TY - JOUR
T1 - Spontaneous complement activation on human B cells results in localized membrane depolarization and the clustering of complement receptor type 2 and C3 fragments
AU - Løbner, Morten
AU - Leslie, Robert G Q
AU - Prodinger, Wolfgang M
AU - Nielsen, Claus H
N1 - Keywords: Annexin A5; Antibodies, Monoclonal; Apoptosis; B-Lymphocytes; Complement Activation; Complement C3; Complement Factor D; Complement Membrane Attack Complex; Complement Pathway, Alternative; Edetic Acid; Humans; Membrane Microdomains; Phosphatidylserines; Receptors, Complement; Receptors, Complement 3d
PY - 2009
Y1 - 2009
N2 - While our previous studies have demonstrated that complement activation induced by complement receptors type 2 (CR2/CD21) and 1 (CR1/CD35) results in C3-fragment deposition and membrane attack complex (MAC) formation in human B cells, the consequences of these events for B-cell functions remain unknown. In the present study, we show that CR2-induced complement activation results in membrane depolarization, as indicated by annexin V binding, with kinetics similar to those of C3-fragment deposition and different from those of MAC formation. On the other hand, like MAC formation, depolarization requires activation of complement via the alternative pathway, as indicated by total inhibition upon neutralization of factor D, and is abrogated by combined blockade of CR1 and CR2, but not of either receptor alone. The membrane depolarization is not associated with the apoptosis of B cells, as examined by co-staining with APO-2.7 or by the TdT-mediated biotin-dUTP nick-end labelling (TUNEL) assay. Confocal microscopy revealed that depolarization and C3 deposition, unlike MAC deposition, are limited to restricted areas on the B-cell surface. Double staining revealed a close association between the C3-fragment patches and membrane depolarization, as well as redistribution of lipid rafts to these areas. We propose that these events may play a role in the regulation of B-cell signalling and cross-talk with T cells.
AB - While our previous studies have demonstrated that complement activation induced by complement receptors type 2 (CR2/CD21) and 1 (CR1/CD35) results in C3-fragment deposition and membrane attack complex (MAC) formation in human B cells, the consequences of these events for B-cell functions remain unknown. In the present study, we show that CR2-induced complement activation results in membrane depolarization, as indicated by annexin V binding, with kinetics similar to those of C3-fragment deposition and different from those of MAC formation. On the other hand, like MAC formation, depolarization requires activation of complement via the alternative pathway, as indicated by total inhibition upon neutralization of factor D, and is abrogated by combined blockade of CR1 and CR2, but not of either receptor alone. The membrane depolarization is not associated with the apoptosis of B cells, as examined by co-staining with APO-2.7 or by the TdT-mediated biotin-dUTP nick-end labelling (TUNEL) assay. Confocal microscopy revealed that depolarization and C3 deposition, unlike MAC deposition, are limited to restricted areas on the B-cell surface. Double staining revealed a close association between the C3-fragment patches and membrane depolarization, as well as redistribution of lipid rafts to these areas. We propose that these events may play a role in the regulation of B-cell signalling and cross-talk with T cells.
U2 - 10.1111/j.1365-2567.2009.03056.x
DO - 10.1111/j.1365-2567.2009.03056.x
M3 - Journal article
C2 - 19740327
SN - 0953-4954
VL - 128
SP - e661-9
JO - Immunology. Supplement
JF - Immunology. Supplement
IS - 1 Suppl
ER -