Elsevier

Molecular Immunology

Volume 44, Issue 8, March 2007, Pages 1827-1834
Molecular Immunology

Candidate inhibitors of porcine complement

https://doi.org/10.1016/j.molimm.2006.10.004Get rights and content

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 C1-inhibitor. 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.

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 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 C3 into C3a and C3b and forms the C5 convertase C4b2a3b. The alternative pathway forms C3b by slow spontaneous hydrolysis of C3 to C3(H2O). C3(H2O) forms complex with factor B, which is then cleaved by factor D to yield C3(H2O)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.

Section snippets

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

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 pig serum as well, provided that higher serum concentration was

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.

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 FUT-175/mL serum.

C1-inhibitor dose-dependently inhibited TCC formation for all three

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

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

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