Abstract P812: Characterizing NMDA Receptor Subunits on B Cells

Abstract

Background: N-methyl-D-aspartate (NMDARs) play a critical role in neuronal excitotoxicity after stroke. The actions of NMDARs have been shown mostly in obligatory GluN1 subunits on neurons and not GluN2A/B subunits. In B cells, these subunits have not been highly characterized though the presence of NMDARs has been shown. The function of the GluN2A/B subunits can be neuroprotective or pro-death in neurons, respectively. We hypothesized that GluN2A and GluN2B subunit presence on B cells would be affected by exposure to extracellular glutamate. Methods: Splenic B cells were isolated from 3-4mo-old C57BL/6 male mice via magnetic separation and treated with physiologic levels of L-glutamate (glu; 1uM) in the presence or absence of 5ug/mL LPS. B cell cytospins were stained for B220, GluN2A, and GluN2B, imaged using confocal microscopy, and quantified in FIJI. An average of 10.7 B cells were quantified per image at 80-157x magnification. RGB channels of the z-stacks were quantified to identify positive B220 expression. The z-stacks were split into 2D images and quantified plane-by-plane to identify GluN2A/B subunit clusters. Each cluster of subunits was recorded per cell in view across all planes of the original z-stack to yield total subunit count. Groups included 14-43 B cells quantified, and the number of subunits per cell were analyzed via ordinary two-way ANOVA, Sidak post-hoc test (Graphpad Prism). Significance was p<0.05. Results: There was an average of 19.3±7.2 GluN2A subunits and 19.0±5.0 GluN2B subunits per cell for unstimulated, untreated B cells. Neither glu treatment (p=0.23) nor LPS stimulation (p= 0.10) impacted the number of GluN2A subunits per B cell. LPS decreased GluN2B subunits when compared to unstimulated B cells (11.1±5.1 subunits; p=0.02). Glu treatment normalized GluN2B subunits per B cell near untreated baseline levels (18.2±11 subunits per cell; p=0.01), resulting in an interaction between LPS stimulation and glu treatment in B cells (F (1, 86) =6.180, P=0.015). Conclusions: Our data suggests activated B cells downregulate GluN2B-containing NMDARs following LPS stimulation. This downregulation mimics that of NMDAR activity on neurons upon excitoxicity (PMID: 24361499) but future studies should confirm GluN2B internalization.