The IgM Flare Following Rituximab and IVIG Administration in Waldenstrom's Macroglobulinemia Is Related to IL-6 Production by Bystander Immune Cells, Possibly through Stimulation of the Fcgriia Receptor.

Rituximab is an important treatment option in Waldenstrom's Macroglobulinemia (WM). We and others have previously reported a paradoxical flare in serum IgM levels following rituximab administration which can occur within hours of its administration, and can affect 40-50% of WM patients leading often to symptomatic hyperviscosity, and or aggravation of other IgM mediated morbidities. We have observed a similar flare phenomenon in 3 WM patients receiving IVIG therapy, suggesting that the IgM flare may result from Fcg receptor signaling. We therefore sought to clarify the mechanism for the IgM flare, in hopes that knowledge of its mechanistic attributes might facilitate development of pre-emptive treatments.

CD19+ sorted BM lymphoplasmacytic cells (LPC) from WM patients were incubated in the presence or absence of rituximab or IVIG for 24-48 hours, alone and in the presence of co-cultured monocytes. IgM levels in supernatant were then determined at 24-48 hours by ELISA. Sera and genomic DNA from 68 WM patients treated with rituximab, of whom 27 (40%) experienced an IgM flare, were also evaluated. Serum IgM and cytokine levels were determined for these patients before and following rituximab. Polymorphisms in FcgRIIA-27, -131, FcgRIIB-187, FcgRIIIA-48, -158, IL-6 Promoter -174, -6331, IL-6R-358, and IL6ST(gp130)-148 were also determined for all 68 WM patients, including both patients who did and did not demonstrate an IgM flare.

We first observed that the direct incubation of WM LPC with either rituximab or IVIG did not induce significant IgM release. We therefore sought to delineate if other bystander immune cells contributed to the IgM flare phenomenon either directly or by cytokine release. We observed that rituximab and IVIG could stimulate both healthy donor and WM patient monocytes to produce IL-6, which was assessed by both RT-PCR and ELISA. IL-6 transcription was increased as early as 3 hours, and was associated with an increase in IL-6 secretion. Since monocytes express various Fcg receptors, we next sought to clarify which receptor was potentially responsible for the induction of IL-6 release. We therefore stimulated monocytes with activating F(ab')₂ fragments directed at FcgRI, FcgRIIA, FcgRIIB, and FcgRIIIA. Only treatment with an FcgRIIA F(ab')₂ activating fragment resulted in robust IL-6 transcription and secretion by monocytes. Since IL-6 plays an important role in stimulating IgM secretion by WM cells, we next examined IL-6 levels in patients who received rituximab and compared their levels over the course of therapy in patients with and without an IgM flare. A spike in IL-6 levels was observed in those patients who experienced an IgM flare, and correlated with the time course of the IgM flare. Given these findings, we co-cultured primary BM WM cells as well as BCWM.1 cells in the presence or absence of monocytes using a Tran swell co-culture system, and observed that overnight incubation with rituximab or IVIG resulted in increased IgM production. This effect could be blocked by simultaneous incubation with an IL-6 blocking antibody. Similar results were obtained by culturing WM LPC with supernatants obtained from rituximab or IVIG stimulated monocytes incubated with and without an IL-6 blocking antibody. Lastly, the frequency of polymorphisms for all 68 WM patients for FcgRIIA-27, -131, FcgRIIB-187, FcgRIIIA-48, -158, IL-6 Promoter -174, -6331, IL-6R-358, and IL6ST(gp130)-148 did not defer from published data bases, and were similar between rituximab treated patients who either experienced or not the IgM flare.

Taken together, the above data suggest that the IgM flare observed in WM patients following rituximab and IVIG administration is likely triggered by IL-6 in response to stimulation of bystander immune cells including monocytes, possibly through FcgRIIA binding. Efforts aimed at blocking IL-6 release should be explored as a pre-emptive means of preventing the IgM flare in WM patients being considered for rituximab and IVIG therapy.

No relevant conflicts of interest to declare.