Induction of Cytosolic Calcium Flux by CD20 Is Dependent upon B Cell Antigen Receptor Signaling

Claire A Walshe,Stephen A Beers,Ruth R French,Claude H.T Chan,Peter W Johnson,Graham K Packham,Martin J Glennie,Mark S Cragg

doi:10.1074/jbc.m708459200

Claire A Walshe, Stephen A Beers + Show 6 more

Open Access

https://doi.org/10.1074/jbc.m708459200

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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.

Highlights

Three main effector mechanisms are available to monoclonal antibody (mAb) to evoke tumor regression: Complement- and antibody-dependent cellular cytotoxicity (CDC and ADCC, respectively) and direct cytotoxic signaling (Refs. 5, 9 –14 and reviewed in Ref. 15)
Type I but Not Type II Anti-CD20 mAbs Induce Calcium Flux— In our first experiments, we assessed the ability of a range of Type I and II anti-CD20 mAbs to evoke a calcium flux in the follicular lymphoma cell line SU-DHL-4, using the anti-B-cell antigen receptor (BCR) mAb SB2H2 as a positive control
Associated after Type I anti-CD20 mAb ligation, and that CD20 and the BCR evoke the same signaling cascade leading to calcium flux through a biphasic response, which can be inhibited by Fc␥RIIb, we wanted to demonstrate formally that Type I-induced calcium flux was dependent upon BCR expression

Summary

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The ability of CD20 to regulate calcium appears to be linked to the generation of calcium flux transduced by ligation of the BCR [27]. It is possible that CD20 facilitates this capacitance calcium flux via an indirect pathway, there is strong evidence to indicate that CD20 itself forms a component of the SOC channel (reviewed in Ref. 15) Together these data indicate that CD20 acts as a SOC channel involved in BCR signal transduction. Two groups working on this problem have arrived at contrasting conclusions Both demonstrate a requirement of Src tyrosine kinases (STK) and raft redistribution in the calcium flux induced after ligation of CD20, one proposes that the calcium flux results from the opening of CD20 as a plasma membrane calcium channel [28], while the other suggests that the calcium flux operates by release of calcium from intracellular stores [21]. These data indicate that CD20 ligation induces a calcium flux through its association with the BCR, rather than through any intrinsic calcium channel capability

EXPERIMENTAL PROCEDURES

RESULTS

DISCUSSION