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Adhesion receptors on bone marrow stromal cells: in vivo expression of vascular cell adhesion molecule-1 by reticular cells and sinusoidal endothelium in normal and gamma-irradiated mice
Abstract
Cell adhesion molecules (CAMs) play a key role in interactions between stromal and hematopoietic cells in bone marrow (BM) and in cell traffic through vascular endothelium. To examine the identity of CAMs involved in these processes in mouse BM, we have investigated the in vivo expression of vascular cell adhesion molecule-1 (VCAM-1) and its counter-receptor, very late antigen-4 (VLA-4). Radioiodinated monoclonal antibodies (MoAbs) detecting VLA-4 and VCAM-1 were injected intravenously. Antibody binding was detected in BM by light and electron microscope radioautography. VCAM-1 labeling was restricted to stromal reticular cells and endothelial cells lining BM sinusoids. VCAM- 1+ reticular cells formed patchy concentrations, especially in subosteal regions, associated with lymphoid, granulocytic, and erythroid cells. After gamma-irradiation to deplete hematopoietic cells, reticular cells and endothelial cells all showed VCAM-1 labeling in apparently increased intensity. VLA-4 labeling was shown by undifferentiated blast cells and lymphohematopoietic cells both in BM cell suspensions and in vivo, especially at reticular cell contact points. The results demonstrate that VCAM-1 is expressed in vivo by certain BM reticular cells, suggesting that the molecule mediates adhesion to multiple lineages of lymphohematopoietic cells. The finding that VCAM-1 is also expressed constitutively by BM sinusoidal endothelium, unlike its inductive expression by endothelia elsewhere, suggests that VCAM-1 and VLA-4 may be involved in regulating the normal cell traffic between BM and the blood stream.
... In this study, we used granulocyte colony stimulating factor (G-CSF), which has been routinely used for therapeutic mobilization of HSCs (27,28); AMD3100 (Plerixafor), which has been shown to mobilize HSCs by antagonizing CXCR4 at the CXCR4/CXCL12 junction (29,30); and a VLA4 inhibitor (Very late antigen 4 inhibitor, BIO5192), as a preparatory conditioning regimen in a mouse model of RAG2 deficiency (13). VLA4, together with vascular cell adhesion molecule-1 (VCAM-1) plays an important role in anchoring HSC progenitors to BM stromal cells and regulates HSC traffic between the BM and peripheral regions (31,32). Inhibition of VLA4 has been shown to mobilize HSCs by interruption of the VCAM-1/VLA-4 axis (33). ...
RAG2 deficiency is characterized by a lack of B and T lymphocytes, causing severe lethal infections. Currently, RAG2 deficiency is treated with a Hematopoietic Stem Cell transplantation (HSCT). Most conditioning regimens used before HSCT consist of alkylating myelotoxic agents with or without irradiation and affect growth and development of pediatric patients. Here, we developed a non-myelotoxic regimen using G-CSF, VLA-4I or AMD3100. These agents are known HSC mobilizers or affect bone marrow (BM) permeability and may support the homing of HSCs to the BM, without inducing major side effects. Female Rag2-/- mice were pre-treated with Busulfan (BU), G-CSF, VLA-4I or AMD3100 and transplanted with male BM cells transduced with a lentiviral vector carrying codon optimized human RAG2 (RAG2co). Peripheral blood cell counts increased significantly after G-CSF, VLA-4I and AMD3100 treatment, but not after BU. Reconstitution of PB lymphocytes was comparable for all groups with full immune reconstitution at 6 months post transplantation, despite different methods of conditioning. Survival of mice pre-treated with non-myelotoxic agents was significantly higher than after BU treatment. Here we show that the non-myelotoxic agents G-CSF, VLA-4I, and AMD3100 are highly effective as conditioning regimen before HSC gene therapy and can be used instead of BU.
... Instead of promoting growth, the membrane-bound stem cell factor (mSCF) is crucial for HSC lodgment, maintenance, and hematopoiesis (Barker, 1994;Driessen et al., 2003), and the membrane-bound version of C-X-C motif chemokine ligand 12 (CXCL12) plays a role in HSC anchorage in the niche rather than chemotaxis (Kollet et al., 2006). Vascular cell adhesion molecule 1 (VCAM-1), a cell-cell adhesion molecule expressed on stromal cells (Jacobsen et al., 1996), regulates HSC homing and retention in the bone marrow (Li et al., 2018;Papayannopoulou et al., 1995). However, it remains unknown on how these membrane-bound factors regulate stem cell behaviors in a localized manner at the cellular level. ...
Membrane-bound factors expressed by niche stromal cells constitute a unique class of localized cues and regulate the long-term functions of adult stem cells, yet little is known about the underlying mechanisms. Here, we used a supported lipid bilayer (SLB) to recapitulate the membrane-bound interactions between hematopoietic stem cells (HSCs) and niche stromal cells. HSCs cluster membrane-bound stem cell factor (mSCF) at the HSC-SLB interface. They further form a polarized morphology with aggregated mSCF under a large protrusion through a synergy with VCAM-1 on the bilayer, which drastically enhances HSC adhesion. These features are unique to mSCF and HSCs among the factors and hematopoietic populations we examined. The mSCF–VCAM-1 synergy and the polarized HSC morphology require PI3K signaling and cytoskeletal reorganization. The synergy also enhances nuclear retention of FOXO3a, a crucial factor for HSC maintenance, and minimizes its loss induced by soluble SCF. Our work thus reveals a unique role and signaling mechanism of membrane-bound factors in regulating stem cell morphology and function.
... After cleavage by thrombin (Grassinger et al., 2009) it binds to a variety of matrix receptors, including CD44 and very late antigen-4 (VLA4, integrin a4b1, both highly expressed on HSCs, Table 1). VLA4 on HSPCs further binds vascular cell adhesion molecule 1 (VCAM1) that is highly expressed by stromal reticular cells and endothelial cells lining bone marrow sinusoids (Jacobsen et al., 1996; Table 1) and integrin engagement mediates intimate cell-to-cell contact required for the support or HSPC function (Jung et al., 2005). Consistently, the conditional deletion of Itga4 from adult HSPCs (Scott et al., 2003) or Vcam1 from endothelial cells (Koni et al., 2001) mobilizes HSPCs to the blood and impairs homing to the bone marrow niche, critically delaying short-term engraftment after transplantation (Koni et al., 2001;Scott et al., 2003). ...
The bone marrow (BM) microenvironment, also called the BM niche, is essential for the maintenance of fully functional blood cell formation (hematopoiesis) throughout life. Under physiologic conditions the niche protects hematopoietic stem cells (HSCs) from sustained or overstimulation. Acute or chronic stress deregulates hematopoiesis and some of these alterations occur indirectly via the niche. Effects on niche cells include skewing of its cellular composition, specific localization and molecular signals that differentially regulate the function of HSCs and their progeny. Importantly, while acute insults display only transient effects, repeated or chronic insults lead to sustained alterations of the niche, resulting in HSC deregulation. We here describe how changes in BM niche composition (ecosystem) and structure (remodeling) modulate activation of HSCs in situ. Current knowledge has revealed that upon chronic stimulation, BM remodeling is more extensive and otherwise quiescent HSCs may be lost due to diminished cellular maintenance processes, such as autophagy, ER stress response, and DNA repair. Features of aging in the BM ecology may be the consequence of intermittent stress responses, ultimately resulting in the degeneration of the supportive stem cell microenvironment. Both chronic stress and aging impair the functionality of HSCs and increase the overall susceptibility to development of diseases, including malignant transformation. To understand functional degeneration, an important prerequisite is to define distinguishing features of unperturbed niche homeostasis in different settings. A unique setting in this respect is xenotransplantation, in which human cells depend on niche factors produced by other species, some of which we will review. These insights should help to assess deviations from the steady state to actively protect and improve recovery of the niche ecosystem in situ to optimally sustain healthy hematopoiesis in experimental and clinical settings.
... The cellular composition and specialized microenvironment of this organ allow the common lymphoid progenitor cell to give rise to B cells, ready to encounter their antigen. This possibility is given by a net of specialized non-lymphoid stromal cells and the surrounding osteoblasts located in intimate contact with the B-cell precursors and the to be, B cells [10][11][12]. As a consequence of this organization, the contribution supplied by stromal cells and osteoblasts is two-fold. ...
Trillions of microorganisms inhabit the mucosal membranes maintaining a symbiotic relationship with the host’s immune system. B cells are key players in this relationship because activated and differentiated B cells produce secretory immunoglobulin A (sIgA), which binds commensals to preserve a healthy microbial ecosystem. Mounting evidence shows that changes in the function and composition of the gut microbiota are associated with several autoimmune diseases suggesting that an imbalanced or dysbiotic microbiota contributes to autoimmune inflammation. Bacteria within the gut mucosa may modulate autoimmune inflammation through different mechanisms from commensals ability to induce B-cell clones that cross-react with host antigens or through regulation of B-cell subsets’ capacity to produce cytokines. Commensal signals in the gut instigate the differentiation of IL-10 producing B cells and IL-10 producing IgA+ plasma cells that recirculate and exert regulatory functions. While the origin of the dysbiosis in autoimmunity is unclear, compelling evidence shows that specific species have a remarkable influence in shaping the inflammatory immune response. Further insight is necessary to dissect the complex interaction between microorganisms, genes, and the immune system. In this review, we will discuss the bidirectional interaction between commensals and B-cell responses in the context of autoimmune inflammation.
... Many molecules have been implicated in adhesion of stem cells to stroma (186,189). These include P-selectins (189); heparan sulfate (24,47); chondroitin sulfate; fibronectin-VCAM-PECAM, and associated receptors (integrins) (69,113,139,177); growth factor receptors, such as c-kit (85) and CD45 (24); and collagen (84). Of particular interest is CD34. ...
... Interestingly, E-selectin plays a key role for homing of T cells to the skin (Kansas, 1996), which may contribute to the persistent warts. On the other hand, E-selectin is highly expressed on bone marrow vascular endothelium (Jacobsen et al., 1996;Schweitzer et al., 1996), and recent data demonstrate a distinct role in this niche: E-selectin expression is required for hematopoietic stem cell (HSC) proliferation, whereas E-selectin deficiency leads to HSC quiescence with greater capacity for self-renewal (Winkler et al., 2012). This ICO SL-mediated effect on adhesion molecules may explain the normocellular marrow with circulating neutropenia in P1. ...
Primary immunodeficiencies represent naturally occurring experimental models to decipher human immunobiology. We report a patient with combined immunodeficiency, marked by recurrent respiratory tract and DNA-based viral infections, hypogammaglobulinemia, and panlymphopenia. He also developed moderate neutropenia but without prototypical pyogenic infections. Using whole-exome sequencing, we identified a homozygous mutation in the inducible T cell costimulator ligand gene ( ICOSLG ; c.657C>G; p.N219K). Whereas WT ICOSL is expressed at the cell surface, the ICOSL N219K mutation abrogates surface localization: mutant protein is retained in the endoplasmic reticulum/Golgi apparatus, which is predicted to result from deleterious conformational and biochemical changes. ICOSL N219K diminished B cell costimulation of T cells, providing a compelling basis for the observed defect in antibody and memory B cell generation. Interestingly, ICOSL N219K also impaired migration of lymphocytes and neutrophils across endothelial cells, which normally express ICOSL. These defects likely contributed to the altered adaptive immunity and neutropenia observed in the patient, respectively. Our study identifies human ICOSLG deficiency as a novel cause of a combined immunodeficiency.
... VCAM-1 is abundantly expressed in the red pulp of the spleen and facilitates integrin-mediated B cell localization in the splenic MZ and peripheral lymphoid tissue compartmentalization (17,18). In addition, the expression of VCAM-1 in BM stromal cells is important for the maturation and retention of PCs in the BM (19)(20)(21)(22)(23). ...
Plasma cell differentiation involves coordinated changes in gene expression and functional properties of B cells. Here, we study the role of Mzb1, a Grp94 cochaperone that is expressed in marginal zone (MZ) B cells and during the terminal differentiation of B cells to antibody-secreting cells. By analyzing Mzb1
-/-
Prdm1
+/gfp
mice, we find that Mzb1 is specifically required for the differentiation and function of antibody-secreting cells in a T cell-independent immune response. We find that Mzb1-deficiency mimics, in part, the phenotype of Blimp1 deficiency, including the impaired secretion of IgM and the deregulation of Blimp1 target genes. In addition, we find that Mzb1
-/-
plasmablasts show a reduced activation of β1-integrin, which contributes to the impaired plasmablast differentiation and migration of antibody-secreting cells to the bone marrow. Thus, Mzb1 function is required for multiple aspects of plasma cell differentiation.
... After maturation, B-lymphocytes become immunocompetent, or capable of binding with a specific antigen (Ag). coding joints are processed imprecisely generating overhangs (P-nucleotides) and the enzyme terminal deoxynucleotidyl transferase (TdT) adds additional non-template nucleotides before the DNA ends are finally joined by ubiquitously expressed DNA repair proteins [23,24,25]. ...
Chronic lymphocytic leukemia (CLL) is one of the most common hematologic malignancy in the western countries and is characterized by the outgrowth of a CD5 positive B cell population. Mature B cells from healthy individuals and B cells from CLL patients usually co-express B-cell receptors (BCRs) of the IgM and IgD isotype and it was recently reported that the isotype influences BCRs signaling strength and lymphoma generation in Eµ-TCL1 transgenic mice. Responsible for the observed signaling differences between IgM and IgD might be the glycosylation of the signal transducing subunit Igα. Further, CLL-derived BCRs have the characteristic capability to induce autonomous, antigen-independent signaling by binding to an internal motif in adjacent BCRs on the same cell. Since interference with BCR signaling is a potential therapeutic approach for the treatment of CLL, knowledge of the exact binding strength required for CLL-receptor signaling is important. Thus, the aims of this study were to investigate (a) the role of Igα-glycosylation in BCRs signaling and (b) to evaluate the exact binding affinity of CLL-BCRs to the BCR-intrinsic epitope. In this study, the Igα glycosylation mutant together with µ or ∂ heavy chain were expressed via biomolecular fluorescence complementation (BiFC) method in B3046 pro-B cell line and flow cytometry were performed. To quantify the binding affinity of CLL-BCRs, soluble IgG-BCRs carrying CLL-derived VH domain were used in the surface plasmon resonance (SPR) based Biacore X100 system. The major outcomes in our experiments demonstrated that, glycosylation defective Igα did not show significant differences in signal strength of BCRs comparing wild type of Igα while expressed with IgM or IgD isotypes. Interestingly, both isotypes with Igα mutant gave lower BCRs surface expression levels than with wild Igα, suggesting that glycosylation influences pre-BCRs expression of IgM and IgD. In binding affinity measurement of CLL-BCRs, the soluble IgG-BCRs as ligand didn't bind to the analyte and yielded nonspecific binding effect, thus affinity could not be determined through Biacore X100 system. From the above findings we conclude that, different glycosylation patterns of Igα are not uniquely responsible regarding signal differences in IgM and IgD isotypes of BCRs. Furthermore, the Biacore X100 system has been proven to be very sensitive and adequate optimizations might necessary to acquire binding affinity data from CLL-BCRs.
... The precise reason why prostate cancer is particularly predisposed to forming bone metastases is as yet unknown. However, it is thought that certain important bone matrix proteins, including vibronectin, bone sialoprotein and collagen type I are facilitated by a number of tethering proteins, such as paxillin, and by reduced blood flow rates which allow prostate cancer cells to adhere, colonise and invade into sites in the long bones (5,6). The inconsistency of metastatic cancer is undoubtedly influenced by the molecular and cellular characteristics of both tumour cells and the tissue which they invade. ...
Background:
Bone metastases are a common feature of advanced prostatic malignancies. They are characterised by a unique prevalence of osteoblastic phenotype and a poor prognosis. Paxillin is a 68-kDa signal transduction adaptor and scaffold protein that contains motifs involved in the mediation of protein-protein interactions. The state of paxillin phosphorylation is central to determining a cell's ability to adhere, detach and migrate and hence has been linked to processes such as wound repair and tumour metastasis. The current study explored the impact of paxillin suppression on prostate and breast cancer cell function and their responsiveness to hepatocyte growth factor (HGF) and bone matrix extract (BME) in order to assess its potential to influence bone colonization and homing.
Materials and methods:
Hammerhead ribozyme transgenes were used to knockdown the expression of paxillin in breast and prostate cancer cell lines. The impact on the cell growth, migration, adhesion and invasion was assessed using in vitro functional assays. In order to explore potential mechanisms, focal adhesion kinase (FAK) inhibitor was also used.
Results:
Knockdown of paxillin expression was observed in all tested cell lines following transfection with the ribozyme transgene. The knockdown of paxillin increased proliferation and invasiveness of LNCaP cells, with no effect on their attachment abilities. The opposite, however, is true for PC-3 cells where, following knockdown, cellular attachment was significantly reduced, while no significant changes in growth and invasiveness were detected. In the MDA-MB-231 breast cancer knockdown model, cells had little difference in proliferative rates and generally increased attachment and reduced invasive abilities. Treatments with HGF and BME had differential effects on targeted cells when compared to controls.
Conclusion:
These data suggest that paxillin appears to influence major cell functions in a diverse range of prostate and breast cancer models. The responsiveness of cells to environmental factors such as HGF or BME may be influenced by paxillin status, although this seems to be dependent on cell type.
... Among them, b-1 and a-4 integrins, as well as other factors such as VCAM-1 and MMP-2, are the most important, but it is possible that their expression levels decrease in subsequent cultures (Karp and Leng Teo, 2009;Ruster et al., 2006;Steingen et al., 2008;Yagi et al., 2010). Some authors have demonstrated that a4/b1 (VLA-4) integrins might be important for the initial capture of MSCs, rolling and transmigration through the endothelial surface after BM transplantation (Jacobsen et al., 1996;Yagi et al., 2010). ...
... Integrins play an important role in the cell-to-cell interaction through adhesion to vascular cell adhesion molecule (VCAM-1) [87]. Integrins α4-β1 mediate the initial capture, rolling and firm attachment of MSCs to the bone marrow [215]. Blockade of integrin β1 inhibited the migration of neutrophils to lung during inflammatory condition and also reduced the number of MSCs in the infarcted myocardium [216,217]. ...
The severity and threat of sepsis is well known and despite several decades of research the mortality continues to be high. Stem cells have great potential to be used in various clinical disorders. The innate ability of stem cells such as pluripotency, self-renewal makes them potential agents for therapeutic intervention. The pathophysiology of sepsis is a plethora of complex mechanisms which include the initial microbial infection, followed by ‘cytokine storm’, endothelial dysfunction, coagulation cascade and the late phase of apoptosis and immune paralysis which ultimately results in multiple organ dysfunction. Stem cells could potentially alter each step of this complex pathophysiology of sepsis. Multiple organ dysfunction associated with sepsis most often leads to death and stem cells have shown their ability to prevent the organ damage and improve the organ function. The possible mechanisms of therapeutic potential of stem cells in sepsis have been discussed in detail. The route of administration, dose level and timing also play vital role in the overall effect of stem cells in sepsis.
... The bone marrow (BM) microenvironment plays a critical role in regulating proliferation and differentiation of hematopoietic cells (HC) [1,2]. This process depends on complex interactions between both compartments that involve growth factors and cytokines [3,4], as well as direct cell-to-cell contacts through surface molecules [5][6][7][8]. In femoral, so-called fatty BM, the microenvironment of residual HCs, which include quiescent stem cells and macrophages (Mo), is composed mainly of adipocytes. ...
Adipocytes are part of hematopoietic microenvironment, even though up to now in humans, their role in hematopoi-esis is still questioned. We have previously shown that accumulation of fat cells in femoral bone marrow (BM) coincides with increased expression of neuropilin-1 (NP-1), while it is weakly expressed in hematopoietic iliac crest BM. Starting from this observation, we postulated that adipocytes might exert a negative effect on hematopoiesis mediated through NP-1. To test this hypothesis, we set up BM adipocytes differentiated into fibroblast-like fat cells (FLFC), which share the major characteristics of primitive unilocular fat cells, as an experimental model. As expected, FLFCs constitutively produced macrophage colony stimulating factor and induced CD34 differentiation into macrophages independently of cell-to-cell contact. By contrast, granulopoiesis was hampered by cell-to-cell contact but could be restored in transwell culture conditions, together with granulocyte colony stimulating factor production. Both functions were also recovered when FLFCs cultured in contact with CD34 cells were treated with an antibody neutralizing NP-1, which proved its critical implication in contact inhibition. An inflamma-tory cytokine such as interleukin-1 or dexamethasone modulates FLFC properties to restore granulopoiesis. Our data provide the first evidence that primary adipocytes exert regulatory functions during hematopoiesis that might be implicated in some pathological processes. STEM CELLS 2008;26:1556 –1564 Disclosure of potential conflicts of interest is found at the end of this article.
... Thus, P-selectin is often involved in early leukocyte recruitment during an inflammatory response. P-selectin is also constitutively expressed at low levels on the endothelium of thymus [33], lung and choroid plexus microvessels [34], bone marrow microvasculature [35,36], in postcapillary venules in skin [37], and on peritoneal macrophages [38]. The transcription of P-selectin can be induced by cytokines such as interleukin (IL)-4 and IL-13 [39,40]. ...
The cell surface represents the interface between the cell and its environment. As such, the cell surface controls cell–cell interactions and functions such as adhesion and migration, and will transfer external cues to regulate processes such as survival, death, and differentiation. Redefining the cell surface by temporarily (or permanently) modifying the molecular landscape of the plasma membrane affects the way in which the cell interacts with its environment and influences the information that is relayed into the cell along downstream signaling pathways. This chapter outlines the role of key enzymes, the glycosyltransferases, in posttranslationally modifying proteins and lipids to fine-tune cells, ability to migrate. These enzymes are critical in controlling the formation of a platform structure, sialyl Lewis x (sLex), on circulating cells that plays a central role in the recognition and recruitment by selectin counter receptors on endothelial cells that line blood vessels of tissues throughout the body. By developing methods to manipulate the activity of these enzymes and hence the cell surface structures that result, treatments can be envisioned that direct the migration of therapeutic cells to specific locations throughout the body and also to inhibit metastasis of detrimental cells such as circulating tumor cells.
... β 1 integrin levels have been shown to become upregulated in the bone marrow of mice in two different models of sepsis (endotoxemia, and cecal ligation & puncture surgery) [43]. Vascular cell adhesion molecule 1 (VCAM-1), a major ligand for α 4 β 1 integrin (VLA-4), is expressed on bone marrow stromal cells, including bone marrow sinusoidal endothelium [44]. Under normal conditions, conditional deletion of α 4 β 1 integrin on hematopoietic stem cells appeared to have no effect on neutrophil trafficking [45]. ...
Neutrophil migration is critical for pathogen clearance and host survival during severe sepsis. Interaction of neutrophil adhesion receptors with ligands on endothelial cells results in firm adhesion of the circulating neutrophils, followed by neutrophil activation and directed migration to sites of infection through the basement membrane and interstitial extracellular matrix. Proteolytic enzymes and reactive oxygen species are produced and released by neutrophils in response to a variety of inflammatory stimuli. Although these mediators are important for host defense, they also promote tissue damage. Excessive neutrophil migration during the early stages of sepsis may lead to an exaggerated inflammatory response with associated tissue damage and subsequent organ dysfunction. On the other hand, dysregulation of migration and insufficient migratory response that occurs during the latter stages of severe sepsis contributes to neutrophils' inability to contain and control infection and impaired wound healing. This review discusses the major steps and associated molecules involved in the balance of neutrophil trafficking, the precise regulation of which during sepsis spells life or death for the host.
... Th e interaction of the integrin -ligand pair VLA-4/VCAM-1 assists in retaining hematopoietic stem cells in the BM and contributes to B cell development therein [4,11]. Th erefore, the observed increase in VCAM-1 expression on a subset of stromal cells and sinusoid endothelial cells within BM from the patient with atypical c-ALL and its respective ligand VLA-4 on c-ALL cells could be responsible for retention of the leukemic cells and, in turn, for deceptive normal blood counts. ...
... Es gibt wenig Untersuchungen, die das Vorkommen von Selektinen auf Vorläuferzellen und Endothel im KM analysiert haben. E-Selektin wird neben VCAM-1 unter physiologischen Bedingungen konstitutiv und selektiv auf dem Endothel von hämatopoetischen Organen exprimiert [112,181,182]. Außerdem gibt es Hinweise auf die konstitutive Expression von E-und P-Selektin in Venülen des murinen KM in Abwesenheit einer Entzündung [183]. ...
... Although the CXCL12/CXCR4 axis works to promote HSC adhesion and retention in the BM, ultimately retention is reliant on specific adhesion molecule interactions within the BM Chapter 1: General Introduction stroma. BM endothelial cells constitutively express the IgSF molecule VCAM-1 (Jacobsen et al., 1996) and interactions between this molecule and VLA-4 are important in the homing of these cells to the BM following systemic administration. Early work illustrated this importance by demostrating that pretreatment of murine haematopoietic progenitors with an α 4 β 1 integrin or VCAM-1 blocking antibody significantly impaired BM homing in lethally irradiated mice by around 50% (Papayannopoulou et al., 1995). ...
... 2X10 7 cells are produced every day in mice and the cells circulate in the blood and lymph (2). B cells arise from common lymphoid progenitors and the earliest B cell precursors require contact with the stromal cells in the BM (3,4), in addition to specific cytokines, particularly IL-7 (5,6). B cells at later stages of development do not require cell contact, but need cytokines for further differentiation. ...
... The first component of the adaptive immune system is that of the B-cell. Similar Within the bone marrow compartment, B-cells progress through stages of pro-B-cells, pre-B-cells, and finally immature B-cells 105 . The characteristic that defines each stage is a successful genetic rearrangement of their immunoglobulin locus 106 . ...
... Overexpression of a4 subunit of the VLA-4 integrin in MSCs, using adenoviral vector, results in increased MSCs homing to bone marrow in a mouse model (Kumar and Ponnazhagan 2007). VLA-4 integrin is composed of CD49d (a4) and CD29 (b1), whose heterodimerization and interaction with VCAM1 results in firm adherence of circulating cells to the endothelium, followed by endothelial transmigration (Springer 1994;Jacobsen et al., 1996;Schweitzer and Drager, 1996). Bobis-Wozowicz et al. (2011) found that hAd-MSCs, with an overexpressed level of CXCR4, showed increased motility, invasiveness and homing to bone marrow of NOD/SCID mice. ...
Stem/progenitor cell based therapeutic approach in clinical practice has been an elusive dream in medical sciences, and improvement of stem cell homing is one of major challenges in cell therapy programs. Stem/progenitor cells have a homing response to injured tissues/organs, mediated by interactions of chemokine receptors expressed on the cells and chemokines secreted by the injured tissue. For improvement of directed homing of the cells, many techniques have been developed either to engineer stem/progenitor cells with higher amount of chemokine receptors (stem cell-based strategies) or to modulate the target tissues to release higher level of the corresponding chemokines (target tissue-based strategies). This review discusses both of these strategies involved in the improvement of stem cell homing focusing on mesenchymal stem cells as most frequent studied model in cellular therapies.
For over half a century, hematopoietic stem cells (HSCs) have been used for transplantation therapy to treat severe hematologic diseases. Successful outcomes depend on collecting sufficient donor HSCs as well as ensuring efficient engraftment. These processes are influenced by dynamic interactions of HSCs with the bone marrow niche, which can be revealed by artificial niche models. Here, a multifunctional nanostructured hydrogel is presented as a 2D platform to investigate how the interdependencies of cytokine binding and nanopatterned adhesive ligands influence the behavior of human hematopoietic stem and progenitor cells (HSPCs). The results indicate that the degree of HSPC polarization and motility, observed when cultured on gels presenting the chemokine SDF‐1α and a nanoscale‐defined density of a cellular (IDSP) or extracellular matrix (LDV) α4β1 integrin binding motif, are differently influenced on hydrogels functionalized with the different ligand types. Further, SDF‐1α promotes cell polarization but not motility. Strikingly, the degree of differentiation correlates negatively with the nanoparticle spacing, which determines ligand density, but only for the cellular‐derived IDSP motif. This mechanism potentially offers a means of predictably regulating early HSC fate decisions. Consequently, the innovative multifunctional hydrogel holds promise for deciphering dynamic HSPC‐niche interactions and refining transplantation therapy protocols.
Under conditions of hypoxia, a change in the morphological and functional state of the components of the microvasculature bed will be accompanied by a change in the adequacy and usefulness of the immune response and hematopoiesis. The purpose of the study was to determine the morphofunctional state of the vascular component of the red bone marrow and spleen at various times of simulated hypoxia and to identify the degree of influence of CD68-positive cells on this state. Chronic hypoxia was modeled on 246 outbred male rats using special chambers containing natural gas from the Astrakhan gas field (Russia) at a concentration not exceeding the maximum allowable. The experiment was continued for 120 days, inhalation was carried out five days a week for four hours a day, the removal of animals from the experiment was carried out every 30 days. The functional activity of the vascular component of the red bone marrow was determined using the method of laser Doppler flowmetry. The degree of expression of inducible (iNOS) and endothelial (eNOS) NO synthases and the distribution of CD68-positive cells in the spleen structures were determined by immunohistochemical method. The study showed that as the duration of chronic simulated hypoxia increases, there is a decrease in the microcirculation index, an increase in myogenic tone and shunting index, which, taken together, indicates a deterioration of organ perfusion and confirms the formation of a hypoxic state. An analysis of the functional activity, carried out using an immunohistochemical study of the expression of iNOS and eNOS in the structures of the spleen, showed that as the duration of the experiment increased, the level of endothelial synthase decreased and the level of inducible synthase increased. Perhaps this is due to the influence of biologically active substances secreted by macrophages activated during hypoxia. This is supported by an increase in the presence of CD68-positive cells in the red pulp and along the connective tissue trabeculae as the experimental exposure increases.
Background
: Bone Marrow (BM) Transplantation has been used to treat malignant and non-malignant BM related disorders. Although an effective strategy, the procedure still suffers from numerous complications like graft rejection and non-specific stem cell distribution. Avoidance of immune rejection of graft has downsized the stem cell quantity available whereas non-specific distribution has necessitated the infusion of increased stem cell dose.
Objective
: A dual purpose approach is needed to reduce the stem cell dose for the transplant. The study has employed cell encapsulation and BM targeting to increase the quantity of transplanted stem cells reaching BM.
Study Design
: Cells were encapsulated with different biomaterials- chitosan (CS), polyallylamine hydrochloride (PAH), polystyrene sulfonate (PSS) and liposomes and; assessed for interaction with immune system components. The biomaterials were conjugated with the peptide that binds to BM specific cell surface molecule, Vascular Cell Adhesion Molecule 1 (VCAM1) and evaluated for a successful transplant.
Results
: Encapsulated cells showed reduced interaction with antibody and cytokine without adverse effects on viability. Constitutive expression of VCAM1 was found to be specific on human bone marrow endothelial cells (hBMECs) and was employed for targeting by conjugating VCAM1 binding peptide to encapsulated cells. Peptide conjugated encapsulated stem cell formulations when transplanted in irradiated mice with and without treatment with radioprotectant amifostine, exhibited an increased percentage of cells reaching bone marrow. Post-transplant survival along with blood count of neutrophils, platelets and leukocytes was also enhanced for VCAM1 binding peptide conjugated formulations with 100% survival demonstrated by peptide conjugated CS/PSS/CS encapsulation formulation in irradiated mice for four weeks.
Conclusion
: Encapsulated cells retained their viability with increased shielding to the immune system ensuring that graft rejection can be avoided. Transplanted encapsulated stem cells distributed in a higher percentage to BM when conjugated to VCAM1 binding peptide which could enable the use of low stem cell dose. Peptide conjugated CS/PSS/CS encapsulation system conferred 100% survival in irradiated mice with increased regeneration demonstrating the ability to treat radiation exposed victims.
Hematopoietic stem cell (HSC) trafficking is regulated by a number of complex mechanisms. Among them are the transmembrane protein Robo4 and the vascular cell adhesion molecule, VCAM1. Endothelial VCAM1 is a well-known regulator of hematopoietic cell trafficking, and our previous studies revealed that germline deletion of Robo4 led to impaired HSC trafficking, with an increase in vascular endothelial cell (VEC) numbers and downregulation of VCAM1 protein on sinusoidal VECs. Here, we utilized two Robo4 conditional deletion models in parallel with Robo4 germline knockout mice (R4 KO ) to evaluate the effects of acute and endothelial cell-specific Robo4 deletion on HSC trafficking. Strikingly similar to the R4 KO , the acute deletion of Robo4 resulted in altered HSC distribution between the bone marrow and blood compartments, despite normal numbers of VECs and wild-type levels of VCAM1 cell surface protein on sinusoidal VECs. Additionally, consistent with the R4 KO mice, acute loss of Robo4 in the host perturbed long-term engraftment of donor wild-type HSCs and improved HSC mobilization to the peripheral blood. These data demonstrate the significant role that endothelial Robo4 plays in directional HSC trafficking, independent of alterations in VEC numbers and VCAM1 expression.
We have previously shown that the HCA/ALCAM (CD166) glycoprotein, a member of the immunoglobulin family that mediates both homophilic and heterophilic cell-cell adhesion, via the CD6 ligand, is expressed at the surface of all of the most primitive CD38−/lo, Thy-1+, rho123lo, CD34+hematopoietic cells in human fetal liver and fetal and adult bone marrow. In the present report we show that HCA is also expressed by subsets of stromal cells in the primary hematopoietic sites that sequentially develop in the human embryo and fetus, ie, the paraaortic mesoderm, liver, thymus, and bone marrow. Adult bone marrow stromal cells established in vitro, including those derived from Stro-1+ progenitors and cells from immortalized cell lines, express HCA. In contrast, no HCA expression could be detected in peripheral lymphoid tissues, fetal spleen, and lymph nodes. HCA membrane molecules purified from marrow stromal cells interact with intact marrow stromal cells, CD34+ CD38−hematopoietic precursors, and CD3+ CD6+peripheral blood lymphocytes. Finally, low but significant levels of CD6 are here for the first time detected at the surface of CD34+ rho123med/lo progenitors in the bone marrow and in mobilized blood from healthy individuals. Altogether, these results indicate that the HCA/ALCAM surface molecule is involved in homophilic or heterophilic (with CD6) adhesive interactions between early hematopoietic progenitors and associated stromal cells in primary blood-forming organs.
Homing of hematopoietic stem cells to the bone marrow (BM) involves sequential interaction with adhesion molecules expressed on BM endothelium (BMEC) and chemokine stromal derived factor-1 (SDF-1). However, the mechanism whereby adhesion molecules regulate the SDF-1–induced transendothelial migration process is not known. E-selectin is an endothelial-specific selectin that is constitutively expressed by the BMEC in vivo. Hence, we hypothesized that E-selectin may mediate SDF-1–induced transendothelial migration of CD34+ cells. We show that CD34+ cells express both E-selectin ligand and fucosyltransferase-VII (FucT-VII). Soluble E-selectin–IgG chimera binds avidly to 75% ± 10% of CD34+ cells composed mostly of progenitors and cells with long-term culture-initiating cell (LTC-IC) potential. To assess the functional capacity of E-selectin to mediate CD34+ cell migration in a transendothelial migration system, CD34+ cells were placed on transwell plates coated with interleukin-1β–activated BMEC. In the absence of SDF-1, there was spontaneous migration of 7.0% ± 1.4% of CD34+ cells and 14.1% ± 2.2% of LTC-IC. SDF-1 induced migration of an additional 23.0% ± 4.4% of CD34+cells and 17.6% ± 3.6% of LTC-IC. Blocking MoAb to E-selectin inhibited SDF-1–induced migration of CD34+ cells by 42.0% ± 2.5% and LTC-IC by 90.9% ± 16.6%. To define the mechanism of constitutive expression of E-selectin by the BMEC in vivo, we have found that vascular endothelial growth factor (VEGF165) induces E-selectin expression by cultured endothelial cells. VEGF-stimulated endothelial cells support transendothelial migration of CD34+ cells that could be blocked by MoAb to E-selectin. These results suggest that trafficking of subsets of CD34+ cells with LTC-IC potential is determined in part by sequential interactions with E-selectin and SDF-1.
The adhesive mechanisms leading to the mobilization of hematopoietic progenitor cells (HPCs) from the bone marrow into the blood are poorly understood. We report on a role for selectins and fucoidan in progenitor mobilization. Baseline levels of circulating HPCs are increased in endothelial selectin-deficient (P/E−/−) mice. Similar levels are observed when E-selectin null (E−/−) mice are treated with anti-P-selectin antibody or with fucoidan (which inhibits P- and L-selectin function). In particular, administration of 2 doses of fucoidan (25 mg/kg) over 6 hours produces profound mobilization of progenitors in wild-type mice and the response is greatly enhanced in E−/− and P/E−/− mice. Competitive reconstitution experiments reveal that fucoidan also elicits long-term (more than 6 months) repopulating stem cells. Mobilization assays using chimeric mice harboring L-selectin–deficient progenitors and wild-type progenitors expressing the green fluorescence protein suggest that L-selectin expression is not required but confers an advantage for fucoidan-induced mobilization. Sulfation is critical as desulfated fucoidan is ineffective. In addition, sulphogalactosylceramide (sulfatide) but not heparin can induce HPC mobilization. Our results indicate that administration of sulfated glycans, especially with concurrent inhibition of E-selectin function, represents a powerful novel method for rapid mobilization of long-term–repopulating stem cells. These findings may help elucidate the mechanisms of HPC trafficking during development and adult life.
To study the role of bone marrow endothelial cells (BMEC) in the regulation of hematopoietic cell trafficking, we have designed an in vitro model of transendothelial migration of hematopoietic progenitor cells and their progeny. For these studies, we have taken advantage of a human BMEC-derived cell line (BMEC-1), which proliferates independent of growth factors, is contact inhibited, and expresses adhesion molecules similar to BMEC in vivo. BMEC-1 monolayers were grown to confluency on 3 μm microporous membrane inserts and placed in 6-well tissue culture plates. Granulocyte-colony stimulating factor (G-CSF )–mobilized peripheral blood CD34+ cells were added to the BMEC-1 monolayer in the upper chamber of the 6-well plate. After 24 hours of coincubation, the majority of CD34+ cells remained nonadherent in the upper chamber, while 1.6 ± 0.3% of the progenitor cells had transmigrated. Transmigrated CD34 cells expressed a higher level of CD38 compared with nonmigrating CD34+ cells and may therefore represent predominantly committed progenitor cells. Accordingly, the total plating efficiency of the transmigrated CD34+ cells for lineage-committed progenitors was higher (14.0 ± 0.1 v 7.8% ± 1.5%). In particular, the plating efficiency of transmigrated cells for erythroid progenitors was 27-fold greater compared with nonmigrating cells (8.0% ± 0.8% v 0.3% ± 0.1%) and 5.5-fold compared with unprocessed CD34+ cells (2.2% ± 0.4%). While no difference in the expression of the β1-integrin very late activation antigen (VLA)-4 and β2-integrin lymphocyte function-associated antigen (LFA)-1 was found, L-selectin expression on transmigrated CD34+ cells was lost, suggesting that shedding had occurred during migration. The number of transmigrated cells was reduced by blocking antibodies to LFA-1, while L-selectin and VLA-4 antibodies had no inhibitory effect. Continuous coculture of the remaining CD34+ cells in the upper chamber of the transwell inserts resulted in proliferation and differentiation into myeloid and megakaryocytic cells. While the majority of cells in the upper chamber comprised proliferating myeloid precursors such as promyelocytes and myelocytes, only mature monocytes and granulocytes were detected in the lower chamber. In conclusion, BMEC-1 cells support transmigration of hematopoietic progenitors and mature hematopoietic cells. Therefore, this model may be used to study mechanisms involved in mobilization and homing of CD34+ cells during peripheral blood progenitor cell transplantation and trafficking of mature hematopoietic cells.
To elucidate the mechanisms by which hematopoietic progenitor cells transmigrate via the bone marrow (BM) endothelial cells, we first established endothelial cell lines from BM and lung, and BM fibroblast cell lines; then we established an in vitro model of transendothelial migration of hematopoietic progenitor cells in the presence of chemoattractants secreted by BM fibroblast cells. The BM endothelial cells expressed vascular cell adhesion molecule-1 (VCAM-1), but the lung endothelial cells did not. The BM fibroblast cells secreted chemoattractants including stroma cell–derived factor (SDF)-1, which could attract hematopoietic progenitor cells to BM and activate the adhesion molecules expressed on hematopoietic progenitor cells after rolling along the endothelial cells. Anti–SDF-1 antibody inhibited the transendothelial migration of a hematopoietic progenitor cell line, FDCP-2. FDCP-2 that expressed very late activation antigen-4 (VLA-4) and normal progenitor cells transmigrated through BM endothelial cells but not lung endothelial cells, even if in the presence of chemoattractants produced by BM fibroblasts. Both anti–VLA-4 and anti–VCAM-1 antibodies inhibited the transendothelial migration of FDCP-2 cells and normal hematopoietic progenitor cells. These findings suggest that the transendothelial migration of hematopoietic progenitor cells is characteristic of BM endothelial cells, and that VLA-4/VCAM-1 and SDF-1 play important roles in the transendothelial migration and, consequently, homing of hematopoietic progenitor cells to BM.
Bone marrow (BM) transplantation still must overcome multiple difficulties and should benefit from better understanding of stem-cell homing and mobilization. Here, we analyzed the involvement of several adhesion molecules in the two processes by treating mice with monoclonal antibodies against these molecules. Treatment of lethally irradiated mice grafted with isogeneic BM cells showed that at least two migration pathways are important for stem-cell homing to the BM, whereas only one of them is involved in lodging of colony-forming unit–spleen (CFU-S) in the spleen. We confirm that the VLA-4/VCAM-1 adhesion pathway is important for stem-cell homing to the BM only and show that CD44 is involved in CFU-S lodging in both BM and spleen. These results show that entry of CFU-S into the spleen is regulated. The observation that when one migration pathway is altered, CFU-S do not enter the BM via the other pathway may indicate that the two mechanisms involved in CFU-S homing into the BM are linked. The adhesion molecules VLA-4 and CD44 are also implied in the mobilization of stem cells into the blood stream of mice injected once with anti–VLA-4 or anti-CD44. Anti–VLA-4 administration led to a significant increase in circulating stem cells as early as 8 hours after treatment. Stem cells mobilized by anti–VLA-4 comprise cells with high self-renewal potential and thus may be used for long-term reconstitution of the hematopoietic tissue.
© 1998 by The American Society of Hematology.
Previously, we have shown that interleukin (IL)-8 induces the rapid (15 to 30 minutes) mobilization of hematopoietic progenitor cells (HPC) in mice. Because integrins are essential for adhesion and transendothelial migration of HPC, we studied the involvement of the β2-integrin leukocyte function-associated antigen-1 (LFA-1) in IL-8–induced mobilization. After a single injection of blocking anti–LFA-1 antibodies, no mobilization of colony-forming cells was observed. In addition, when mice were pretreated with anti–LFA-1 or saline and subsequently injected with 30 μg of IL-8, mobilization of HPC was completely blocked. We showed that this was not due to anti–LFA-1 antibodies affecting colony formation, as addition of anti–LFA-1 antibodies to colony cultures in semisolid medium had no inhibitory activity. Also, anti-intercellular adhesion molecule (ICAM)-1 antibodies, directed to the main ligand of LFA-1 significantly inhibited the IL-8–induced mobilization. Furthermore, IL-1–induced mobilization was significantly inhibited by anti–LFA-1 antibodies. Because LFA-1 is reported to be expressed on more differentiated HPC, it was considered that the IL-8–induced mobilization of more primitive HPC would not be blocked by anti–LFA-1 antibodies. Transplantation of blood-derived mononuclear cells (MNC) from IL-8–mobilized animals pretreated with anti–LFA-1 antibodies protected only 25% of lethally irradiated recipient mice, whereas the radioprotection rate of control mice transplanted with MNC derived from IL-8-mobilized animals was 86% (P < .01). Anti-LFA–1 antibodies did not interfere with stem cell homing, as transplantation of IL-8-mobilized blood MNC, incubated in vitro with these antibodies resulted in 100% radioprotection. We conclude that anti–LFA-1 antibodies completely prevent the rapid mobilization of colony-forming cells and of cells with radioprotective capacity induced by IL-8. These results indicate a major role for the β2-integrin LFA-1 in the IL-8–induced mobilization of hematopoietic stem cells.
Bone marrow (BM) transplantation still must overcome multiple difficulties and should benefit from better understanding of stem-cell homing and mobilization. Here, we analyzed the involvement of several adhesion molecules in the two processes by treating mice with monoclonal antibodies against these molecules. Treatment of lethally irradiated mice grafted with isogeneic BM cells showed that at least two migration pathways are important for stem-cell homing to the BM, whereas only one of them is involved in lodging of colony-forming unit–spleen (CFU-S) in the spleen. We confirm that the VLA-4/VCAM-1 adhesion pathway is important for stem-cell homing to the BM only and show that CD44 is involved in CFU-S lodging in both BM and spleen. These results show that entry of CFU-S into the spleen is regulated. The observation that when one migration pathway is altered, CFU-S do not enter the BM via the other pathway may indicate that the two mechanisms involved in CFU-S homing into the BM are linked. The adhesion molecules VLA-4 and CD44 are also implied in the mobilization of stem cells into the blood stream of mice injected once with anti–VLA-4 or anti-CD44. Anti–VLA-4 administration led to a significant increase in circulating stem cells as early as 8 hours after treatment. Stem cells mobilized by anti–VLA-4 comprise cells with high self-renewal potential and thus may be used for long-term reconstitution of the hematopoietic tissue.
© 1998 by The American Society of Hematology.
The timely mobilization of hematopoietic stem and progenitor cells (HSPCs) is essential for maintaining hematopoietic and tissue leukocyte homeostasis. Understanding how HSPCs migrate between bone marrow (BM) and peripheral tissues is of great significance in the clinical setting, where therapeutic strategies for modulating their migration capacity determine the clinical outcome. Here, we identify an epigenetic regulator, Phc2, as a critical modulator of HSPC trafficking. The genetic ablation of Phc2 in mice causes a severe defect in HSPC mobilization through the derepression of Vcam1 in bone marrow stromal cells (BMSCs), ultimately leading to a systemic immunodeficiency. Moreover, the pharmacological inhibition of VCAM-1 in Phc2-deficient mice reverses the symptoms. We further determine that Phc2-dependent Vcam1 repression in BMSCs is mediated by the epigenetic regulation of H3K27me3 and H2AK119ub. Together, our data demonstrate a cell-extrinsic role for Phc2 in controlling the mobilization of HSPCs by finely tuning their bone marrow niche.
Mesenchymal stem cells (MSC) comprise a heterogeneous population of rapidly proliferating cells that can be isolated from adult (e.g., bone marrow, adipose tissue) as well as fetal (e.g., umbilical cord) tissues (termed bone marrow (BM)-, adipose tissue (AT)-, and umbilical cord (UC)-MSC, respectively) and are capable of differentiation into a wide range of non-hematopoietic cell types. An additional, unique attribute of MSC is their ability to home to tumor sites and to interact with the local supportive microenvironment which rapidly conceptualized into MSC-based experimental cancer cytotherapy at the turn of the century. Towards this purpose, both naïve (unmodified) and genetically modified MSC (GM-MSC; used as delivery vehicles for the controlled expression and release of antitumorigenic molecules) have been employed using well-established in vitro and in vivo cancer models, albeit with variable success. The first approach is hampered by contradictory findings regarding the effects of naïve MSC of different origins on tumor growth and metastasis, largely attributed to inherent biological heterogeneity of MSC as well as experimental discrepancies. In the second case, although the anti-cancer effect of GM-MSC is markedly improved over that of naïve cells, it is yet apparent that some protocols are more efficient against some types of cancer than others. Regardless, in order to maximize therapeutic consistency and efficacy, a deeper understanding of the complex interaction between MSC and the tumor microenvironment is required, as well as examination of the role of key experimental parameters in shaping the final cytotherapy outcome. This systematic review represents, to the best of our knowledge, the first thorough evaluation of the impact of experimental anti-cancer therapies based on MSC of human origin (with special focus on human BM-/AT-/UC-MSC). Importantly, we dissect the commonalities and differences as well as address the shortcomings of work accumulated over the last two decades and discuss how this information can serve as a guide map for optimal experimental design implementation ultimately aiding the effective transition into clinical trials.
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Hematopoiesis is the process by which cells that are undifferentiated and uncommitted proliferate, differentiate, and mature resulting in the production of terminally differentiated blood cells that serve a wide variety of functions, including hemostasis, oxygen delivery to tissues, and defense against a multitude of foreign antigens and microorganisms. Bone marrow is the principal site of hematopoiesis and must maintain homeostatic production of blood cells as well as be rapidly and specifically responsive to changing demands. Cytologic and histologic preparations of bone marrow reveal the complexity of this tissue, exhibiting hematopoietic cells of multiple lineages in varied stages of maturation, stromal cells, and vasculature all encased by bone. In the conduct of nonclinical studies that evaluate the safety of drug candidates, it is imperative to adequately assess potential treatment-related effects on hematopoiesis through assessment of both peripheral blood and bone marrow evaluation.
The task of protecting the body from a neverending barrage of infectious agents and pathogens is a daunting one indeed. Bacteria, viruses, parasites and foreign substances can penetrate protective barriers such as the skin and mucous membranes, and are thus capable of entering the body virtually anywhere. Lymphocytes, which form the backbone of the adaptive immune response, confront this challenge with both singularity of purpose and boundless tenacity.
Several types of myeloproliferative syndrome (MPS) can involve the hepatobiliary tract, with a wide array of pathologic conditions. In MPSs with infiltration of liver parenchyma, hepatomegaly is a general feature and may be massive, often in conjunction with marked splenomegaly. Primary myelofibrosis (agnogenic myeloid metaplasia) is a clonal myeloproliferative neoplasm characterized by a growth of predominantly granulocyte precursors and megakaryocytes. Involvement of the liver shows accumulation of these cells mainly within sinusoids. This intrahepatic proliferation is frequently associated with liver fibrosis, mainly perisinusoidal fibrosis. The neoplastic cells can also form myeloid tumors or granulocytic sarcomas in the liver. Chronic myelogenous leukemia can also cause proliferation of immature myeloid cells in the liver, with massive hepatomegaly, together with signs of extramedullary hepatic hematopoiesis. In chronic neutrophilic leukemia, maturing and mature neutrophils can accumulate in the liver. In essential thrombocythemia, a myeloproliferative neoplasm involves primarily the megakaryocyte lineage; abnormal megakaryocytes accumulate in hepatic sinusoids. In this MPS, thrombotic complications of liver veins are a well-known phenomenon.
Hematopoietic stem and progenitor cells (HSPC) express at their surface an array of cell adhesion receptors that enables them to interact with extracellular matrix components and transmembrane cell adhesion molecules expressed within stem cell niches in the bone marrow. Herein, we review the different cell adhesive interactions taking place within the bone marrow between HSPC and their niches (via integrins, CD44, selectin ligands, and Eph receptors) and how they support HSPC anchoring in the bone marrow and regulate HSPC fate during hematopoiesis. We review how malignant hematopoietic cells utilize adhesive interactions to resist chemotherapy treatments. Finally, we review how these adhesive interactions can be therapeutically targeted to increase HSPC mobilization for transplantation and to chemosensitize malignant hematopoietic cells to chemotherapy.
The bone marrow and thymus are remarkable factories for blood cells. Although impressively large numbers of cells of various types are produced, the output is carefully controlled. Lymphocytes that emerge from these organs are essential to life, but some are potentially capable of inducing autoimmune disease or malignancy. For that reason, intricate mechanisms have evolved for checking the maturing cells for quality and functional capability. Blood cells of most types can be made in other organs, as is particularly obvious during embryonic life, or when the marrow is ablated. However, this is not the case during normal adult circumstances, and it has long been a goal to determine what is special about central lymphoid tissues. Extraordinary progress has been made in discovering cytokines and cell interaction molecules produced in those sites, and we are beginning to understand how each delivers positive and negative signals for lymphocyte formation. However, additional interesting molecules are still being found, and new roles are being ascribed to previously known ones. The focus of this chapter will be on several classes of molecules expressed within the bone marrow environment. Their study may reveal mechanisms that control the movement of lymphocyte precursors within and from bone marrow, the rate of lymphocyte production, and how defective or potentially harmful cells are eliminated.
Aufgrund der kurzen Lebensspanne vieler lymphatischer und myeloischer Zellen muss die Hämatopoese eine kontinuierlich hohe Produktion unterschiedlich differenzierter Blutzellen und gewebeständiger Zellen sicherstellen (1010 Erythrozyten/h, bzw. 4×l08 Granulozyten/h). Zusätzliche Anforderungen an die Hämatopoese sind eine exakte Regulation, um enge physiologische Grenzbereiche nicht zu über- oder unterschreiten, und eine rasche Anpassung an Situationen mit erhöhtem Zellverbrauch.
Inflammatory responses often involve the selective accumulation in tissues of complex mixtures of leukocytes. In order to understand the processes governing migration and accumulation of mature leukocytes, it is useful to begin by considering the development of leukocytes in adult bone marrow, as well as the earliest migrations made by hematopoietic cells during prenatal development.
To improve treatment of acute lymphoblastic leukaemia (ALL), a better understanding of disease development is needed to tailor new therapies. Connective tissue growth factor (CTGF/CCN2) is highly expressed in leukaemia cells from the majority of paediatric patients with B-lineage ALL (pre-B ALL). CTGF is a matricellular protein and plays a role in aggressive cancers. Here we have genetically engineered leukaemia cells to modulate CTGF expression levels. Elevated CTGF levels accelerated disease dissemination and reduced survival in NOD/SCID mice. In vitro studies showed that CTGF protein induces stromal cell proliferation, promotes adhesion of leukaemia cells to stromal cells and leads to overexpression of genes associated with cell cycle and synthesis of extracellular matrix (ECM). Corresponding data from our leukaemia xenograft models demonstrated that CTGF leads to increased proliferation of non-leukaemia cells and deposition of ECM in the bone marrow. We document for the first time a functional role of CTGF in altering disease progression in a lymphoid malignancy. The findings provide support for targeting the bone marrow microenvironment in aggressive forms of leukaemia.Oncogene advance online publication, 25 January 2016; doi:10.1038/onc.2015.525.
The discovery of vascular cell adhesion molecule-1 (VCAM-1) depended largely on the development of an efficient and reproducible technique for culturing human umbilical vein endothelial cells and on the demonstration that treatment with inflammatory cytokines, interleukin-1 (IL-1), and tumor necrosis factor (TNF) α activates endothelium (Pober & Cotran, 1990). Activated endothelium becomes hyperadhesive for leukocytes through a process dependent on protein synthesis. VCAM-1 was identified in activated endothelium through the use of monoclonal antibodies (Rice & Bevilacqua, 1989, Carlos et al., 1990) and expression cloning (Osborn et al., 1989). The former studies involved immunizing mice with activated endothelium and screening for monoclonal antibodies that recognized cytokine-inducible epitopes. The monoclonal antibodies were then used to identify a unique protein by immunoprecipitation and leukocyte adhesion function with adhesion-blocking assays. Expression cloning was done with a subtracted cytokine-activated human umbilical vein endothelial cell library packaged in a eukaryotic expression vector that was expressed in transiently transfected COS cells. Complementary DNA (cDNA) was extracted for reamplification and rescreening from cells exhibiting increased leukocyte adhesion (Osborn et al., 1989).
In order to function as effector cells of the innate or specific immune system, leukocytes must leave the intravascular compartment. In the course of this process, circulating cells are exposed to significant hydrodynamic forces exerted by rapidly moving blood components.1 Consequently, an intricate system of specialized surface molecules has evolved on leukocytes and endothelial cells (EC) that confers the mechanical stability necessary for interactions between blood cells and the vessel wall in the presence of flow. These receptors include a group of endothelial immunoglobulin superfamily (IgSF) members, several leukocyte integrins that function as IgSF counter-receptors, the three selectins and their sialomucin ligands, and a number of other molecules on leukocytes, platelets, EC, and within the extracellular matrix (reviewed in refs. 2 –5).
The development of bone metastasis is an organized multi-step process in which some organs are preferential targets. In patients with advanced cancer, bone is the third most common site for metastases after lung and liver. Some cancers are particularly osteophilic, such as cancers of the breast and prostate where bone metastases occur in 60 to 70% of patients. Bone metastases occurrence can be explained by the expression of different malignant factors such as the chemokine receptors CXCR4 or CX3CR1, or the integrin αVβ3 promoting cancer cell homing to bone. Another critical parameter of tumor cell development in bone is their ability to survive and grow in the bone microenvironment. Cancer cells (the seeds) arriving in the bone marrow cavity will not grow if they are not properly responsive or adapted to the bone environment (the soil). Bone is a unique and rich environment whose integrity relies on the balance between osteoclastic bone resorption and osteoblastic bone formation to renew and adapt. The arrival of tumor cells and their interactions with bone cells disrupts normal bone remodeling causing abnormal new bone formation or bone destruction that characterizes osteoblastic and osteolytic metastases, respectively. Osteoblastic metastases are common with advanced prostate cancer while breast cancer bone metastases are typically osteolytic. However, many bone metastases are mixed and contain both osteoblastic and osteolytic characteristics. These skeletal complications have significant clinical consequences (hypercalcemia, intractable pain, fractures, nerve compression syndromes) that reduce the quality of life of these patients. More importantly, unbalanced bone remodeling provides metastatic tumor cells the means to grow and survive in bone.
Hematopoiesis occurs in a specific microenvironment formed by the stroma of hematopoietic organs characterized by variable cellular composition. The combined action of stromal elements controls the proliferation and differentiation of hematopoietic cells by means of direct cell-cell interactions and the production of humoral factors. The role of different cell populations of transient and definitive hematopoietic organs in the maintenance of hematopoiesis, as well as the stromal mechanisms of the regulation of this process, is considered in the present review. Particular attention is paid to the contribution of mesenchymal stromal cells, which are capable both of differentiation into more specialized stromal components and direct regulation of the functioning of hematopoietic cells, to the formation of hematopoietic microenvironment.
Postnatal bone marrow tissue is known to contain multipotential bone marrow mesenchymal stem cells (MSCs) with the capacity to develop into various mature marrow stromal elements and associated skeletal tissues. This chapter reviews the properties of clonogenic stromal cell types within postnatal bone marrow tissue and addresses a poorly understood aspect of their cell biology: namely, the developmental relationships between the different bone marrow stromal cell lineages. In particular, evidence for the existence of a multipotential bone marrow MSC is discussed, including an examination of the cellular and molecular characteristics of MSC and potential clinical applications.
Polymers have been utilized to deliver the drug to targeted site in controlled manner, achieving
the high-therapeutic efficacy. Polymeric drug conjugates having variable ligands as attachments
have been proved to be biodegradable, stimuli sensitive and targeted systems.
Numerous polymeric drug conjugates having linkers degraded by acidity or intracellular
enzymes or sensitive to over expressed groups of diseased organ/tissue have been synthesized
during last decade to develop targeted delivery systems. Most of these organs have number of
receptors attached with different cells such as Kupffer cells of liver have mannose-binding
receptors while hepatocytes have asialoglycoprotein receptors on their surface which mainly
bind with the galactose derivatives. Such ligands can be used for achieving high targeting and
intracellular delivery of the drug. This review presents detailed aspects of receptors found in
different cells of specific organ and ligands with binding efficiency to these specific receptors.
This review highlights the need of further studies on organ-specific polymer–drug conjugates
by providing detailed account of polymeric conjugates synthesized till date having organspecific
targeting.
Human umbilical cord blood, an alternate source of haematopoietic stem cells (HSC),
has been successfully used to reconstitute haematopoiesis in both related and unrelated
transplant recipients. However, because CB has fewer total cells (and as a consequence
fewer HSC and progenitor cells) CB transplant recipients often experience delayed
engraftment as compared with that seen in bone marrow or mobilized peripheral blood
transplant recipients. Delayed engraftment exposes patients to an increased risk of
infection and bleeding. Cytokine-mediated expansion has been investigated to improve
engraftment after CB HSC transplantation as a means to expand the total cell number
and both the HSC and progenitors populations. However, its effect on HSC function
remains controversial. We hypothesise that if cytokine-mediated expansion promotes
divisional recruitment and multilineage differentiation it causes changes in phenotype
and cell cycle related gene expression which may be detrimental to the engraftment
capacity of haematopoietic cells. Therefore we investigated the relationship between
cell division, phenotype and engraftment potential of CB CD34+ cells following
cytokine-mediated expansion. High resolution cell division tracking using the
fluorescent dye CFSE was used to monitor changes as a consequence of cytokine mediated
expansion in phenotype and function in CB CD34+ cells. Cytokine-mediated
expansion caused upregulation of lineage and proliferation markers and adhesion
molecules and downregulation of putative stem cell markers with concomitant cell
division. However, these changes in phenotype as a consequence of cytokine-mediated
expansion may not reflect or be predictive of a functional change in the expanded
population. Cytokine-mediated expansion of CB CD34+ also caused changes in cell cycle related gene expression of G1 phase regulators. CB CD34+ cells exhibited
expression of all D cyclins, albeit at different levels and p21WAF1 was differentially
expressed across CB samples. The effect of cell division on the engraftment potential as
a consequence of cytokine-mediated expansion was examined in CB CD34+. Cytokine mediated
expansion of CB CD34+ cells reduced, but did not completely eliminate
engraftment potential, as a proportion of the expanded and divided cell populations
retained their ability to engraft the NOD-SCID mouse. Overall, this study confirms
reports in the literature that cytokine-mediated expansion induces changes in the
phenotype of HSC and compromises their in vivo function.
Human bone marrow (BM) is a relevant site for immunoglobulin (Ig) generation in vivo. The occurrence of BM cells capable of spontaneous and high rate Ig secretion for 14 d in vitro has been described previously. Accordingly, these cells provide a suitable model for studying terminal B cell maturation within the BM. We have reported recently that these BM cells are not totally differentiated when isolated from the body, as they require inductive signals from adherent stromal BM cells to complete their maturation. Interleukin (IL)-6 produced by these adherent BM cells was identified as one such signal. The present work shows that IL-6 was necessary, but not sufficient, for the induction of BM Ig-secreting cells, since the cytokine was unable to restore missing IgG in nonadherent BM cell cultures. Supernatants (SN) obtained from cultures of stromal adherent BM cells, either freshly isolated or derived from long-term BM culture (LTBMC), restored Ig secretion by nonadherent BM cells, suggesting that additional soluble factors from BM stromal cells were required. Fibronectin (FN) was identified as that factor, as can be deduced from the following findings: (a) stromal, but not nonadherent, BM cells constitutively produced FN; (b) anti-FN antibodies markedly reduced the IgG secretion in cultures of BM mononuclear cells (BMMC), and blocked the inductive effect of stromal cell SN on nonadherent BM cells, and such a blockade could be reversed by exogenous FN; and (c) finally, although neither IL-6 nor FN alone exerted any effect, the combination of both factors induced optimal Ig secretion by nonadherent BM cells. Furthermore, VLA-4 molecules seemed to be the FN receptor that was active in this culture system, as indicated by: (a) BM Ig-secreting cells exhibited the phenotype VLA-4+ VLA-5-; (b) mAbs directed to VLA-4 (anti-CD29 and anti-CD49d), but not those directed to other adhesion molecules, inhibited Ig secretion by BMMC cultures, and this effect was reversed by FN; (c) the inductive role of the entire FN molecule could be replaced by a fragment containing the CS-1 region, but not by a fragment containing the RGDS sequence; and (d) only mAbs anti-CD49d capable of blocking VLA-4-FN interaction inhibited induction by either the FN or the CS-1-containing fragment of FN.(ABSTRACT TRUNCATED AT 400 WORDS)
Mutant Sl/Sld mice exhibit decreased marrow hematopoiesis. The defect is known to reside in the marrow microenvironment of these animals, which is reproduced in vitro by primary marrow explants as well as by cloned marrow stromal cell lines. Bone marrow progenitor cells are incapable of adhering to primary Sl/Sld stromal cells or cloned stromal cell lines derived from them to form cobblestone-islands and proliferate. The role of hemonectin, a marrow-specific adhesion protein in the defective hematopoiesis of the Sl/Sld mice, was studied. Indirect immunoperoxidase staining of marrow in situ from Sl/Sld mice showed little specific staining while specific staining was seen in a pericellular distribution in marrow from +/+ mice. Hemonectin expression in several cloned stromal cell lines from Sl/Sld mice was compared by immunoblotting with that in cloned stromal cell lines from normal +/+ littermates. Cell line Sld3, which has the least hematopoiesis supportive capacity in vitro, showed no detectable hemonectin by immunoblotting, while Sld1 and Sld2 showed detectable but greatly reduced amounts compared with normal +/+ 2.4, GBI/6, and D2XRII. Confluent cultures incubated with purified hemonectin and engrafted with enriched progenitors showed a significant increase in the cumulative number of cobbleston-islands and day 14 spleen colony-forming units (CFU-s) forming progenitors (39.15 +/- 3.6/dish; 16.3 +/- 3.1/dish, respectively), compared with untreated Sld3 cultures (cobblestone-islands 8.1 +/- 3.6/dish; CFU-s forming progenitors 8.8 +/- 0.05/dish). Hemonectin-mediated progenitor cell binding to the Sld3 stromal cells was specifically inhibited by antihemonectin but not by preimmune serum. These data support the role of hemonectin in early progenitor-stromal cell interactions.
A cell adhesion model was previously used to select a series of monoclonal antibodies (mAbs), which were subsequently found to recognize CD44/Pgp-1. Interest in these reagents increased with the finding that they totally inhibited production of lymphoid or myeloid cells in long-term bone marrow cultures. Further investigation has now revealed that hyaluronate is a potential ligand for CD44 and that hyaluronate recognition accounts for the adhesion between B lineage hybridoma and stromal cells. The hybridoma cells adhered to hyaluronate-coated plastic wells as well as to monolayers of stromal cells. The adhesion in both cases was inhibited by treatment with hyaluronidases, and did not require divalent cations. Addition of exogenous hyaluronate also diminished binding of lymphoid cells to stromal cells. One of several mAbs to Pgp-1/CD44 was particularly effective at blocking these interactions. Since hyaluronate and Pgp-1/CD44 were present on both cell types, experiments were done to determine the cellular location of interacting molecules required for the adhesion process. Treatment of lymphoid cells with an anti-Pgp-1/CD44 antibody was more inhibitory than antibody treatment of the stromal cells. Conversely, hyaluronidase treatment of stromal cells reduced subsequent binding more than treatment of the lymphoid cells. Adhesive interactions that involve hyaluronate and CD44 could contribute to a number of cell recognition processes, including ones required for normal lympho-hemopoiesis.
A new panel of mAbs was prepared to a stromal cell line known to support lymphocytes in Whitlock-Witte type long-term bone marrow cultures. These antibodies were then screened with a cell adhesion assay and four were selected that inhibited the binding of B lineage cells to stromal cell monolayers. Immunofluorescent and biochemical analyses revealed that these new antibodies detected epitopes of the previously described Pgp-1/CD44 antigen complex. Addition of Pgp-1/CD44 antibodies to Dexter-type long-term bone marrow cultures completely prevented emergence of myeloid cells and they also blocked lymphocyte growth in Whitlock-Witte type cultures. mAbs MEL-14, LFA-1, and CD45R did not inhibit under the same conditions and there was no apparent relationship to Ig isotype. Adherent layers in treated cultures were not unusual in terms of morphology and the antibodies did not affect factor-dependent replication of lymphoid or myeloid progenitor cells. Therefore, the mechanism of inhibition may not involve direct toxicity to precursors or microenvironmental elements. Previous studies in humans and mice have implicated Pgp-1/CD44-related glycoproteins in the migration of peripheral lymphoid cells, as well as interactions of cells with the extracellular matrix. These findings suggest that they may also be critical for formation of lymphoid and myeloid cells within bone marrow.
Stromal cells are believed to regulate lympho-hematopoiesis through direct cell-cell interactions and the release of growth factors. Many questions remain, however, about their lineage derivation and functional heterogeneity. We previously prepared a panel of stromal cell lines from murine spleen and bone marrow and characterized them based on their ability to support lymphocyte growth in long-term cultures. These cells are now compared with respect to their expression of various immunoglobulin superfamily and cytokine genes by Northern blot analysis. These results indicate that although stromal cells appear to be mesodermal in origin, they are not closely related developmentally to the hematopoietic progenitor cells they support. The potential production of at least six cytokines was demonstrated. All clones constitutively expressed mRNA for macrophage colony stimulating factor, interleukin-6, transforming growth factor beta and neuroleukin. The most potent lymphocyte supporting clones also made interleukin 7 constitutively. Previous findings had suggested that these clones responded to exogenous stimuli and this has now been demonstrated in terms of induced expression of IL-6 and G/M-CSF mRNA. Interleukin 6 mRNA levels were markedly upregulated by exposure of cells to LPS, TNF, IL-1, IL-6, IL-7, and EGF. G/M-CSF mRNA levels were "superinduced" by the combination of LPS and cycloheximide, a protein synthesis inhibitor. These responses are similar to ones documented by investigators working with endothelial cells and fibroblasts. Together, these data suggest that stromal cells are a multifunctional component of the lymphopoietic microenvironment and may be active participants in a complex, cytokine-mediated regulatory network.
Erythroid differentiation of murine erythroleukemia (MEL) cells is far more extensive when the cells are attached to fibronectin-coated dishes than in suspension culture. Cells induced in suspension culture for 4 d become arrested at a late erythroblast stage and do not undergo enucleation. Incubation of cells in suspension beyond 4 d results in lysis. In contrast, cells induced by DMSO on fibronectin-coated dishes for 7 d differentiate into enucleating cells, reticulocytes, and erythrocytes. As determined by quantitative immunoblotting, cells induced in suspension culture accumulate approximately 33% of the amount of the major erythroid membrane protein Band 3 present in erythrocyte, whereas cells induced on fibronectin-coated dishes accumulate 80-100% of the amount present in erythrocytes. Both suspension-induced cells and cells induced on fibronectin-coated dishes accumulate approximately 90% of the amount of spectrin and ankyrin present in erythrocytes. As revealed by immunofluorescence microscopy during enucleation of MEL cells, both Band 3 and ankyrin are sequestered in the cytoplasmic fragment of the emerging reticulocyte. Enucleated and later-stage cells detach from the fibronectin matrix, due to the loss of the surface fibronectin receptor; this mimics the normal release of reticulocytes from the matrix of the bone marrow into the blood. Thus a fibronectin matrix provides a permissive microenvironment within which erythroid precursor cells reside, proliferate, migrate, and express their normal differentiation program.
Haematopoietic progenitor cells proliferate and mature in semisolid media when stimulated by exogenous haematopoietic cell growth factors (HCGFs) such as granulocyte-macrophage colony-stimulating factor (GM-CSF). They also proliferate in association with marrow-derived stromal cells although biologically active amounts of HCGFs cannot be detected in stromal culture supernatants. It is possible that HCGFs are synthesized in small amounts by stromal cells but remain bound to the stromal cells and/or their extracellular matrix (ECM). This interpretation accords with haematopoietic progenitor cell proliferation in close association with stromal layers in long-term cultures. Glycosaminoglycans (GAGs) are found in the ECM produced by stromal cells. They are prime candidates for selectively retaining HCGFs in the stromal layer; they influence embryonic morphogenesis and cyto-differentiation and they may regulate haematopoiesis. We now report that granulocyte-macrophage colony-stimulating activity can be eluted from cultured stromal layers and that exogenous GM-CSF binds to GAGs from bone marrow stromal ECM. Selective compartmentalization of HCGFs in this manner may be an important function of the marrow microenvironment and may be involved in haematopoietic cell regulation.
To examine potential mechanisms by which hematopoiesis may be regulated by endothelial cells within the bone marrow (BM) microenvironment, we have devised a technique for the in vitro study of the interaction of human BM microvascular endothelial cells (BMEC) with hematopoietic cells. Microvessels isolated by collagenase digestion of spicules obtained from filtered BM aspirate were plated on gelatin-coated plastic dishes, and colonies of endothelial cells grown from microvessel explants were further purified by Ulex europaeus lectin affinity separation. BMEC monolayers isolated by this technique grew in typical cobblestone fashion, stained positively with antibody to factor VIII/von Willebrand factor, and incorporated acetylated LDL. Immunohistochemical studies showed that BM microvessels and BMEC monolayers express CD34, PECAM, and thrombospondin. Incubation of resting BMEC with BM mononuclear hematopoietic cells resulted in the selective adhesion of relatively large numbers of CD34+ progenitor cells and megakaryocytes. The binding of purified BM-derived CD34+ progenitor cells to BMEC was dependent on divalent cations and was partially blocked by antibodies to CD34. IL-1 beta treatment of BMEC monolayers resulted in an increase of CD34+ progenitor cell adhesion by mechanisms independent of CD34 or divalent cations. BMEC exhibit specific affinity for CD34+ progenitor cells and megakaryocytes, suggesting that the BM microvasculature may play a role in regulating the trafficking, proliferation, and differentiation of lineage specific hematopoietic elements, and possibly of pluripotent stem cells within the CD34+ population.
Transverse histologic sections of bone marrow obtained from mice that were sacrificed by perfusion fixation at intervals following tritiated thymidine injection were studied by means of radioautography. A kinetic gradient was demonstrated across the marrow section, with the highest proliferative rate in the subendosteal region. Megakaryocytes were shown to originate from the rapidly proliferating subendosteal cells. The immediate proliferating precursors of mature granulocytes were slowly proliferating cells found predominantly in the central region of the marrow. It was concluded that in the steady state there must be a migration of cells from the subendosteal region to the central region with concomitant growth retardation of the migrating cells.
The endothelium of bone marrow sinuses is a continuous layer which is selective in its cellular transport. It is not known how selective and massive seeding of hemopoietic progenitor cells after intravenous transplantation of marrow cells occurs. We postulate that the conditioning irradiation could disrupt the endothelial barrier, thus permitting the "homing" of progenitor cells to occur. To demonstrate this phenomenon, we irradiated mice with doses ranging from 100-2000 cGy-total body and studied perfusion-fixed marrow by transmission electron microscopy. The major finding was sloughing and denudation of plasma membrane, particularly on the luminal side of endothelium. Membrane vesiculation was also frequently seen in this border. Moreover, dilatation of the perinuclear space and rough endoplasmic reticulum was commonplace and testified to instability and fragility of the membrane system. Focal cytoplasmic swelling of endothelium was seen reflecting increased permissiveness of the endothelial barrier. Endocytosis and phagocytosis were increased in the marrow; and the endothelium, normally quiescent with regard to phagocytosis, was now overtly phagocytic. A dipogenecity of the adventitial layer was increased as hemopoietic function of marrow decreased. The end result of membrane alterations in the endothelium was the appearance of discontinuities in these cells, which form the essential element of bone marrow-blood barrier. Consequent to these discontinuities, the permissiveness of the endothelial barrier was enhanced and those cellular elements, such as mature, nonreticulated erythrocytes that are normally confined to the vascular space, now appeared in large number in the hemopoietic compartment. With low doses, these findings were transient and repair set in by 1-2 weeks. With higher doses, total disruption of marrow-blood barrier occurred and the process did not seem to be repairable. We conclude that the conditioning irradiation before bone marrow transplantation is essential in disrupting the endothelial barrier and permitting large-scale entry of transplanted cells into the hemopoietic compartment.
Studies in animal models suggest that the integrin adhesion protein VLA-4 may play an important role in lymphopoiesis. The relationship between cell adhesion and lymphopoiesis in humans has been difficult to study because of the relative rarity and stringent in vitro growth requirements of lymphoid progenitors from normal adult human bone marrow. To determine the functional significance of VLA-4-mediated adhesion in human lymphopoiesis, we developed a culture system in which a bone marrow-derived adherent layer supports the formation of colonies of terminal deoxynucleotidyl transferase (TdT)-positive lymphoid precursor cells from normal adult human bone marrow. Limiting dilution studies were consistent with clonal origin of these colonies. CFU-TdT were enriched in the CD34+ bone marrow fraction, consistent with CD34 expression by other hematopoietic progenitors. CD34 expression and lack of lineage-specific markers in a significant proportion of the TdT+ colony cells suggest that the TdT+ CFU may represent an uncommitted lymphoid progenitor cell. Development of TdT+ colonies required direct contact with the adherent layer and was significantly inhibited by specific anti-VLA-4 alpha chain antibody, suggesting a functional role for the previously reported VLA-4-dependent adhesion of human B cell precursors to bone marrow-derived fibroblasts.
Expression of CD2 on developing and mature murine B cells was examined by using an antipeptide antiserum (L50). Most Ig-bearing splenic B cells were found to express CD2. Anti-CD5 and anti-B220 mAb divided the peritoneal B cells into two populations expressing high and low levels of these proteins; both populations were found to express uniform levels of CD2. Abelson murine leukemia virus-transformed pre-B cell lines derived from fetal liver and adult bone marrow were analyzed to delineate the ontogeny of CD2 in the B cell lineage. The results show that onset of CD2 expression correlates with the presence of cytoplasmic mu-chain. Therefore, the earliest CD2+ pre-B cell in the developing B cell population appears to be the classical pre-B cell.
The action of human rIL-1 beta on confluent, quiescent monolayers of human umbilical vein endothelial cells (HUVEC) has been studied for the induction of new membrane proteins. Two approaches have been taken. The first is a quantitative two-dimensional gel analysis of [35S]cysteine-labeled membrane proteins of HUVEC with and without cytokine treatment. This analysis indicates that there are a restricted number of new membrane proteins synthesized in the first 6 h of IL-1 treatment, on the order of 19 out of a total of over 600 detectable proteins. Second, we have prepared two mAb (1E7 and 2G7) to different epitopes of a major inducible sialoglycoprotein with molecular mass of 114 kDa and an isoelectric point of 4.6 to 4.8. These antibodies were compared with two additional antibodies, 3B7 and 7A9, which were shown to react with the endothelial leukocyte adhesion molecule-1 (ELAM-1) protein as expressed in COS cells. The 1E7/2G7 protein is distinct from ELAM-1, based upon biochemical comparisons as well as the inability of the 1E7 and 2G7 antibodies to react with ELAM-1-transfected COS cells. The protein defined as 1E7/2G7 is neither expressed constitutively nor in an inducible manner on PBMC, granulocytes, platelets, fibroblasts, or keratinocytes. The 7A9 and 3B7 antibodies are shown to block granulocyte binding to IL-1-activated HUVEC. The 2G7 antibody is effective at inhibiting the binding of T cells but not granulocytes to IL-1-activated endothelium, suggesting this new protein is an adhesion protein that may be active in vivo in T cell-endothelial cell adhesion-related events such as inflammation or lymphocyte recirculation. In addition, T cells were shown to utilize the ELAM-1 protein in binding to cytokine-activated HUVEC. Antibodies directed to both proteins had additive effects on inhibition of T cell adhesion.
The expression and function of a new cytokine-induced endothelial cell adhesion protein, vascular cell adhesion molecule-1 (VCAM-1), was characterized in vitro by using a monoclonal antibody, MoAb 4B9, which recognizes a functional epitope on this protein. As determined by enzyme-linked immunosorbent assay and radioimmunoprecipitation of metabolically labeled cells, VCAM-1 was minimally expressed on unstimulated human umbilical vein endothelium (HUVE), but was rapidly induced by recombinant human tumor necrosis factor-alpha (rhTNF-alpha), rh interleukin-1, and lipopolysaccharide. In contrast to intercellular adhesion molecule-1, VCAM-1 was not induced on dermal fibroblasts or arterial smooth muscle cells after stimulation with rhTNF, or on keratinocytes after stimulation with rh interferon-gamma. MoAb 4B9 significantly inhibited the adherence of peripheral blood lymphocytes (PBL) and lymphocytic cell lines, but not neutrophils, to rhTNF-activated HUVE. The inhibitory effect of MoAb 4B9 on PBL adherence to HUVE was additive to that produced by the CD18 MoAb 60.3. These results show that VCAM-1 mediates a CD18-independent pathway of peripheral blood lymphocyte adherence to cytokine-stimulated HUVE. We propose that lymphocyte binding to VCAM-1, induced on endothelium by cytokines, may be an important component of lymphocyte emigration at sites of inflammation or immune reaction.
Marrow stromal elements produce as yet uncharacterized soluble growth factors that can stimulate the proliferation of murine pre-B cells, although close contact between these two cell types appears to ensure a better pre-B cell response. We have now shown that freshly isolated normal pre-B cells (ie, the B220+, surface mu- fraction of adult mouse bone marrow) adhere to fibronectin (FN) via an RGD cell-attachment site, as shown in a serum-free adherence assay, and they lose this functional ability on differentiation in vivo into B cells (ie, the B220+, surface mu+ fraction). Similarly, cells from an immortalized but stromal cell-dependent and nontumorigenic murine pre-B cell line originally derived from a Whitlock-Witte culture were also found to adhere to fibronectin (FN) via an RGD cell-attachment site. Moreover, in the presence of anti-FN receptor antibodies, the ability of this immortalized pre-B cell line to proliferate when co-cultured with a supportive stromal cell line (M2-10B4 cells) was markedly reduced (down to 30% of control). This suggests that pre-B cell attachment to FN on stromal cells may be an important component of the mechanism by which stromal cells stimulate normal pre-B cell proliferation and one that is no longer operative to control their more differentiated progeny. Two differently transformed pre-B cell lines, both of which are autocrine, stromal-independent, tumorigenic in vivo, and partially or completely differentiation-arrested at a very early stage of pre-B cell development, did not bind to FN. In addition, anti-FN receptor antibodies were much less effective in diminishing the ability of these tumorigenic pre-B cells to respond to M2-10B4 cell stimulation, which could still be demonstrated when the tumorigenic pre-B cells were co-cultured with M2-10B4 cells at a sufficiently low cell density. Analysis of cell surface molecules immunoprecipitated from both the nontumorigenic and tumorigenic pre-B cell lines by an anti-FN receptor antibody showed an increase in very late antigen (VLA) alpha chain(s) in both tumorigenic pre-B cell lines and a decrease in the beta 1 chain in one. Interestingly, all of the pre-B cell lines expressed similar amounts of messenger RNA for the beta 1 chain of the FN receptor. These results suggest that alteration of FN receptor expression on pre-B cells may represent a mechanism contributing to the outgrowth of leukemic pre-B cells with an autocrine phenotype and capable of stromal cell-independent, autonomous growth.
Studies involving normal and genetically defective experimental animals, monoclonal antibodies, inducible cell lines, soluble mediators, and innovations in long-term culture are focused in the chapter. The name “B lymphocyte” denotes the origin of these cells in birds and mammals (bone marrow), and the review in the chapter focuses on events taking place in the latter. Progress in this field is being achieved by rapidly converging technologies and experimental approaches as the same general questions attract molecular, cellular, and developmental biologists. While some multipotential stem cells can be found in the circulation, these may have limited proliferative potential, and it has not been proved that they are capable of becoming B lymphocytes. More likely, a relatively small pool of stem cells is retained within specialized “niches” within bone marrow and maintained by minimal self-renewal. Monoclonal antibodies can be used to identify normal cells that are committed to become B lymphocytes, and this has opened many investigative possibilities. The transformed counterparts of normal B cell precursors can undergo spontaneous or induced changes in culture. Different variations on the culture system permit the long-term propagation of stem cells, B lineage precursors, or intermediate cell types in absolute dependence on adherent stromal cells.
The production of B lymphocytes and myeloid cells occurs in the bone marrow in association with a supporting population of stromal cells. To determine whether these processes are dependent upon the same or different populations of stromal cells, stromal cell lines were generated from the adherent layer of a Dexter type long-term bone marrow culture. These cultures support myeloid cells and their precursors, a B cell precursor, and the adherent layer cells with support B cell differentiation under appropriate conditions. Two of the lines examined, S10 and S17, express class I histocompatibility antigens but not other hemopoietic cell surface determinants such as Thy-1, Lyt-1, Ig, Ia, Mac-1, or BP-1. Both lines could support myelopoiesis under Dexter conditions upon seeding with nylon wool-passed bone marrow. The nylon wool passage depletes stromal cells capable of forming adherent layers in vitro but retains hemopoietic precursors. The number of cells and colony-forming units-granulocytes/macrophages in the nonadherent cell population recovered 3 wk post-seeding had increased 19-fold and 10-fold, respectively, in the reseeded cultures of S10 and S17. After 3 wk of growth in Dexter conditions, the reseeded cultures were transferred to conditions optimal for B cell differentiation described by Whitlock and Witte. After 4 wk of growth, hemopoietic cells were consistently recovered from S17 cultures but not those of S10. A proportion of these cells from S17 cultures expressed the 14.8 antigen and were surface IgM positive. Surviving hemopoietic cells present in cultures of S10 were primarily macrophages. These findings indicate that S17 but not S10 can support both myelopoiesis and B lymphopoiesis and suggest that one stromal cell population has the capacity to form a hemopoietic microenvironment for both lineages.
The aim of this study was to investigate the relative contribution of direct contact with stromal cells v stromal cell-derived soluble mediators to the differentiation of B lymphocytes and cells from other hematopoietic lineages. This was investigated by making a comparison between hemopoietic cells grown in direct contact with stroma to those in diffusion chambers (DCs) placed over purified populations of stroma. The source of stromal cells was adherent layers from myeloid or lymphoid long-term bone marrow cultures that had been treated with mycophenolic acid, an antibiotic that depletes hemopoietic cells from the cultures but retains a functional stroma. The cells seeded into the chambers were fresh marrow cells that had been passed through two consecutive nylon wool columns to deplete cell populations capable of forming an adherent cell layer in vitro. DCs were placed in wells in which the adherent stroma, growing under myeloid or lymphoid conditions, was present. The results indicate that progenitors of granulocytes and macrophages survived and differentiated in DCs under myeloid culture conditions, as the number of cells and absolute number of CFU-GM increased over that present in the reseed population. These levels, however, were markedly less than in parallel cultures in which the cells were seeded directly onto stroma. Hematopoiesis in DCs placed over hemopoietically active stroma was not optimal, suggesting that factors were used by those hemopoietic cells closest to the stroma. A B lymphocyte precursor survived in DCs under myeloid but not lymphoid conditions, and its differentiation into B lymphocytes was dependent on close association with stromal cells; B lymphopoiesis initiated when cells from DCs grown under myeloid conditions were harvested from the chambers and seeded directly onto stroma initiated and maintained under lymphoid bone marrow culture conditions. B lymphopoiesis did not initiate if the DC from the myeloid conditions was left intact and placed directly over a lymphoid stromal cell layer in lymphoid conditions.
This paper reviews ultrastructural studies of the intercellular contacts or junctions between cells of the bone marrow. Studies using tannic acid and glutaraldehyde as a fixative have shown pentalaminar complexes between many types of cells in marrow of mice and chicks. These intercellular contacts occur between adjacent stromal cells, between stromal cells and developing blood cells and, in marrow of mice, between migrating blood cells and cells of the sinusoidal wall. Because of their location and widespread occurrence, it is believed these contacts may represent a type of adherent junction helping to maintain an orderly arrangement of blood cells and stromal cells in the marrow. Migrating blood cells may use these contacts as anchoring sites during locomotion toward the sinusoids and in crossing the sinusoidal wall. On the other hand, since these junctions resemble gap junctions of other tissues, one should not exclude the possibility that they are involved in cellular communication. Freeze-fracture and lanthanum impregnation studies have failed to demonstrate these junctions in marrow. Studies using ruthenium red have shown apparent sites of attachment between cells of the marrow, but it is not known whether these sites correspond to the intercellular contacts seen in tannic acid preparations.
An in vivo cell surface labelling technique using radioautography has been developed to visualise the distribution of IgM-bearing B lymphocytes within the bone marrow. Anaesthetized 3-week-old mice were perfused via the common iliac artery with: (1) serum-containing medium (SCM), (2) 125I-labelled anti-IgM antibody in SCM, (3) SCM, and (4) fixative. In radioautographic sections of femoral marrow labelled surface IgM+ cells were observed either singly or in small clusters throughout the extravascular haemopoietic marrow cords. Binding specificity was demonstrated by the displacement of 125I-anti-IgM labelling by excess anti-IgM and by the binding of perfused 125I-anti-H-2Kk antibody in CBA/J (H-2Kk) mice but not in C57BL/6 (H-2Kb) mice. Quantitative analysis of radioautographic sections revealed an even distribution of labelled cells throughout CBA/J marrow perfused with 125I-anti-H-2Kk, indicating a uniform accessibility of perfused antibody to cells in the haemopoietic cords. This labelling pattern contrasted with that in 125I-anti-IgM perfused animals in which surface IgM+ cells, although widely distributed in the bone marrow, showed areas of concentration, speculatively clones of maturing B lymphocytes. This method of labelling surface IgM and other cell markers in situ provides an approach to study the microenvironment of B lymphocyte genesis in the bone marrow.
The role of mammalian bone marrow in generating surface IgM (sIgM)-bearing B lymphocytes is reviewed. Precursor cells in the marrow give rise to large, rapidly dividing cells bearing free cytoplasmic μ chains (cμ). The progeny of the large cμ⁺ cells form a population of small, nondividing cμ⁺ cells that mature into small lymphocytes, progressively expressing sIgM and other B-cell surface membrane components. Newly formed sIgM⁺ cells soon migrate through the bloodstream to the spleen and other lymphoid tissues, where they may die after a short lifespan or be activated to produce antibody molecules. The large-scale lymphocytopoiesis in the bone marrow thus maintains a population of rapidly renewed virgin B lymphocytes in the peripheral lymphoid tissues. This process continuously creates and selects B cell clones with the wide range of antibody specificites necessary to mediate primary humoral immune responses through postnatal life.
By using a technique that combines metaphase arrest with immunofluorescence labeling, the proliferation of specifically identified pre-B cells in mouse bone marrow has been analyzed under physiological conditions in vivo. Pre-B cells bearing cytoplasmic mu-chains and no surface mu-chains constituted 12% of marrow nucleated cells, or 27 X 10(5) cells/femur, whereas surface mu-bearing B lymphocytes totaled 33 X 10(5) cells/femur. Pre-B cells measured 7 to 14 micron in diameter, the small number seen in metaphase (1 to 2%) being large cells (greater than 10 microns). After vincristine injection, the metaphase incidence (Imet) of pre-B cells increased with cell size; a broad-dose range of vincristine gave similar Imet values. Mitoses were arrested for 4 hr with no apparent cell death. Linear regression analysis of the increase in Imet of pre-B cells 2 to 4 hr after vincristine revealed a rate of entry into mitosis of 6.3%/hr, relative to all pre-B cells (average compartment turnover time, 16 hr), and 15.3%/hr for the large proliferating pre-B cell subset. This represented a pre-B cell production of 1.3 X 10(5) cells/femoral shaft/hr or 0.5 X 10(8) cells/whole bone marrow organ/day, emphasizing the magnitude of B lymphocyte genesis in normal bone marrow. Combined with reported renewal rates for small pre-B cells and small B lymphocytes, these values form a kinetic model of B lymphocyte development. The results reveal an apparent overproduction of large pre-B cells, consistent with a speculative post-mitotic loss of some immature primary B lymphocytes.
Growth of mature B cells and their precursors from mouse bone marrow was maintained in culture for greater than 1 year. Feeder layers of adherent bone marrow cells, established in medium containing fetal calf serum and no exogenous steroids, were used to provide an in vitro environment that supported continuous growth and development of these cells. In such bone marrow cultures, the number of cells bearing membrane immunoglobulin increased gradually for 4 wk and then decreased. Between 10 and 14 wk, some of the cultures gave rise to continuously dividing B-cell populations that contained pre-B cells (producing mu heavy chains only and sensitive to transformation by Abelson murine leukemia virus) as well as mature B cells (synthesizing both light and heavy chains of IgM). Immunoglobulin molecules synthesized by cells in these populations were heterogeneous in two-dimensional gel analysis. This suggests that mature B cells arose via maturation of pre-B cells in the cultures that involved rearrangement and expression of different variable region gene segments.
The full hemopoietic competence in the fetus occurs after the development of the marrow, implying that the environment can be critical for the expression of progenitor cell potential. Ultrastructural studies of marrow have highlighted the close physical association of the cytoplasmatic progress of stromal cells with developing blood cells. The intimacy and prevalence of these relationships fostered the thought that there is chemical information transferred from stromal to hemopoietic cells. The ability of hemopoietic cell proliferation, differentiation and maturation to occur in culture dishes in vitro has raised questions regarding the apparent independence of hemopoiesis from stromal elements. Several explanations are possible for this phenomenon. Firstly, this apparent independence is not true for suspension cultures in which stromal cells are necessary for long term progenitor cell sustenance. Thus, semi-solid media may create conditions that permit short term growth in the absence of an ideal environment. Secondly, macrophages or their secretory product may be capable of substituting for stromal cell and there may be small numbers of stromal cells in the marrow cultures, inadvertently. Most importantly, semi-solid cultures may not be quantitatively representative of the proliferative potential of marrow. It is difficult to put aside the must compelling argument for the importance of the highly specialized nature of the marrow stroma in the support of hemopoiesis. (Szirmai, Stuttgart, GFR)
The homeostatic mechanisms which control B lymphocyte renewal in the bone marrow are unknown. Mouse bone marrow produces many small lymphocytes which develop surface IgM and other B lymphocyte properties. Putative precursors show cytoplasmic mu chains but earlier progenitors have been characterized. Some marrow small lymphocytes are long-lived recirculating B and T cells. [3H]Thymidine and IgM labelling in femoral marrow sections suggest that recirculating lymphocytes migrate mainly through the marrow periphery while indigenous lymphocytes may be formed peripherally and migrate centrally as they mature. Thus, the localization of lymphocytes appears to be non-random. The effects of possible regulatory factors on bone marrow small lymphocytes production have been examined by [3H]thymidine labelling and radioautography. Administration of anti-IgM antibodies in vivo eliminates all B lymphocytes but the marrow lymphocyte production rate remains unchanged. After sublethal X-irradiation the marrow shows an over-shoot B lymphocyte production, while the lymphocyte numbers in shielded marrow remain stable. In neonatally thymectomized or congenitally athymic mice marrow lymphocyte production is unaffected. Studies in germ-free and antigen-stimulated mice reveal a basal level of marrow lymphocyte production, normally stimulated non-specifically by environmental factors. Thus, marrow lymphocyte production appears to be basically independent of feedback control from the peripheral B lymphocyte pool or of specific humoral factors, but fluctuates widely after perturbation or when amplified by exogenous stimuli. These findings suggest the importance of microenvironmental factors, as yet undefined, in the regulation of bone marrow lymphocytes.
In the germinal center (GC), B cells are either selected to become memory cells or are eliminated through the process of programmed cell death. FDC which are intimately associated with the GC B cells are thought to be important in this selection process. Previously, we have shown that the LFA-1 (CD11a/CD18)-ICAM-1 (CD54) and VLA-4 (CD49d)-VCAM-1 (CD106) adhesion pathways are involved in FDC-B cell interaction. In the present study, we have explored whether these adhesive interactions contribute to the process of B cell selection by studying the effects on apoptosis of GC B cells. Using FDC and B cells derived from human tonsils, we found that only B cells adherent to FDC remain viable: disruption of FDC-B-cell clusters with mAb against LFA-1 alpha (CD11a), VLA-4 (CD49d), ICAM-1 (CD54), or VCAM-1 (CD106) results in apoptosis of the B cells. Furthermore, we found that GC B cells, upon adhesion to plastic-coated purified ICAM-1 (CD54) or VCAM-1 (CD106), show diminished apoptosis. Importantly, we observed that, at low concentration, ICAM-1 (CD54) and VCAM-1 (CD106) act synergistically with anti-IgM, in inhibiting apoptosis. Together, our data strongly suggest that adhesion of B cells via the LFA-1 (CD11a/CD18)-ICAM-1 (CD54) pathway and VLA-4 (CD49d)-VCAM-1 (CD106) pathway contributes to B cell selection.
We have utilized two well-characterized human leukemia/lymphoma (LL) cell lines, UTMB-460 and CEM, to determine the role of integrin very late antigen-alpha 4 beta 1 (VLA-4) and its ligand vascular cell adhesion molecule-1 (VCAM-1) in the adherence of the LL cells to marrow stromal cells (MSC). Both these LL cell lines express alpha and beta subunits of VLA-4. VCAM-1 is constitutively expressed by human MSC and its expression can be upregulated by interleukin-4 (IL-4) and recombinant human tissue necrosis factor-alpha (rTNF-alpha). IL-4 and rTNF-alpha stimulation of MSC is associated with a significant increase in the adherence of both UTMB-460 and CEM LL cells to the cytokine-stimulated MSC. Monoclonal antibodies directed against the alpha and beta subunits of VLA-4 and VCAM-1 significantly inhibit adherence of the LL cells to unstimulated and cytokine-treated MSC. The data reported indicate that VCAM-1 and integrin VLA-4 are obligatory adhesion proteins in the heterotypic adherence between human LL cells and MSC. The constitutive expression of VCAM-1 by MSC may be partially responsible for retention of leukemia cells in th bone marrow and metastasis of lymphomas to the bone marrow.
Studies of cell population dynamics and microenvironmental organization of B lymphopoiesis in the bone marrow of normal mice and in various genetically modified states have shown that cell loss, involving processes of apoptosis and macrophage-mediated cell deletion, is a prominent feature of the primary genesis of B lymphocytes. Balanced against the influence of proliferative stimulants, the programmed death of precursor B cells provides a quantitative control, determining the magnitude of the final output of functional B lymphocytes to the peripheral immune system. The cell loss mechanisms can be readily set in motion by external or systemic influences, making the B-cell output particularly vulnerable to suppression by ionizing irradiation, stress or other systemic mediators. In addition, however, cell loss exerts an important quality control in the formation of the primary B-cell repertoire. The combination of apoptosis and macrophage-mediated deletion, acting at successive stages of B-cell differentiation, efficiently eliminates many precursors having non-productive Ig gene rearrangements, cell cycle dysregulations, and certain autoreactive Ig specificities. Outstanding areas of further work abound. Important questions concern the nature of mechanisms which underlie the processes of B-cell apoptosis and macrophage deletion in bone marrow, the microenvironmental signals involved in B-cell life or death decisions and genetic factors which may override these B-cell culling mechanisms. The answers will be relevant to problems of autoimmune disease, humoral immunodeficiency and B-cell neoplasia.
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