Rituxan (anti-CD20 antibody)-induced translocation of CD20 into lipid rafts is crucial for calcium influx and apoptosis

Abstract

SummaryRituxan, a chimeric anti-CD20 antibody, is the first antibody approved for immunotherapy in non-Hodgkin's B-cell lymphoma and other B-cell lymphoproliferative disorders. Additionally, efficacy of Rituxan treatment has been reported in nonmalignant autoimmune diseases such as rheumatoid arthritis. Crosslinking of CD20 molecules by Rituxan induces therapeutic Bcell depletion. CD20 is a B-lymphocyte specific integral membrane protein, proposed to function as a store-operated calcium channel, which is activated upon receptor-stimulated calcium depletion of intracellular stores. Crosslinking of CD20 by antibodies has been reported to induce a redistribution of CD20 molecules to specialized microdomains at the plasma membrane known as lipid rafts. Here, we report that in the absence of Rituxan, CD20 exhibits a low affinity to lipid rafts. However, binding of Rituxan significantly increases the affinity of CD20 for lipid rafts resulting in its redistribution to a fraction resistant to Triton X-100 solubilization. Furthermore, we demonstrate that disturbing the raft integrity by cholesterol extraction results in dissociation of CD20 from a Triton X-100 resistant fraction followed by complete inhibition of Rituxan-induced calcium entry and apoptosis. The integrity of lipid rafts seems to play a crucial role for CD20-induced caspase activation. These data show, for the first time, that Rituxan-induced translocation of CD20 to lipid rafts is important for increased intracellular Ca 2 + + + + levels and downstream apoptotic signalling.

Imaging and measuring the rituximab-induced changes of mechanical properties in B-lymphoma cells using atomic force microscopy

“…Unruh et al [37] have demonstrated that the translocation and hypercrosslinking of CD20 are necessary for rituximab to activate src family kinases (SFK)-dependent signaling and apoptosis. Janas et al [38] have shown that rituximab-induced translocation of CD20 to lipid rafts is crucial for increased intracellular Ca 2+ levels and downstream apoptotic signaling. Also the rituximab binding to CD20 significantly interferes with B-cell receptor (BCR) signaling, resulting in a time-dependent inhibition of the BCR-signaling cascade involving Lyn, Syk, PLCc2, Akt, and ERK, and calcium mobilization [39].…”

Recombinant Anti-CD4 Antibody 13B8.2 Blocks Membrane-Proximal Events by Excluding the Zap70 Molecule and Downstream Targets SLP-76, PLCγ1, and Vav-1 from the CD4-Segregated Brij 98 Detergent-Resistant Raft Domains

“…Residual CD4, LAT, and p56 Lck molecules were still observed upon OKT4 binding/hypercross-linking in the nonraft fraction (33), whereas rIgG 1 13B8.2 treatment, without hyper-cross-linking, completely localized CD4 into Brij 98 DRM together with LAT and p56 Lck proteins. These results raise several questions concerning the influence of the detergent (65), the temperature of extraction, and hyper-cross-linking requirement (5), not yet resolved, but of critical importance to ascertaining the raft "physiology" (3). In our case, the fact that no hyper-cross-linking was necessary for rIgG 1 13B8.2-induced raft partitioning correlates with the fact that the monovalent baculovirus-expressed rFab 13B8.2 is sufficient to induce CD4 accumulation/retention in Brij 98 DRM.…”

“…Numerous unusual immunomodulatory or anti-HIV effects have already been ascribed for ligands directed against this CDR3-like region in the D1 domain of CD4 (63). Epitope specificity governing raft localization and further biological effects have mainly been defined for anti-CD20 reagents (5,6,16). It would be interesting to compare the epitope specificity of anti-CD4 Abs YNB.46 (30), KT6, YTS177, and YTA3.1 (31,32), all of which are known to induce inhibition of Zap70 phosphorylation, with the CDR3-like epitope specificity (residues Glu 87 and Asp 88 in the D1 domain of CD4) of Ab 13B8.2 (64), which reorganizes Zap70 outside the rafts and modulates its phosphorylation profile.…”

“…On the basis of these observations, one emerging hypothesis is that the biological effects of new biotechnological drugs such as therapeutic Abs are linked to their ability to quantitatively and qualitatively modulate the clustering of target membrane receptors, signaling kinases, apoptosis-, proliferation-, and cytotoxicity-involved molecules, and lipids into these raft domains. Concerning the therapeutic Ab anti-CD20 rituximab, but also other anti-CD20 Abs, there is a correlation between the ability of these Abs to elicit CD20 translocation into Triton DRM (4) and their capacity to initiate kinase-dependent calcium mobilization, apoptosis induction (5), and complementdependent cytolysis (6) of B lymphoma cells. Rituximab also induces ceramide accumulation together with CD20 translocation into Triton DRM, leading to activation of the ceramide-triggered signaling pathway, which mediates proliferation inhibition of B lymphoma cells (7).…”

Rituximab therapy in malignant lymphoma

“…CD20 binding by rituximab is followed by homotypic aggregation, rapid translocation of CD20 into specialized plasma membrane microdomains known as rafts and induction of apoptosis. Membrane rafts concentrate src family kinases and other signaling molecules (phospholipases, caspases y), and the anti-CD20-induced apoptotic signals are though to occur as a consequence of CD20 accumulation in rafts (Janas et al, 2005). The role of complement-dependent cytotoxicity (CDC) is suggested by the consumption of complement observed after rituximab administration but in vitro CDC does not correlate with clinical response in lymphomas (Winkler et al, 1999;Weng and Levy, 2001).…”