title: Induction of cytosolic calcium flux by CD20 is dependent upon B Cell antigen receptor signaling.
abstract: The anti-CD20 monoclonal antibody (mAb) rituximab is now routinely used for the treatment of non-Hodgkins lymphoma and is being examined in a wide range of other B-cell disorders, such as rheumatoid arthritis. Despite intensive study, the mechanism of action still remains uncertain. In the current study, anti-CD20 mAb-induced calcium signaling was investigated. Previously, we grouped anti-CD20 mAbs into Type I (rituximab-like) and Type II (B1-like) based upon various characteristics such as their ability to induce complement activation and redistribute CD20 into detergent-insoluble membrane domains. Here we show that only Type I mAbs are capable of inducing a calcium flux in B cells and that this is tightly correlated with the expression of the B-cell antigen receptor (BCR). Inhibitor analysis revealed that the signaling cascade employed by CD20 was strikingly similar to that utilized by the BCR, with inhibitors of Syk, Src, and PI3K, but not EGTA, p38, or ERK1/2, completely ablating calcium flux. Furthermore, binding of Type I but not Type II mAbs caused direct association of CD20 with the BCR as measured by FRET and resulted in the phosphorylation of BCR-specific adaptor proteins BLNK and SLP-76. Crucially, variant Ramos cells lacking BCR expression but with unchanged CD20 expression were completely unable to induce calcium flux following ligation of CD20. Collectively, these data indicate that CD20 induces cytosolic calcium flux through its ability to associate with and "hijack" the signaling potential of the BCR.
authors.affiliation: Cancer Sciences Division, General Hospital, Southampton University School of Medicine, Southampton SO16 6YD, United Kingdom.
authors.forename: Claire A
authors.initials: CA
authors.lastname: Walshe
authors.forename: Stephen A
authors.initials: SA
authors.lastname: Beers
authors.forename: Ruth R
authors.initials: RR
authors.lastname: French
authors.forename: Claude H T
authors.initials: CH
authors.lastname: Chan
authors.forename: Peter W
authors.initials: PW
authors.lastname: Johnson
authors.forename: Graham K
authors.initials: GK
authors.lastname: Packham
authors.forename: Martin J
authors.initials: MJ
authors.lastname: Glennie
authors.forename: Mark S
authors.initials: MS
authors.lastname: Cragg
mesh_terms: D000911:Antibodies, Monoclonal / Q000737:chemistry; D018951:Antigens, CD20 / Q000378:metabolism*; D001402:B-Lymphocytes / Q000378:metabolism*; D002118:Calcium / Q000737:chemistry / Q000378:metabolism; D020013:Calcium Signaling; D002450:Cell Communication; D002469:Cell Separation; D002614:Chelating Agents / Q000494:pharmacology; D003600:Cytosol / Q000378:metabolism*; D006801:Humans; D008954:Models, Biological; D010766:Phosphorylation; D011947:Receptors, Antigen, B-Cell / Q000378:metabolism*; D015398:Signal Transduction; D048051:p38 Mitogen-Activated Protein Kinases / Q000378:metabolism
chemical_list: D000911:Antibodies, Monoclonal; D018951:Antigens, CD20; D002614:Chelating Agents; D011947:Receptors, Antigen, B-Cell; D048051:p38 Mitogen-Activated Protein Kinases; D002118:Calcium