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.

Hodgkin's lymphoma (HL) is among the most frequent nodal lymphomas in the Western world and is classified into two disease entities: nodular lymphocyte-predominant Hodgkin's lymphoma (NLPHL) and classical Hodgkin's lymphoma (cHL, 95% of all HL). HL lesions are characterised by a minority of clonal neoplastic cells, namely Hodgkin and Reed-Sternberg (HRS) cells and their variants in cHL and lymphocyte-predominant (LP) cells in NLPHL, both occurring within a microenvironment of, for example, reactive T and B cells, macrophages and granulocytes that are assumed to support the proliferation and maintenance of neoplastic cells through cytokines, chemokines and growth factors. Insulin-like growth factor I (IGF-I) is an important growth factor involved in proliferation, differentiation, apoptosis and cell survival of numerous (including immune) tissues and probably has a role in tumour pathogenesis and maintenance. Although HL is characterised by disturbed cell differentiation and apoptosis mechanisms, with the involvement of the IGF-I receptor (IGF-1R), the distinct location of IGF-I in HL has not yet been defined. We localise IGF-I by double-immunofluorescence in frequent neoplastic cells of all cHL and NLPHL cases investigated. Additionally, IGF-I immunoreactivity is detected in high endothelial venules and various immune cells within the surrounding tissue of cHL including neutrophils and macrophages. IGF-1R immunoreactivity of variable intensity is found in HRS cells and high endothelial venules within the microenvironment in cHL. We assume that autocrine and paracrine IGF-I plays an anti-apoptotic role in tumour pathogenesis and in shaping the tumour microenvironment.

CD20 is a B-lymphocyte-specific integral membrane protein, implicated in the regulation of transmembrane calcium conductance, cell-cycle progression and B-lymphocyte proliferation. CD20 is proposed to function as a SOCC (store-operated calcium channel). SOCCs are activated by receptor-stimulated calcium depletion of intracellular stores. Sustained calcium conductivity across the plasma membrane mediated by SOCC activity is required for long-term calcium-dependent processes, such as transcriptional control and gene expression. Cross-linking of CD20 by antibodies (e.g. Rituxan) has been reported to induce a rapid redistribution of CD20 into specialized microdomains at the plasma membrane, known as lipid rafts. Recruitment of CD20 into lipid rafts and its homo-oligomerization are suggested to be crucial for CD20 activity and regulation. This review outlines recent biochemical studies characterizing the role of CD20 in calcium signalling in B-lymphocytes and evaluates an engagement of lipid rafts in the regulation of CD20-mediated calcium conductivity.