CR2-mediated activation of the complement alternative pathway results in formation of membrane attack complexes on human B lymphocytes

Abstract

Normal human B lymphocytes activate the alternative pathway of complement via complement receptor type 2 (CR2, CD21), that binds hydrolysed C3 (iC3) and thereby promotes the formation of a membrane-bound C3 convertase. We have investigated whether this might lead to the generation of a C5 convertase and consequent formation of membrane attack complexes (MAC). Deposition of C3 fragments and MAC was assessed on human peripheral B lymphocytes in the presence of 30% autologous serum containing 4.4 mM MgCl2/20 mM EGTA, which abrogates the classical pathway of complement without affecting the alternative pathway. Blockade of the CR2 ligand-binding site with the monoclonal antibody FE8 resulted in 56 +/- 13% and 71 +/- 9% inhibition of the C3-fragment and MAC deposition, respectively, whereas the monoclonal antibody HB135, directed against an irrelevant CR2 epitope, had no effect. Blockade of the CR1 binding site with the monoclonal antibody 3D9 also resulted in a minor reduction in MAC deposition, while FE8 and 3D9, in combination, markedly reduced deposition of both C3 fragments (91 +/- 5%) and C9 (95 +/- 3%). The kinetics of C3-fragment and MAC deposition, as well as the dependence of both processes on CR2, indicate that MAC formation is a consequence of alternative pathway activation.

Figure 1

Deposition of C3 fragments and C9 on peripheral B cells. Histograms displaying the fluorescence intensity of B cells stained with FITC-conjugated polyclonal anti-human C3d (upper panel) and the anti-C9 mAb, E11, following incubation for 90 min in normal human serum (NHS)/Mg/EGTA (dark grey peak), NHS/EDTA (black peak) or RPMI-1640 (light grey peak). The white peak depicts non-B lymphocytes in the PBMC preparation incubated in NHS/Mg/EGTA, which was used in all subsequent experiments as the background value.

Figure 2

The kinetics of AP-mediated C3-fragment deposition and C9 incorporation. The mean fluorescence intensity (MFI) of B cells stained with polyclonal anti-C3d (♦) or mAb E11 (▪) following incubation for various times with normal human serum (NHS)/Mg/EGTA (solid lines) or NHS/EDTA (broken lines). The data shown are for a representative experiment from the four performed.

Figure 3

Influence of CR2 and CR1 blockade on C3-fragment deposition and MAC formation on B cells. The percentage of C3d-specific (open columns) and C9-specific (solid columns) fluorescence, respectively, relative to incubation in normal human serum (NHS)/Mg/EGTA, in the absence of blocking antibodies, is given. B cells were incubated under the given conditions in the presence of the CR2-blocking mAb FE8, the CR1-blocking mAb 3D9 or the control mAbs HB135, for CR2 and HB8592, for CR1. The error bars display the 95% confidence intervals for the values obtained with sera from six donors.