Complement regulation in innate immunity and the acute-phase response: inhibition of mannan-binding lectin-initiated complement cytolysis by C-reactive protein (CRP)

Abstract

Mannan-binding lectin (MBL) is an acute-phase protein which activates complement at the level of C4 and C2. We recently reported that the alternative pathway also is required for haemolysis via this 'lectin pathway' in human serum. CRP is another acute-phase reactant which activates the classical pathway, but CRP also inhibits the alternative pathway on surfaces to which it binds. Since serum levels of both proteins generally increase with inflammation and tissue necrosis, it was of interest to determine the effect of CRP on cytolysis via the lectin pathway. We report here that although CRP increases binding of C4 to MBL-sensitized erythrocytes, which in turn enhances lectin pathway haemolysis, it inhibits MBL-initiated cytolysis by its ability to inhibit the alternative pathway. This inhibition is characterized by increased binding of complement control protein H and decreased binding of C3 and C5 to the indicator cells, which in turn is attributable to the presence of CRP. Immunodepletion of H leads to greatly enhanced cytolysis via the lectin pathway, and this cytolysis is no longer inhibited by CRP. These results indicate that CRP regulates MBL-initiated cytolysis on surfaces to which both proteins bind by modulating alternative pathway recruitment through H, pointing to CRP as a complement regulatory protein, and suggesting a co-ordinated role for these proteins in complement activation in innate immunity and the acute-phase response.

Fig. 1

CRP inhibition of mannan-binding lectin (MBL)-initiated haemolysis. Indicator sheep erythrocytes optimally coated with phosphocholine-conjugated bovine serum albumin (PC–BSA) and mannan were reacted with MBL (1 μg) and increasing amounts of CRP prior to the addition of agammaglobulinaemic human sera; lysis was determined as described. No lysis was observed with E-PC sensitized with CRP alone.

Fig. 2

Binding of C4, C3 and C5 to CRP- and mannan-binding lectin (MBL)-sensitized indicator cells. Sheep erythrocytes (E) were coated with phosphocholine-conjugated bovine serum albumin (PC–BSA), mannan or both ligands, sensitized with CRP (50 μg), MBL (1 μg) or both molecules, and reacted with C7-deficient human serum as described; after appropriate washes, the cells were reacted with biotinylated antisera to C4, C3 and C5, respectively, and the mean channel fluorescence (MCF) was calculated in the usual way.

Fig. 3

H binding to CRP- and mannan-binding lectin (MBL)-sensitized indicator cells. Indicator cells were prepared as described, and reacted with C7-deficient human serum; H binding was determined as mean channel fluorescence (MCF) after 5 and 30 min incubation, respectively.

Fig. 4

Effect of CRP on mannan-binding lectin (MBL)-initiated haemolysis in H-depleted serum. Lysis of E-M-MBL was greatly increased in agammaglobulinaemic human sera (AHS) that was depleted of H (H-R) (centre panel), and addition of CRP did not inhibit haemolysis in H-depleted serum (right panel). H depletion also led to a small, but significant, degree of haemolysis of CRP-sensitized E-PC.

Fig. 5

Diagrammatic sketch of the effects of CRP and mannan-binding lectin (MBL) on complement activation, indicating the classical pathway (thin solid line), alternative pathway (dotted line) and lectin pathway (heavy solid line); arrows other than the directional indicators from CRP indicate enzymatic cleavages. CRP activates the classical pathway via C1q, but inhibits the alternative pathway by enhancing the interaction between H and C3b, displacing B from C3b and increasing inactivation of C3b by I. This latter effect predominates during complement activation by both MBL and CRP, and hence CRP inhibits C-dependent lysis initiated by MBL via the lectin pathway. It is not yet clear whether a divalent cation (e.g. Mg²⁺) is required for interactions of MBL with the MASP-1 and MASP-2 (M1 and M2).