Effect of supraphysiologic levels of C1-inhibitor on the classical, lectin and alternative pathways of complement

Abstract

C1-inhibitor is increasingly used experimentally and clinically in inflammatory conditions like septicemia and ischemia-reperfusion injury. Several mechanisms may account for the anti-inflammatory effects of C1-inhibitor, including inhibition of complement. The aim of the present study was to investigate and compare the supraphysiologic effect of C1-inhibitor on the three complement pathways. Novel assays for specific evaluation of the classical, lectin and alternative pathways were employed using normal human serum supplemented with increasing concentrations of C1-inhibitor. Solid-phase classical- and lectin pathway activation was dose-dependently and significantly reduced up to 85% in the range of 2–28 times physiologic C1-inhibitor concentration. The lectin pathway was more potently inhibited than the classical at low doses. A functional lectin pathway assay demonstrated a significant reduction of C4 deposition up to 86% even at low concentration of C1-inhibitor and documented the effect to be at the level of MBL/MASPs. In contrast, C1-inhibitor had no effect on solid-phase alternative pathway activation, but significantly reduced cobra venom factor-induced fluid-phase activation up to 88%. The negative controls albumin and IgG had no effect on complement activation. The positive inhibitory controls compstatin (C3 inhibition), EDTA- or MBL-deficient sera reduced complement activation by 82–100%. We conclude that C1-inhibitor in high physiologic doses differentially inhibits all three-complement pathways. The inhibition pattern was strikingly different in the classical and lectin pathway, compared to the alternative. Previous studies interpreting the effects of C1-inhibitor as only due to classical pathway inhibition needs reconsideration. The data has implications for the therapeutic use of C1-inhibitor.

Introduction

C1-inhibitor (C1-INH) was named according to its ability to inhibit the C1 subcomponents C1r and C1s in the classical pathway and is the only known physiologic inhibitor of these proteases (Pensky et al., 1961). Early studies of patients suffering from hereditary angioedema (HAE) shed further light on C1-INH's role in the classical pathway (Landerman et al., 1962, Donaldson and Evans, 1963). These patients’ heterozygous C1-INH deficiency leads to an increased spontaneous activation of C4 and C2 (Donaldson and Rosen, 1964). As this breakdown takes place in the fluid phase, and not on a solid surface like, e.g. bacteria, C4b is rapidly inactivated by factor I. This has been the main explanation as to why the classical C3 convertase C4bC2a does not assemble in HAE patients (Klein, 1990). A higher ratio of C4b-binding protein to C4 may also hinder activation beyond C4 and C2 (Gronski et al., 1988). Later, sensitive assays showed a very modest activation of C3 in HAE patients (Nielsen et al., 1995). During attacks of HAE even a minor increase in the terminal complement complex (TCC) was revealed (Nielsen et al., 1996).

In the lectin pathway, discovered in the late 1980's, mannose-binding lectin (MBL) or ficolins bind structures containing sugar on the surface of foreign particles and microbes (Kawasaki et al., 1989, Lynch et al., 2004). MBL share similarities with C1q in the classical pathway. Complexes of MBL and a C1s-like enzyme named MBL-associated serine protease (MASP)-2 become activated when bound to the target. The resulting sequential cleavage of C4 and C2 creates a C4bC2a complex indistinguishable from the C3 convertase of the classical pathway (Matsushita and Fujita, 1992, Thiel et al., 1997). MASP-2 is inhibited by C1-INH and alpha-2 macroglobulin (Matsushita et al., 2000, Terai et al., 1995). Furthermore, a role for C1-INH in regulating the alternative pathway by a non-covalent binding to C3b was recently postulated (Jiang et al., 2001).

Patients suffering from C1-INH deficiency experience bouts of edema in nearly every organ in the body (Donaldson and Evans, 1963). Many of these patients have had their edema effectively reversed by intravenous infusion of C1-INH concentrate (Agostoni et al., 1980). The concentrate is well tolerated (De Serres et al., 2003). The recognition of C1-INH's pivotal role in controlling the complement system sparked the idea of infusing supraphysiologic doses of C1-INH in conditions where complement activation contributes to the pathophysiology. Such conditions are sepsis, cytokine-induced vascular leak syndrome, acute myocardial infarction, trauma, burns, multiple organ failure and graft rejection (Caliezi et al., 2000, Kirschfink and Mollnes, 2001).

The specific effect of supraphysiologic C1-INH concentration in serum on each of the three initiating complement pathways is largely unknown and has not previously been compared under similar conditions. By use of newly developed assays we therefore examined and compared these effects. The data indicate that C1-INH has at least the same inhibitory effect on the lectin pathway as on the classical pathway. Inhibition of the alternative pathway was observed only for the fluid-phase. The data has implications for the use of C1-INH as a therapeutic reagent and for interpretation of previously published observations.