Sustained Activation of Cell Adhesion Is a Differentially Regulated Process in B Lymphopoiesis

It is largely unknown how hematopoietic progenitors are positioned within specialized niches of the bone marrow microenvironment during development. Chemokines such as CXCL12, previously called stromal cell–derived factor 1, are known to activate cell integrins of circulating leukocytes resulting in transient adhesion before extravasation into tissues. However, this short-term effect does not explain the mechanism by which progenitor cells are retained for prolonged periods in the bone marrow. Here we show that in human bone marrow CXCL12 triggers a sustained adhesion response specifically in progenitor (pro- and pre-) B cells. This sustained adhesion diminishes during B cell maturation in the bone marrow and, strikingly, is absent in circulating mature B cells, which exhibit only transient CXCL12-induced adhesion. The duration of adhesion is tightly correlated with CXCL12-induced activation of focal adhesion kinase (FAK), a known molecule involved in integrin-mediated signaling. Sustained adhesion of progenitor B cells is associated with prolonged FAK activation, whereas transient adhesion in circulating B cells is associated with short-lived FAK activation. Moreover, sustained and transient adhesion responses are differentially affected by pharmacological inhibitors of protein kinase C and phosphatidylinositol 3-kinase. These results provide a developmental cell stage–specific mechanism by which chemokines orchestrate hematopoiesis through sustained rather than transient activation of adhesion and cell survival pathways

Linkage of Reduced Receptor Affinity and Superinfection to Pathogenesis of TR1.3 Murine Leukemia Virus

TR1.3 is a Friend murine leukemia virus (MLV) that induces selective syncytium induction (SI) of brain capillary endothelial cells (BCEC), intracerebral hemorrhage, and death. Syncytium induction by TR1.3 has been mapped to a single tryptophan-to-glycine conversion at position 102 of the envelope glycoprotein (Env102). The mechanism of SI by TR1.3 was examined here in comparison to the non-syncytium-inducing, nonpathogenic MLV FB29, which displays an identical BCEC tropism. Envelope protein expression and stability on both infected cells and viral particles were not statistically different for TR1.3 and FB29. However, affinity measurements derived using purified envelope receptor binding domain (RBD) revealed a reduction of >1 log in the K(D) of TR1.3 RBD relative to FB29 RBD. Whole-virus particles pseudotyped with TR1.3 Env similarly displayed a markedly reduced binding avidity compared to FB29-pseudotyped viral particles. Lastly, decreased receptor affinity of TR1.3 Env correlated with the failure to block superinfection following acute and chronic infection by TR1.3. These results definitively show that acquisition of a SI phenotype can be directly linked to amino acid changes in retroviral Env that decrease receptor affinity, thereby emphasizing the importance of events downstream of receptor binding in the cell fusion process and pathology

Focal Adhesion Kinase (FAK) Is Required for CXCL12-Induced Dhemotaxis and Adhesion to VCAM-1 in B Lymphocytes and Hematopoietic Stem/Progenitor Cells

Abstract The chemokine CXCL12 (SDF-1) and its receptor CXCR4 play a critical role in hematopoietic progenitor cell migration and positioning within the bone marrow (BM). However, CXCL12/CXCR4-induced signaling pathways in blood cell migration are poorly characterized. Based on previous studies of BM progenitor B cells indicating a strong correlation between FAK signaling and CXCL12 induced adhesion to VCAM-1, we speculated that FAK might be an important signaling component in CXCL12-induced chemotaxis and integrin-mediated adhesion. Here, we used two approaches to reduce FAK expression in (human and mouse) progenitor B cells and mouse Sca-1+, Kit+, Lin− stem/progenitor cells. FAK specific siRNAs reduced FAK expression by 80% and abolished both CXCL12-induced chemotaxis and adhesion to VCAM-1 in the pro-B cell line, REH. FAK knock-down did not change expression levels of CXCR4 and VLA-4 integrin. FAK expression was rescued by transfection with wild type, chicken FAK, which also restored both CXCL12-induced chemotaxis and adhesion. Furthermore, we found that in FAK deficient cells CXCL12-induced activation of the GTP-ase Rap1 was reduced, suggesting the importance of FAK in CXCL12-mediated inside-out integrin activation. CXCL12-mediated chemotaxis was also impaired in primary progenitor B cells and hematopoietic stem/progenitor cells (HSC/P) isolated from FAK floxed mice, in which FAK was deleted by Cre-mediated excision of FAK floxed alleles. Cre-mediated FAK deletion did not affect cell viability or induce apoptosis. These studies suggest that FAK may function as a key intermediary in signaling pathways controlling hematopoietic cell migration and lineage development

Evidence for a protective role of the Gardos channel against hemolysis in murine spherocytosis

It has been shown that mice with complete deficiency of all 4.1R protein isoforms (4.1-/-) exhibit moderate hemolytic anemia, with abnormal erythrocyte morphology (spherocytosis) and decreased membrane stability. Here, we characterized the Gardos channel function in vitro and in vivo in erythrocytes of 4.1-/- mice. Compared with wild-type, the Gardos channel of 4.1-/- erythrocytes showed an increase in Vmax (9.75 ± 1.06 vs 6.08 ± 0.09 mM cell × minute; P < .04) and a decrease in Km (1.01 ± 0.06 vs 1.47 ± 1.02 μM; P < .03), indicating an increased sensitivity to activation by intracellular calcium. In vivo function of the Gardos channel was assessed by the oral administration of clotrimazole, a well-characterized Gardos channel blocker. Clotrimazole treatment resulted in worsening of anemia and hemolysis, with decreased red cell survival and increased numbers of circulating hyperchromic spherocytes and microspherocytes. Clotrimazole induced similar changes in 4.2-/- and band 3+/- mice, indicating that these effects of the Gardos channel are shared in different models of murine spherocytosis. Thus, potassium and water loss through the Gardos channel may play an important protective role in compensating for the reduced surface-membrane area of hereditary spherocytosis (HS) erythrocytes and reducing hemolysis in erythrocytes with cytoskeletal impairments. (Blood. 2005;106:1454-1459

Functional receptor for C3a anaphylatoxin is expressed by normal hematopoietic stem/progenitor cells, and C3a enhances their homing-related responses to SDF-1.

Complement has recently been implicated in developmental pathways and noninflammatory processes. The expression of various complement components and receptors has been shown in a wide range of circulating myeloid and lymphoid cells, but their role in normal hematopoiesis and stem cell homing has notyet been investigated. We report that normal human CD34 cells and lineage differentiated hematopoietic progenitors express the complement anaphylatoxin C3a receptor (C3aR) and respond to C3a. Moreover, C3a, but not the biologically inactive desArg-C3a, induces calcium flux in these cells. Furthermore, we found thatC3 is secreted by bone marrow stroma and that, although C3a does not influence directly the proliferation/survival of hematopoietic progenitors, it (1) potentiates the stromal cell–derived factor 1 (SDF-1)– dependent chemotaxis of human CD34cells and lineage-committed myeloid, erythroid, and megakaryocytic progenitors; (2) primes SDF-1–dependent trans-Matrigel migration; and (3) stimulates matrix metalloproteinase-9 secretion and very late antigen 4 (VLA-4)–mediated adhesionto vascular cell adhesion molecule 1 (VCAM-1). Furthermore, we found that murine Sca-1 cells primed by C3a engrafted faster in lethally irradiated animals. These results indicate that normalhuman hematopoietic stem and progenitor cells express functional C3aR and that the C3aR-C3a axis sensitizes the responses of these cells to SDF-1 and thus may be involved in promoting their homing into the bone marrow via cross talkwith the SDF–CXC chemokine receptor-4 (CXCR4) signaling axis. C3a is the first positive regulator of this axis to be identified