Fas receptor clustering and involvement of the death receptor pathway in rituximab-mediated apoptosis with concomitant sensitization of lymphoma B cells to fas-induced apoptosis

J Immunol. 2007 Feb 15;178(4):2287-95. doi: 10.4049/jimmunol.178.4.2287.


Ab binding to CD20 has been shown to induce apoptosis in B cells. In this study, we demonstrate that rituximab sensitizes lymphoma B cells to Fas-induced apoptosis in a caspase-8-dependent manner. To elucidate the mechanism by which Rituximab affects Fas-mediated cell death, we investigated rituximab-induced signaling and apoptosis pathways. Rituximab-induced apoptosis involved the death receptor pathway and proceeded in a caspase-8-dependent manner. Ectopic overexpression of FLIP (the physiological inhibitor of the death receptor pathway) or application of zIETD-fmk (specific inhibitor of caspase-8, the initiator-caspase of the death receptor pathway) both specifically reduced rituximab-induced apoptosis in Ramos B cells. Blocking the death receptor ligands Fas ligand or TRAIL, using neutralizing Abs, did not inhibit apoptosis, implying that a direct death receptor/ligand interaction is not involved in CD20-mediated cell death. Instead, we hypothesized that rituximab-induced apoptosis involves membrane clustering of Fas molecules that leads to formation of the death-inducing signaling complex (DISC) and downstream activation of the death receptor pathway. Indeed, Fas coimmune precipitation experiments showed that, upon CD20-cross-linking, Fas-associated death domain protein (FADD) and caspase-8 were recruited into the DISC. Additionally, rituximab induced CD20 and Fas translocation to raft-like domains on the cell surface. Further analysis revealed that, upon stimulation with rituximab, Fas, caspase-8, and FADD were found in sucrose-gradient raft fractions together with CD20. In conclusion, in this study, we present evidence for the involvement of the death receptor pathway in rituximab-induced apoptosis of Ramos B cells with concomitant sensitization of these cells to Fas-mediated apoptosis via Fas multimerization and recruitment of caspase-8 and FADD to the DISC.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Antibodies, Monoclonal / pharmacology*
  • Antibodies, Monoclonal / therapeutic use
  • Antibodies, Monoclonal, Murine-Derived
  • Antigens, CD20 / immunology
  • Antineoplastic Agents / pharmacology*
  • Antineoplastic Agents / therapeutic use
  • Apoptosis / drug effects*
  • Apoptosis / immunology
  • Burkitt Lymphoma / drug therapy
  • Burkitt Lymphoma / immunology*
  • CASP8 and FADD-Like Apoptosis Regulating Protein / genetics
  • CASP8 and FADD-Like Apoptosis Regulating Protein / immunology
  • Caspase 8 / immunology
  • Cell Line, Tumor
  • Fas Ligand Protein / immunology*
  • Fas-Associated Death Domain Protein / immunology
  • Humans
  • Membrane Microdomains / immunology
  • Protein Transport / drug effects
  • Protein Transport / immunology
  • Receptor Aggregation / drug effects*
  • Receptor Aggregation / immunology
  • Rituximab
  • fas Receptor / immunology*


  • Antibodies, Monoclonal
  • Antibodies, Monoclonal, Murine-Derived
  • Antigens, CD20
  • Antineoplastic Agents
  • CASP8 and FADD-Like Apoptosis Regulating Protein
  • FADD protein, human
  • Fas Ligand Protein
  • Fas-Associated Death Domain Protein
  • fas Receptor
  • Rituximab
  • CASP8 protein, human
  • Caspase 8