The Rituximab and IVIG Related IgM Flare In Waldenstrom's Macroglobulinemia Is Associated with Monocytic Activation of Fcgriia Signaling, and Triggering of IL-6 Release by the PI3K/AKT and MAPK Pathways
Authors: Yang Guang , Xu Lian , Hunter Zachary , Liu Xia , Cao Yang , Jiang Jingrui , Tseng Hsiuyi , Zhou Yangsheng , Ioakimidis Leukothea , Hanzis Christina , Sheehy Patricia , Patterson Christopher , Treon Steven P. 
Published: October 2019

Abstract
Abstract 2870

Background:
We and others have previously reported a paradoxical flare in serum IgM levels following rituximab and/or IVIG administration which can occur within hours of its administration, and can affect 40–70% of WM patients. The paradoxical flare can often lead to symptomatic hyperviscosity, and/or aggravation of other IgM mediated morbidities. Direct stimulation of WM cells by rituximab or IVIG does not lead to further IgM release, and IL-6 levels rise in those patients experiencing a rituximab induced IgM flare (Yang et al, ASH 2009). We therefore sought to clarify the molecular mechanisms permitting the rituximab or IVIG mediated IgM flare in WM.

Patients and Method:
We performed co-culture studies of WM cells utilizing a transwell system with granulocytes or monocytes in the presence or absence of rituximab or IVIG. Granulocyte and monocyte cell lysates and supernatants were collected after rituximab stimulation for further analysis. IL-6 levels were assessed by multiplex assays and real-time RT-PCR. siRNAs targeting FcgRIA, FcgRIB, FcgRIIA, FcgRIIB, and FcgRIIIA were used to knockdown Fcγ receptors on primary monocytes. Fcγ receptor expression following siRNA transfection was confirmed by real-time RT-PCR and flow cytometry. Proteins from Rituximab stimulated monocytes were analyzed with phospho-antibody arrays and confirmed with western blotting.

Results:
A significant increase in IgM release was observed following co-culture of primary WM cells with monocytes and co-incubation with either rituximab or IVIG; was associated with IL-6 release, and could be blocked by anti-IL6 antibodies. The induction of IL-6 following rituximab stimulation of monocytes was significantly reduced following siRNA knockdown of FcgRIIA. Phospho-antibody array and ingenuity pathway analyses revealed that both PI3K/AKT and MAPK pathways were involved in signaling initiated by rituximab stimulation of monocytes through FcgRIIA. Increased phosphorylation of PI3K, AKT, p38MAPK and ERK1/2 was confirmed by western blotting with phospho-specific antibodies. In addition, inhibitors specific for PI3K, AKT, p38MAPK and ERK1/2 also reduced IL-6 production.

Conclusion:
Taken together, the above data suggest that the rituximab and/or IVIG related IgM flare observed in WM patients is triggered by IL-6 in response to stimulation of bystander monocytes through FcgRIIA binding and activation of the PI3K/AKT and MAPK pathways. Inhibitors aimed at these pathways block IL-6 release from rituximab stimulated monocytes. The results provided a framework for investigation of pre-emptive therapies for blocking the IgM flare in WM patients being considered for rituximab and/or IVIG therapy.

Disclosures:
No relevant conflicts of interest to declare.