The B cell antigen receptor (BCR) serves dual, interrelated functions in B cell activation. The first is to initiate signal cascades that result in the transcription of a variety of genes associated with B cell activation. The second is to traffic bound antigen into the cell to deliver the antigen into intracellular compartments where the antigen is proteolytically cleaved and the resulting peptides are assembled into MHC class II-peptide complexes. The MHC class II-peptide complexes are expressed on the B cell surface for recognition by helper T cells that will as a consequence be activated to provide essential growth and differentiation factors to the B cell. The signaling and antigen transport functions of the BCR are interdependent in that the BCR signaling is necessary for the correct and rapid intracellular targeting of the antigen. Our current evidence indicates that the BCR enters the class II-containing compartment as an intact, signaling competent, receptor. However, at present it is an open question as to whether the BCR signals intracellularly and if so what role intracellular signaling might play in B cell activation. The long term goal of this project is to gain a better understanding of the molecular mechanisms that underlie the correct targeting of the BCR to the class II-containing compartments including determining where in the pathway discrete signaling events occur and to further define the role of intracellular signaling on B cell activation with particular focus on the synergistic interaction of the BCR with the intracellular Toll like receptors (TLRs).? ? Studies to delineate the components of the BCR signaling cascade necessary for correct targeting of the BCR provided evidence that the BCR-induced activation of phospholipase D (PLD) is necessary for the correct targeting of the BCR into MHC class II-containing intracellular compartments. PLD is a signal-transduction-activated enzyme that hydrolyzes phosphatidylcholine to generate the second messenger phosphatic acid and choline. PLD has been implicated in controlling a wide variety of cellular processes including the endocytosis and vesicular trafficking of cellular receptors. We showed that the activity of PLD was not required for endocytosis of the BCR from the plasma membrane but was required for subsequent steps in the BCRs trafficking pathway. ? ? We have mapped the cellular location where components of the BCR signaling pathway are activated by confocal microscopy using antibodies specific for the phosphorylated forms of signaling components including Syk, p38, ERK and JNK. Our results indicated that components of the BCR signaling pathway are activated sequentially and in defined subcellular locations. We observed that pSyk appeared on the plasma membrane immediately following BCR crosslinking, whereas the phosphorylated forms of p38, ERK and JNK were not detected until the BCR had internalized from the plasma membrane and trafficked to the class II-containing compartments. We showed that blocking BCR internalization by dansylcadaverine blocked the phosphorylation of both ERK and JNK. The internalized BCR colocalized in LAMP-positive compartments with pERK and pJNK. ? ? We also investigated the function and regulation of the intracellular signaling of the BCR. BCR signaling is regulated and fine tuned by an array of B cell coreceptors many of which are present on the cell surface where they presumably interact with the BCR. However, members of one family of receptors, the TLRs, that have been shown to influence BCR signaling are located in intracellular endosomes and not on the plasma membrane. One endosomal TLR, TLR9, that recognizes unmethylated CpG-DNA motifs has been shown to enhance BCR signaling resulting in hyperactivation of B cells to proliferate and differentiate into antibody secreting plasma cells. Recent evidence indicates that synergistic signaling between the BCR and TLR9 underlies the production of self-reactive antibodies in systemic autoimmune diseases and partly explains the preponderance of auto-antibodies that react with DNA-containing antigens. ? ? Both the TLR9- and BCR-signaling pathways ultimately result in activation of the p38, JNK and NFKB. Although the BCR and TLR9 initiate signaling from two spatially distinct sites, the BCR from the plasma membrane, and TLR9 from endosomes, our observation that the BCR-induced activation of p38 and JNK occurs inside the cell in large intracellular compartments suggested that such compartments may be sites of synergistic signaling between the BCR and TLR9 in response to DNA-containing antigens. Using confocal microscopy and antibodies specific for the BCR, TLR9, p38 and pJNK we showed that following the binding and internalization of DNA-containing antigens, the BCR signals for the recruitment of TLR9 from multiple small endosomes to a large LAMP1-positive compartments into which the BCR trafficks antigen and where synergistic signaling through to p38 and JNK occurs. The recruitment of TLR9 to the BCR was by a dynein-mediated, microtubule-network dependent process and was necessary for B cell hyper-responses to DNA-containing antigens. Of considerable interest was the observation that recruitment of TLR9 was dependent on the BCRs activation of PLD. Thus, PLD plays a role in both the correct targeting of the BCR to intracellular compartments and the recruitment of TLR9 to the same compartments. The BCRs PLD-activation dependent recruitment of the TLR9 to the MVB represents a novel mechanism underlying the hyper-response of B cells to DNA-containing antigens. Recent results indicate that two additional intracellular TLRs, TLR3 and TLR7 are similarly recruited by the BCR from endosomes to the large, signaling active compartment. Thus, this recruitment mechanism appears to have been conserved to allow the synergistic interaction of the intracellular TLRs with the internalized BCR.? ? We have used a combination of electron and confocal microscopy to better characterize the compartment into which the BCR trafficks and to which the BCR recruits TLRs. By electron microscopy the compartment has the double membranes and multiple vesicular bodies and characteristics of autophagosomes. By confocal microscopy the compartment expressed the autophagosome marker LC3 and in B cells from LC3-difficient mice the BCR failed to recruit TLRs. Collectively these results indicate that the autophagosome is the site of BCR-TLR synergistic signaling. The autophagosomes are of interest as they have been recently shown to play a role in MHC-class II antigen processing by facilitating the transport of antigens in the cytoplasm to the MHC class II molecules.
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