candidates were tested: serine protease inhibitors (FUT-175 and C1-inhibitor), monoclonal antibodies (anti-factor B (fB) and anti-factor D (fD)) and a recombinant regulatory protein (vaccinia virus complement control protein (VCP)). Read-out was the terminal C5b-9 complement complex (TCC). The serine protease inhibitors FUT-175 and C1-inhibitor dose-dependently inhibited TCC formation in zymosan-, HAIGG- and E. coli-activated porcine sera, but with different efficacy. Complete inhibition of TCC was obtained using 0.2 mg/mL FUT-175, but required 16 mg/mL of C1inhibitor. The monoclonal anti-fB and -fD antibodies both inhibited TCC formation dose-dependently, but in different ways. Anti-fB at high dose (1 mg/mL) completely inhibited TCC formation in sera activated with zymosan and virtually completely in sera activated with HAIGG, but not in sera activated with E. coli. Anti-fD inhibited all three activators at low dose (0.05 mg/mL), and approximately 50% TCC reduction was obtained. The recombinant complement regulatory protein VCP efficiently and dose-dependently inhibited TCC formation with a complete inhibition found at 0.05 mg/mL for all three activators. All candidates tested inhibited porcine complement activation, but in different ways and to different degrees. Of the complement-specific candidates, VCP inhibited all activators completely at low doses. © 2006 Elsevier Ltd. All rights reserved. Keywords: Anti-factor B; Anti-factor D; C1-inhibitor; Complement; Complement inhibitors; Escherichia coli; FUT-175; Porcine; Vaccinia virus complement control protein (VCP)
1. Introduction In response to invading pathogens, the innate immune system plays an important role in protecting the host. The complement system is part of the innate immune defence. The system arose early in the evolution of species and is both preserved and
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Corresponding author. Tel.: +47 48144121; fax: +47 23073510. E-mail address: [email protected] (E.B. Thorgersen).
0161-5890/$ – see front matter © 2006 Elsevier Ltd. All rights reserved. doi:10.1016/j.molimm.2006.10.004
developed later, reflecting the importance in protection against non-self (Zarkadis et al., 2001). Complement is a cascade system in blood. It consists of three known initiating pathways; the classical (CP), the lectin (LP) and the alternative pathway (AP) and one common final pathway. Recognition of opsonised microorganisms by the classical pathway, via antibodies that bind to C1q, and the lectin pathway, via MBL-MASP complexes, leads to activation of serine proteases that cleave complement components C4 and C2. This leads to the formation of the protease complex C4b2a, which then cleaves
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C3 into C3a and C3b and forms the C5 convertase C4b2a3b. The alternative pathway forms C3b by slow spontaneous hydrolysis of C3 to C3(H2 O). C3(H2 O) forms complex with factor B, which is then cleaved by factor D to yield C3(H2 O)Bb. This complex is able to cleave C3 to C3a and C3b. C3b binds factor B and the second alternative pathway C3 convertase, C3bBb, is formed and upon stabilization with properdin generates the C5 convertase C3b3bBb. After the cleavage of C5 by either C5 convertase, C5b binds to C6 and the terminal sequence then forms the terminal complement complex (TCC), either as the membrane-inserted C5b-9 (membrane attack complex) or as the soluble sC5b-9. A number of soluble and cell-bound regulatory proteins act to inhibit activation at different levels, keeping the system under tight control (Mollnes et al., 2002). The complement system plays an essential role in host defence, in particular by opsonising microbes (Goldfarb and Parrillo, 2005). However, increasing evidence has underscored the pathogenic side of excessive complement activation leading to tissue damage in several inflammatory conditions, including sepsis (Ward, 2004), trauma and shock (Yao et al., 1998), nephritis (Welch, 2002), arthritis (Linton and Morgan, 1999; Wouters et al., 2006), ischemia/reperfusion injury (Arumugam et al., 2004) and respiratory distress syndrome (Robbins et al., 1987). Thus, inhibitors of the complement system may be useful in the treatment of these conditions (Mollnes and Kirschfink, 2006). Inhibitors that target broadly several of the cascade proteins (such as serine proteases), complement specific monoclonal antibodies, and recombinant regulatory proteins targeting specific sites in the complement cascade are all candidate inhibitors. The pig is widely used as a model animal for human diseases. Only occasionally though, complement inhibitors have been used in pig studies, e.g. with a soluble complement receptor type 1 (sCR1) (Pierre et al., 1998; Szebeni et al., 1999) and an anti-C5a antibody (Amsterdam et al., 1995; Mohr et al., 1998; Tofukuji et al., 1998). However, a detailed evaluation and comparison of the effects of various candidate inhibitors on the three different complement activation pathways in pigs is missing. The current study was therefore undertaken to address the need.
2. Material and methods 2.1. Sera and reagents Sera from pigs (Norsvin, Norwegian farm pigs (landrace), out-bred stock) were prepared and frozen in aliquots at −70 ◦ C. Zymosan A (Z-4250) was purchased from Sigma (St. Louis, MO). Gamma globulin (human, 165 mg/mL, Vno. 027490) was purchased from Biovitrum (Stockholm, Sweden) and prepared to heat-aggregated immunoglobulin G (HAIGG) by heating a dilution of 10 mg/mL of gamma globulin to 63 ◦ C for 10 min. Escherichia coli was purchased from American Type Culture Collection (ATCC; Manassas, VA). Human serum albumin (HSA; 200 mg/mL, Vno. 478172) was purchased from Octapharma AG (Lachen, Switzerland). Sterile phosphatebuffered saline (PBS; cat no. 14040-083) was purchased from Gibco, Invitrogen Corporation (Paisley, Scotland).
2.2. Serine protease inhibitors FUT-175 (nafamostat mesilate, Futhan) was purchased from Torii Pharmaceutical Co. (Tokyo, Japan). Human C1-inhibitor was purchased from ZLB Behring AB (Danderyd, Sweden). 2.3. Monoclonal antibodies Mouse monoclonal antibody (mAb) to mouse factor B (clone 1379 5a IgG1), shown to cross-react with pig (Thurman et al., 2005), and pig factor D (clone 230A IgG1) (Fung unpublished) were produced in the co-authors laboratories and described in detail previously. A mouse-anti-human CD22 (clone IS7 IgG1) mAb was purchased from Diatec (Oslo, Norway). Anti-CD22 was tested in flow cytometry and showed no cross-reactivity with porcine antigens and could therefore be used as an isotype control. 2.4. Recombinant complement regulatory protein Vaccinia complement control protein (VCP) described previously (Kotwal and Moss, 1988; Kotwal et al., 1990) was produced using the Pichia pastoris yeast expression system as described before (Murthy et al., 2001; Ghebremariam et al., 2005). 2.5. Enzyme immunoassays The soluble terminal C5b-9 complement complex (TCC), was measured in an enzyme immunoassay (EIA), as described previously (Mollnes et al., 1985) and later modified (Mollnes et al., 1993b). Briefly, the monoclonal antibody aE11 reacting with a neoepitope exposed in C9 after incorporation in the C5b9 complex was used as capture antibody, the final concentration in the wells was 3 g/mL. A biotinylated monoclonal anti-C6 (Quidel Corporation, San Diego, CA) was used as detection antibody in a final concentration of 4 g/mL. Both antibodies cross-react with specific pig epitopes and the assay can be used to detect porcine TCC (Mollnes et al., 1993a). The standard was normal human serum activated with zymosan and defined to contain 1000 AU/mL. Zymosan-activated porcine serum was used as a positive control and the buffer used as a diluent for the standards and samples, containing PBS, ethylenediaminetetraacetic acid (EDTA) and a detergent (Tween), was used as a negative control. 3. In vitro experimental model 3.1. Test of complement pathways in serum Porcine serum from three different pigs were tested using a commercial available EIA (Wielisa, Wieslab, Lund, Sweden), for assessment of complement functional activity in classical, lectin, and alternative pathway (Seelen et al., 2005). The kit is developed for human use but because it contains the mAb aE11 as detection antibody, which cross-react with the porcine epitope (Mollnes et al., 1993a), it was here found to work with
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pig serum as well, provided that higher serum concentration was used. Porcine sera diluted 1/10 for CP and LP and 1/2 for AP gave similar results as for human serum diluted according to the recommendation of the kit instruction (1/101 for CP and LP and 1/18 for AP). 3.2. Serum experiments Porcine sera were incubated with three complement activators: zymosan, a polysaccharide from the yeast Saccaharomyces cerevisiae cell membrane, known to be a potent activator of the lectin pathway (Brouwer et al., 2006) and the alternative pathway (Fearon and Austen, 1977) to a final concentration of 1 mg/mL serum, heat-aggregated immunoglobulin G (HAIGG), known to activate the classical pathway of complement (Fust et al., 1978) to a final concentration of 1 mg/mL serum, and E. coli to a final concentration of 1 × 108 /mL serum. PBS was used as background control. Inhibition experiments were performed by pre-incubation for 5 min with the different candidate inhibitors and their controls. An isotype-matched control mAb and HSA were used as controls. The complement activators were added and incubated for 30 min at 37 ◦ C. Complement activation was stopped by adding EDTA to a final concentration of 20 mM. The samples were kept on ice and then centrifuged for 15 min at 3000 × g (4 ◦ C), for removal of E. coli and zymosan from the samples. The T0 baseline sample was processed immediately. The sera were stored at −70 ◦ C until analyzed. 3.3. Data presentation Data are presented as ratios (indicated as “normalized values” in the figure legends) between the measured value at each concentration of inhibitor and the baseline uninhibited value. Median and range of these values are presented. The quantities are presented as mass throughout the text and in the figures. In order to compare the efficacy on a molar basis, the lowest molar concentration of each agent giving maximum inhibition of complement activation is presented (Table 1). 3.4. Ethics The study was approved by the The Norwegian Animal Experimental Board.
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4. Results 4.1. Test of the complement pathways in serum The Wielisa kit, measuring the functional activity of the classical, lectin and alternative complement pathway, cross-reacted with porcine complement and all three pathways of the complement system were found to be intact in the sera used. 4.2. Effect of candidate inhibitors on complement activation in serum 4.2.1. Serine protease inhibitors FUT-175 dose-dependently inhibited complement activation, as measured by TCC, for all three activators, whereas HSA had no effect (Fig. 1). Inhibition was complete at 0.2 mg FUT175/mL serum. C1-inhibitor dose-dependently inhibited TCC formation for all three activators, whereas HSA had no effect (Fig. 2). Inhibition was complete at 16 mg (64 Units) C1-inhibitor/mL serum. HAIGG and E. coli were inhibited more efficiently at lower doses than zymosan (Fig. 2). 4.2.2. Monoclonal antibodies Anti-factor B dose-dependently and completely inhibited zymosan- and almost completely inhibited HAIGG-induced TCC formation, at a concentration of 1 mg/mL (Fig. 3). In serum incubated with E. coli, the antibody had virtually no effect. A marginal inhibition at low doses was observed, which was not seen at higher doses (Fig. 3). The isotype-matched control mAb showed no inhibitory effect (Fig. 3). Anti-factor D dose-dependently inhibited TCC formation for all three activators (Fig. 4). Maximal inhibition was observed using low doses (0.05 mg/mL), but was limited to a reduction of TCC formation of approximately 50% (Fig. 4). The isotypematched control mAb showed no inhibitory effect (Fig. 4). 4.2.3. Recombinant complement regulatory protein Vaccinia virus complement control protein (VCP) dosedependently inhibited TCC formation for all three activators, whereas HSA had no effect (Fig. 5). Inhibition was complete at 0.05 mg VCP/mL serum, but inhibited markedly at
Table 1 Molarity of the lowest concentration of each porcine complement candidate inhibitor giving maximum inhibitiona Candidate inhibitor
FUT-175 C1-inhibitor Anti-factor B Anti-factor Dd VCP a b c d
Molecular weight (Da)
540 105,000 150,000b 150,000b 28,800
Molarity (M) Zymosan
HAIGG
Escherichia coli
2.2 × 10−3
2.2 × 10−3
2.2 × 10−3 9.2 × 10−4 –c 2.0 × 10−6 1.1 × 10−5
9.2 × 10−4 4.0 × 10−5 2.0 × 10−6 1.1 × 10−5
9.2 × 10−4 4.0 × 10−5 5.1 × 10−7 1.1 × 10−5
Maximum inhibition varied considerably between the different inhibitors (see Section 4). Approximate molecular weight for anti-factors B and D. Anti-factor B had almost no inhibitory effect on E. coli-induced complement activation. The highest obtainable anti-factor D concentration used did not permit determination of maximum inhibition (see Section 5).
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Fig. 1. Effect of FUT-175 on complement activation in porcine serum. Sera from three different pigs were pre-incubated with FUT-175 or HSA for 5 min, and then complement activators (zymosan, HAIGG, Escherichia coli) were added and incubated for 30 min at 37 ◦ C. Read-out was TCC measured using ELISA. Median and range of normalized values of three separate experiments are shown.
Fig. 2. Effect of C1-inhibitor on complement activation in porcine serum. Sera from three different pigs were pre-incubated with C1-inhibitor or HSA for 5 min, and then complement activators (zymosan, HAIGG, E. coli) were added and incubated for 30 min at 37 ◦ C. Read-out was TCC measured using ELISA. Median and range of normalized values of three separate experiments are shown. One Unit of C1-inhibitor corresponds to 0.25 mg.
lower concentrations. Thus, at 10-fold lower concentration (0.00625 mg/mL) VCP inhibited TCC formation by approximately 40% in zymosan-activated sera, 70% in HAIGGactivated sera and 60% in E. coli-activated sera (Fig. 5). 4.2.4. Comparison of the inhibitors on a molar basis Due to the different molecular weights of the inhibitors, quantities are expressed as molarity as well in order to compare the efficacy on a molar basis. The lowest molar concentration which gave a maximum inhibition for each of the activators is therefore shown (Table 1).
5. Discussion Three groups of candidate complement inhibitors (serine protease inhibitors, monoclonal antibodies, and a recombinant complement regulatory protein) were examined in vitro in porcine serum. Sera were tested using the Wielisa functional kit for human complement, which was found to cross-react with porcine serum, and all three initiating pathways of complement were found to be intact. The synthetic serine protease inhibitor FUT-175 (nafamostat mesilate, Futhan) is known to inhibit several proteins in
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Fig. 3. Effect of anti-factor B on complement activation in porcine serum. Sera from three different pigs were pre-incubated with anti-factor B or an isotype matched control for 5 min, and then complement activators (zymosan, HAIGG, E. coli) were added and incubated for 30 min at 37 ◦ C. Read-out was TCC measured using ELISA. Median and range of normalized values of two separate experiments are shown.
the human complement cascade (C1r, C1s, C3, MASPs, factor B, factor D) and other serine proteases in the contact system and the coagulation system (Fujii and Hitomi, 1981; Ikari et al., 1983; Pfeifer et al., 1999). FUT-175 is a commercial available substance and is used in Japan for the treatment of pancreatitis (Imaizumi et al., 2004). In the present study it showed a dose-dependent inhibition to baseline TCC values at 0.2 mg/mL in porcine serum incubated with zymosan, HAIGG or E. coli. FUT-175 is easily available at a low price and thus a promising
candidate for inhibition of porcine complement. These advantages are counteracted by the fact that it is not a specific complement inhibitor. Furthermore, the molecular weight is low (approximately 540 Da (Miyata et al., 2006)) and therefore the bioavailability is probably limited due to its rapid elimination from circulation. C1-inhibitor is a serine protease inhibitor and naturally occurring regulator of the complement and the contact systems and, thus, like FUT-175 it is not a complement specific inhibitor. It is
Fig. 4. Effect of anti-factor D on complement activation in porcine serum. Sera from three different pigs were pre-incubated with anti-factor D or an isotype matched control for 5 min, and then complement activators (zymosan, HAIGG, E. coli) were added and incubated for 30 min at 37 ◦ C. Read-out was TCC measured using ELISA. Median and range of normalized values of three separate experiments are shown.
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Fig. 5. Effect of VCP on complement activation in porcine serum. Sera from three different pigs were pre-incubated with VCP or HSA for 5 min, and then complement activators (zymosan, HAIGG, E. coli) were added and incubated for 30 min at 37 ◦ C. Read-out was TCC measured using ELISA. Median and range of normalized values of three separate experiments are shown.
a glycosylated peptide of 478 amino acid residues with a molecular weight of approximately 105 kDa, consistent with a probable good bioavailability and prolonged retention. Physiological plasma concentration in man is 0.24 mg/mL corresponding to 1 U/mL (Caliezi et al., 2000). We used human C1-inhibitor in our experiments. According to a recent study the reactive site of porcine and human C1-inhibitor are highly homologous with similar complement inhibitory function in pig and human sera (Kobayashi et al., 2006). In porcine serum, the HAIGG-induced complement activation was inhibited in a dose-dependent manner, as expected since C1-inhibitor is a regulator of the classical pathway (Caliezi et al., 2000). In E. coli-activated serum C1inhibitor showed a similar dose-dependent inhibition of complement, whereas the inhibition of complement activation by zymosan was less profound. Jiang and co-authors have reported an inhibitory effect of C1-inhibitor on the alternative complement pathway by binding to C3b (Jiang et al., 2001). Therefore the effect observed in our study may be explained concurrently. Nevertheless, inhibition of TCC induced by all three activators required substantial amounts of the protein, which hardly would be attainable in vivo. Anti-factor B has previously been shown to be a promising inhibitor of the complement system, protecting against antiphospholipid antibody-induced pregnancy loss (Thurman et al., 2005) and renal ischemia/reperfusion injury in mice (Thurman et al., 2006). Recently it has been shown to be effective in reducing airway hyperresponsiveness in a murine model of allergic airway disease (Taube et al., 2006). Consistently, the present study documented a dose-response inhibition of TCC induced by zymosan and HAIGG, showing that mouse anti-factor B cross-reacts with pig factor B. The zymosan-induced complement activation was completely inhibited, whilst the HAIGG-induced activation was reduced by approximately 80%. This effectiveness of antifactor B on classical pathway induced TCC formation probably reflects the importance of the alternative pathway as an amplifi-
cation loop for this pathway (Harboe et al., 2004). A complete inhibition required a relatively high concentration, though corresponding well with theoretical dose needed for neutralization of factor B in serum. However, the effect on E. coli induced complement activation was surprising. A small inhibitory effect was seen at the lower doses of the mAb, which was not seen at higher doses. An unspecific activating effect of the antibody on complement in high doses is ruled out by the fact that the same dose efficiently reduced HAIGG- and zymosan-induced TCC formation. It is therefore tempting to speculate that E. coli has properties, on the surface or by released products to the fluid-phase, which abolish the effect of anti-factor B. Anti-factor B might therefore not be a suitable candidate for treatment of sepsis caused by E. coli, although it is promising in the treatment of the conditions mentioned above, as well as for inhibition of meconium-induced complement activation (own unpublished data). Factor D is the rate limiting protease of the alternative complement pathway because of its low concentration in blood (Volanakis and Narayana, 1996). It is therefore not surprising that a lower concentration was required for complement inhibition than that of anti-factor B. The alternative pathway is an amplification loop for the other two pathways, making it very important in the cascade for response to invading microorganisms (Harboe et al., 2004). This and other studies have shown that a very efficient inhibition of the human alternative complement pathway is obtained at concentrations below 0.01 mg anti-human factor D/mL serum. In the present study the anti-porcine factor D tested inhibited all three activators, but with a maximal inhibition limited to approximately 50% at 0.05 mg/mL. The anti-porcine factor D used in this study was produced by E. coli, and is therefore not glycosylated in contrast to the anti-human factor D mAb 166-32 (Fung et al., 2001). This may explain that the maximum inhibitory effect obtained in porcine serum was restricted. We cannot, however, fully exclude that an additional
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effect could have been obtained using higher concentration of the antibody since no plateau was reached. The amount of protein in the stock solution did not allow higher final concentration in the experiment. VCP is a 28.8 kDa 243 amino acid protein secreted by vaccinia virus infected mammalian cells. It is structurally related to C4b-binding protein, and functionally closely related to CR1. It binds to C3b and C4b in the rodent and human complement cascade and thereby arrests the C3 convertases (Kotwal and Moss, 1988; Jha and Kotwal, 2003). In a study of VCP’s ability to protect xenoendothelial cells from damage by the complement system, Al-Mohanna and colleagues found that VCP might bind to ␣-galactose residues on pig aortic endothelial cells thereby competitively blocking binding of xenoreactive natural antibodies, showing additional properties of this protein (Al-Mohanna et al., 2001). Tests under various and harsh conditions show that VCP is a robust protein and therefore easy to ship, store and administer (Smith et al., 2002). Several studies have demonstrated the beneficial effects of VCP as complement inhibitor in rodent disease models: collagen-induced arthritis (Jha et al., 2005), spinal cord injury (Reynolds et al., 2004), traumatic brain injury (Hicks et al., 2002; Pillay et al., 2005), experimental peritonitis (Scott et al., 2003), atherosclerosis (Thorbjornsdottir et al., 2005) and mouse-to-sensitized rat and guinea pig-to-rat heart xenotransplantations (Anderson et al., 2002, 2003). The effect of VCP on porcine complement was however not known prior to this study, which clearly document a potent complement inhibitory effect by all activators tested. A marked reduction in TCC formation was observed at 0.006 mg/mL and a complete inhibition was obtained at 0.05 mg/mL with all three activators. Provided that VCP can be retained in circulation after intravenous administration in pigs, it will probably be an excellent candidate for future in vivo studies. In summary, the serine protease inhibitors FUT-175 and C1inhibitor both inhibited porcine complement, FUT-175 being more efficient in inhibiting complement activation. Monoclonal antibodies to factor B and porcine factor D inhibited porcine complement differentially with respect to activation mechanism and dose required. VCP inhibited all activators of complement at low doses and emerges to be a promising candidate for future porcine studies.
Acknowledgements We thank Anne Pharo and Merethe Sanna Borgen for excellent laboratory technical assistance and Dorthe Christiansen for growing and preparing the bacteria. Financial support was kindly provided from The Norwegian Research Council, The Norwegian Council on Cardiovascular Disease, The Norwegian Foundation for Health and Rehabilitation, The Odd Fellow Foundation, The Research Council of Rikshospitalet, The Sonneborn Charitable Trust, Family Blix Foundation and Sigvald Bergesen d.y. and wife Nanki’s Foundation. GJK is senior International Wellcome Trust fellow for biomedical sciences in South Africa. YTG is a recipient of the poliomyelitis research foundation (PRF) and UCT fellowships and scholarships.
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