Synergistic interaction between complement receptor type 2 and membrane IgM on B lymphocytes

Abstract

We sought biochemical evidence for a role of C receptors types 1 (CR1) and 2 (CR2) in B cell activation. A flow cytometer was used to measure the fluorescence of tonsillar cells that had been loaded with the calcium-dependent indicator indo-1, and cells were stimulated by cross-linking cell-bound DA4.4 anti-IgM, Yz-1 anti-CR1 or HB5 anti-CR2 with goat anti-mouse IgG. There was a direct dose-response relationship between the proportion of cells having increased cytoplasmic free calcium concentration (Cai) after addition of second antibody and the amount of cell-bound Fab' DA4.4. In contrast, no rise in Cai was observed after cross-linking bound Yz-1 or HB5. To determine whether CR1 or CR2 could modify the increase in Cai induced by cross-linking membrane IgM, Cai was monitored after addition of second antibody to cells bearing combinations of either Yz-1 or HB5 with a limited amount of DA4.4. The combination of Yz-1 with DA4.4 yielded little or no further increase in the percentage of cells responding to cross-linking with elevated Cai compared with DA4.4 alone. However, the combination of HB5 with limited DA4.4 synergistically enhanced this response, resulting in stimulation that was equivalent to that obtained with optimal concentrations of DA4.4. The synergistic effect of CR2 was also observed with avidin as the cross-linking reagent for bound biotinylated HB5 and DA4.4, occurred in the presence of EGTA, and did not require T cells. Studies of the proliferation of B cell-enriched PBMC demonstrated that, whereas HB5 coupled to Sepharose alone induced little or no DNA synthesis, the combination of HB5 with limited DA4.4 on Sepharose induced a dose-related synergistic increase in the incorporation of [3H]thymidine.