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We previously reported that complement-dependent haemolysis of sheep erythrocytes (E) coated with mannan (M) and sensitized with human mannan-binding lectin (MBL) via the lectin pathway in man occurs in Mg-EGTA and requires alternative pathway amplification. Calcium was required for MBL binding to E-M, but once the E-M-MBL intermediate was formed, MBL was retained and haemolysis occurred in the absence of calcium. Comparable or greater lectin pathway haemolysis in the absence of calcium was observed upon incubation of E-M-MBL in guinea-pig, rat, dog and pig sera, and was further investigated in the guinea-pig, in which titres were much higher ( approximately 14-fold) than in man, and in contrast to humans, greater than classical pathway haemolytic activity. As in human serum, no lysis was observed in C4- or C2-deficient guinea-pig serum until purified C4 or C2, respectively, were restored. However, lectin pathway haemolytic activity in the guinea-pig did not require the alternative pathway. Removal (>98%) of factor D activity by three sequential passages through Sephadex G-75, resulting in serum which retained a normal classical pathway but no alternative pathway haemolytic activity, did not reduce the ability of guinea-pig serum to mediate haemolysis via the lectin pathway. Further, the C3-convertase formed via the lectin pathway (E-M-MBL-C4,2) lysed in C2-deficient guinea-pig but not human serum chelated with EDTA, a condition which precludes alternative pathway amplification. Thus, lectin pathway haemolysis occurs efficiently in guinea-pig serum, in the absence of calcium and without requirement for alternative pathway amplification. The guinea-pig provides a model for studying the assembly and haemolytic function of a lectin pathway which contrasts with the lectin pathway of man, and allows for comparisons that may help clarify the role of this pathway in complement biology.

Lysis via the newly discovered lectin pathway of complement activation is reviewed. Mannan-coated erythrocytes sensitized with MBL are lysed in human serum containing Mg-EGTA via the lectin pathway by a process which requires alternative pathway amplification. The inhibitory activities of C4bp and factor H, which are augmented in the presence of MBL, regulate this hemolysis. Lectin pathway activity is enhanced by IgG, which inhibits H activity, and is inhibited by C-reactive protein, which enhances the activity of H. Lectin pathway hemolysis in Mg-EGTA also is seen in other species, and is particularly intense and does not require alternative pathway amplification in the guinea pig. New investigations using E-RaLPS as the MBL-binding agent allowed comparison with classical pathway activation by rabbit anti-RaLPS using the same indicator cell. E-RaLPS-MBL are lysed in human serum-Mg-EGTA, and alternative pathway amplification is required. The addition of rabbit anti-E to E-RaLPS-MBL leads to significant enhancement of lysis in Mg-EGTA, much greater than Ig enhancement of hemolysis via the alternative pathway. Lectin pathway activation also enhances the antibody-independent C activation of the classical C pathway via C1q by ReLPS, as well as the direct activation of the alternative C pathway by wild type LPS. Thus, potentiation of reactions initiated at sites of IgG deposition and Ig-independent complement activation represents another characteristic of the lectin pathway.

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.