The requirement of localized, CR2-mediated, alternative pathway activation of complement for covalent deposition of...

We have shown previously that normal B cells share, with Epstein-Barr virus-transformed and malignant B cells, the ability to activate the alternative pathway (AP) of complement in vitro, resulting in the deposition of C3 fragments on the cell surface. Complement receptor type 2 (CR2, CD21) has been implicated directly as the site for formation of an AP convertase, which provides nascent C3b for deposition at secondary sites on the B-cell surface. In the present study, we have examined C3 fragment deposition in vitro in more detail by (1) assessing the importance of locally generated C3b for the deposition process, (2) investigating whether CR2 is the sole requirement for conferring AP activation capacity on a cell, and (3) determining whether CR2's function, as an AP activator, has different structural requirements from ligand binding. Increasing the availability of native C3, by increasing the serum (NHS) concentration, resulted in enhanced C3 fragment deposition on the B cells, whereas use of factor 1-depleted NHS, which showed massive fluid phase C3 conversion during the incubation, diminished the deposition. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting of untreated and hydroxylamine-treated lysates from B cells, after in vitro activation, revealed that the majority of C3 fragments (primarily iC3b and C3dg) had been covalently bound to the cell surface. Transfection of COS cells with wild-type CR2 or a deletion mutant lacking 11 of the molecule's 15 homologous domains, but retaining the ligand-binding site, revealed that expression of intact CR2 conferred a 12-fold increase in AP-activating capacity on these cells, while no increase in AP activity was apparent on cells transfected with the mutant CR2.