CCR2 KO mice exhibit up-regulated BCR proximal signaling, including positive and negative signals
Surface BCRs stimulated by antigens initiate B cell signaling transduction, and the intensity of this signaling was enhanced by CD19. CD19 recruits Bruton’s tyrosine kinase (Btk) by activating PI3K, which is critical for amplifying downstream signaling. To explore the molecular mechanism through which CCR2 mediates the regulation on BCR signaling, we examined the aggregation of phosphorylated CD19 (pCD19) following stimulation with sAg for 5, 10, and 30 min. Compared to WT B cells, the colocalization between BCR and pCD19 in CCR2 KO B cells was significantly increased at 5 min and gradually decreased to the 30 min time-point when pCD19 was internalized along with BCR. Similar results were seen using western blotting to examine the pCD19 protein levels after 5 min of activation, with CCR2 KO B cells showing significantly higher pCD19 levels than those of WT B cells (Fig. 2A-D ). Notably, although the colocalization between pCD19 and BCR after 10 min of stimulation decreased in CCR2 KO B cells, the aggregation of pCD19 remained at higher levels than that observed in WT B cells. This indicates that CCR2 deficiency might affect the spatial organization of CD19 also play a potential role in B cell activation. Next, we evaluated the total levels of BCR signaling-protein phosphorylated tyrosine (pY); typical BCR related negative signal molecule-phosphorylated Src homology 2 (SH2) domain-containing inositol polyphosphate (pSHIP); and phosphorylated Btk (pBtk). Compared to WT B cells, the pY and pBtk levels were significantly increased from 5 to 10 min post-stimulation and then decreased after 30 min of stimulation (Fig. 2E-H ). This was consistent with the immunoblotting results (Fig. 2I ). Likewise, the colocalization between pY or pBtk and BCR was increased in CCR2 KO B cells (Fig. 2J-K ). Similarly, the pSHIP level in CCR2 KO B cells also increased after 5 and 10 min of activation (Fig. 2L-M ), and its colocalization with BCR peaked at 10 min, which was also confirmed by immunoblotting (Fig. 2N-O ).
Since the phospholipase C gamma 2 (PLCγ) induced-calcium (Ca2+) mobilization is responsible for the BCR signaling pathways,26and CCR2, as G protein-coupled receptor, can trigger a flux of intracellular Ca2+, we investigated the Ca2+mobilization using FCM. We discovered that the Ca2+mobilization was enhanced in CCR2 KO B cells upon sAg stimulation (Fig. 2P ). These findings suggest that the CCR2 deficiency leads to an up-regulation of BCR proximal signaling molecules.