| Leukocyte residence in lymphoid organs is controlled by a balance between retention and egress-promoting chemoattractants sensed by pertussis toxin (PTX)-sensitive Galphai protein-coupled receptors (GPCRs). Here, we use two-photon intravital microscopy to show that immature B cell retention within bone marrow (BM) was strictly dependent on amoeboid motility mediated by CXCR4 and CXCL12 and by VLA-4 integrin-mediated adhesion to VCAM-1. However, B lineage cell egress from BM is independent of PTX-sensitive GPCR signaling. B lineage cells expressing PTX rapidly exited BM even though their motility within BM parenchyma was significantly reduced. Our experiments reveal that when immature B cells are near BM sinusoids their motility is reduced, their morphology is predominantly rounded, and cells reverse transmigrate across sinusoidal endothelium in a largely nonamoeboid manner. Immature B cell egress from BM was dependent on a twofold CXCR4 down-regulation that was antagonized by antigen-induced BCR signaling. This passive mode of cell egress from BM also contributes significantly to the export of other hematopoietic cells, including granulocytes, monocytes, and NK cells, and is reminiscent of erythrocyte egress.;B-intrinsic mechanisms that control the downregulation of CXCR4 in immature B cells are still not clear. SOCS3 (Suppressor of Cytokine Signaling 3), traditionally known as a negative regulator of JAK/STAT signaling in immune cells, is expressed at the immature BM B cell stage. SOCS3 deficient immature BM B cells are increased in bone marrow, exhibit prolonged FAK signaling, and increased adhesion to VCAM-1. However, it is still not known whether B-cell intrinsic or extrinsic SOCS3 deficiency results in a defect in immature BM B cell egress. Using a B-cell specific cre recombinase system as well as combination of flow histological and 2-photon intravital microscopy techniques, we unveil that B-cell intrinsic SOCS3 deficiency does not impair bone marrow egress, regulation of CXCR4, or migration of immature BM B cells in vivo. This study rules out a B cell-intrinsic role for SOCS3 in regulating CXCR4 mediated immature B cell egress from the bone marrow and are consistent with findings by Tarlinton and colleagues using the same B-cell specific cre recombinase system. |
