The integrin leukocyte function-associated antigen-1 (LFA-1) is important in the promotion of B cell adhesion, thereby facilitating immunological synapse (IS) formation and B cell activation. Despite this significance, the associated signaling mechanisms regulating LFA-1 activation remain elusive. Here, we show that both isoforms of the small GTPase Rac expressed by primary B cells, Rac1 and Rac2, were activated rapidly downstream of Src-family kinases, guanine-nucleotide exchange factors Vav1 and Vav2, and phosphoinositide-3 kinase (PI3K) after BCR engagement. We identify Rac2, but not Rac1, as critical for B cell adhesion to intercellular adhesion molecule-1 (ICAM-1) and IS formation. Furthermore, B cells expressing constitutively active Rac2 are highly adhesive. We observe that Rac2-deficient B cells exhibit lower amounts of Rap1-GTP and severe actin polymerization defects, identifying a potential mechanism underlying their behavior. We postulate that this critical role for Rac2 in mediating B cell adhesion and IS formation might apply in all lymphocytes.
Journal of Cell Science 2280 activation (Lanzavecchia, 1985;Rock et al., 1984). Subsequently activated B cells can develop into plasma cells that are competent for large-scale production of soluble antibodies, or memory cells that provide long-lasting immunological memory.In this Commentary we highlight recent investigations of the mechanism underlying inside-out integrin activation in response to antigen stimulation through the BCR. Intriguingly, we observe a number of important differences in the mechanism that underlies inside-out activation according to the particular integrin activated and the cellular context of integrin expression. We discuss recent evidence that identifies key signalling molecules that govern the activity and distribution of LFA-1 and VLA-4 following engagement of the BCR. Finally, we propose a mechanism whereby these regulatory molecules mediate the cytoskeleton rearrangements that are required for the integrin activation and subsequent IS formation that occur during the antigen-recognition process. Antigen-induced BCR signallingThe BCR can be described as a receptor tyrosine kinase, and its stimulation by a specific antigen results in the proliferation and differentiation of B cells. The BCR comprises a membrane immunoglobulin (Ig) in complex with the heterodimeric Igα-Igβ sheath (Fig. 1B), which allows its stable expression in the membrane (Reth, 1989;Venkitaraman et al., 1991). The constituents of the BCR act in concert to couple the recognition of extracellular antigens with the initiation of intracellular signalling through the immunoreceptor tyrosine-based activation motifs (ITAMs) of the Igα-Igβ sheath (Hombach et al., 1990;Reth, 1989). These activated ITAMs subsequently act as recruitment sites for the assembly of a complex array of intracellular adaptors and signalling molecules in a structure known as the signalosome (DeFranco, 2001;Fruman et al., 2000). The precise composition of a signalosome is dictated by the nature and context of the antigenic stimulant, and determines the outcome of signalling through the BCR (Depoil et al., 2008;Weber et al., 2008). In the specific case of inside-out activation of integrins it would be expected that this outcome involves the direct targeting of the cytoplasmic domains of the integrin itself, in addition to the extensive reorganisation of the cytoskeleton that is necessary for the recognition of antigens that are presented on a spatially constrained surface.The mechanism of inside-out activation of integrins has been investigated extensively in T cells (reviewed in Kinashi, 2005). The Journal of Cell Science 121 (14) Fig. 1. Molecular mechanisms required for the activation of integrin-mediated B-cell adhesion in response to membrane-bound antigens. (A) Prior to antigen stimulation, the 'resting' B cell contains BCRs and inactive integrins that are distributed throughout the membrane. Following BCR engagement with antigen on the surface of an antigen-presenting cell (APC) that expresses integrin ligands, crosslinking of BCRs initiates int...
Human B-cell studies in vitro have routinely used B lymphocytes purified from spleen, blood or tonsils irrespective of potential differences in their immunological traits. In this study, we compared the functional responses of total (CD19(+)) and memory B cells (Bmem; CD19(+)/CD27(+)) isolated from blood and tonsils to different stimuli. Peripheral B cells showed enhanced survival and proliferation compared with their tonsillar equivalents when stimulated for 10 days. Stimulated B cells from both tissues secreted significantly greater amounts of cytokines than unstimulated controls demonstrating their functional responsiveness. Analysis of CD27 expression over time indicated that the conditions that promoted survival and proliferation of peripheral Bmem, caused massive tonsillar Bmem death. Purified tonsillar Bmem failed to expand but rapidly differentiated in antibody secreting cells and subsequently underwent apoptosis. In contrast, circulating Bmem showed delayed activation and differentiation, but exhibited a longer lifespan and active proliferation. In addition, short-term stimulation of tonsillar Bmem resulted in the production of more immunoglobulin G (IgG) than their peripheral counterparts. At later time points, however, IgG production from the different B cells was reversed. Our findings imply that the tissue located and peripheral Bmem have distinct behaviors, indicating organ dependent functional responses that should not be generalizable to all Bmem. This work provides a greater understanding of how Bmem location is coupled to specialized roles of B lymphocytes.
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