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Keywords:

  • stem cells;
  • progenitor cells;
  • haematopoiesis;
  • adhesion receptors;
  • ligands

The importance of adhesion receptors in haematopoiesis

  1. Top of page
  2. The importance of adhesion receptors in haematopoiesis
  3. The sialomucins in haematopoiesis
  4. The selectin family
  5. The integrin family
  6. CD44
  7. In vivo functions and summary
  8. Acknowledgment
  9. References

Stem cells are the basic cellular units responsible for the development of all tissue and organ systems in the body. It therefore follows that an understanding of stem cell biology has enormous implications for the treatment and cure of diseases affecting many millions of individuals worldwide (Fuchs & Segre, 2000; Watt & Hogan, 2000; Weissman, 2000a,b). This review will be restricted to haematopoietic stem cells and their progeny. Haematopoietic stem cells first appear at two sites in the developing embryo: in the yolk sac and from splanchnopleural mesoderm in the aorta-gonad-mesonephros (AGM) region of the embryo proper (reviewed in Dieterlen-Lièvre, 1998; Godin et al, 1999; Keller et al, 1999; Medvinksy & Dzierzak, 1999). They then migrate in order to colonize a series of haematopoietic sites that include the fetal liver, thymus, spleen, omentum and, eventually, the bone marrow, giving rise to sequential generations of blood cells. Under steady-state conditions, haematopoiesis in the adult takes place within the bone marrow microenvironment, where a rare population of slowly cycling pluripotent haematopoietic stem cells are programmed to ‘self renew’ and proliferate or to undergo commitment to lymphoid or myeloid progenitors prior, to becoming increasingly restricted to differentiating along one of nine haematopoietic cell lineages (Fig 1). These stem cells are capable of producing more than one billion blood cells per day and of generating the entire blood system throughout an adult lifespan. Recent studies also suggest that the haematopoietic stem cell may have remarkable versatility, with its ability to be reprogrammed into non-haematopoietic lineages such as muscle cells (reviewed in Keller et al, 1999; Fuchs & Segre, 2000; Weissman, 2000a).

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Figure 1. Schematic representation of haematopoietic lineage development in adult bone marrow.

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During adult life, circulating haematopoietic stem cells and their progeny also have the ability to migrate into the bone marrow by interacting with the luminal surface of bone marrow venules and sinusoidal endothelia under conditions of shear, albeit at much lower shear forces than found in the vascular supply to other tissues such as lymph nodes, spleen and Peyer's patches (reviewed in Mazo & von Andrian, 1999). They must then transit across the endothelial barrier and come into contact with bone marrow stromal niches. The reciprocal process probably occurs with the mobilization of haematopoietic progenitor cells. The survival, quiescence, proliferation, commitment, differentiation, migration, specific gene expression, reprogramming and death of haematopoietic stem cells and their progeny are controlled by external cues that are provided by the microenvironmental niches in which they reside or become located (Lemischka, 1997; reviewed in Verfaillie, 1998; Whetton & Graham, 1999). These niches are composed of microenvironmental or stromal cells (macrophages, endothelial cells, adipocytes, reticular cells and T cells) and the cell-associated or cell-secreted molecules (cytokines and extracellular matrix molecules) that these stromal cells produce. They may also contain more than one type of stem cell, as is evident in bone marrow niches with the presence of mesenchymal haematopoietic and endothelial stem cells (reviewed in Fuchs & Segre, 2000). Recent studies implicate cytokine, adhesion and signalling receptors and also proteinases on haematopoietic stem cells and their progeny as key elements in detecting and translating the extrinsic cues provided by the haematopoietic microenvironmental niches. Thus, these niches or microenvironments supply a specific set of molecules that determine or regulate the haematopoietic stem cell fate (Fig 2). Despite this, it is not entirely clear if similar molecular mechanisms are used for the migration of haematopoietic stem cells and their progeny in and out of the different haematopoietic tissues during adult and fetal life, and for homing of haematopoietic stem cells within microenvironmental niches in haematopoietic organs, or if these processes are dependent on the regulated function of an entirely different array of adhesion receptor/ligand interactions. Cell adhesion molecules (CAMs) on haematopoietic stem/progenitor cells and their microenvironmental cells include members of the integrin family, CD44, the immunoglobulin superfamily (IgSF), the selectins and the sialomucins (Table I). They differ in their avidities for a series of cognate ligands and, therefore, in the relative strength and duration of their adhesive interactions. They also transmit positive or negative signals via specific motifs located in their cytoplasmic domains, either directly or by cross-talk with other membrane-associated receptors. This review will only attempt to summarize a selection of the major adhesion receptors, particularly sialomucins, selectins and integrins, that have been implicated in human and murine haematopoietic development and cytokine-induced mobilization, both in in vitro experimental systems and in vivo.

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Figure 2. The haematopoietic stem cell microenvironment. Interactions between haematopoietic stem cells and components of their microenvironmental niche regulate stem cell fate.

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Table I.  Cell adhesion receptors and their ligands.
ReceptorLigands involved in cell homing, trafficking & adhesion References
  1. Other members of the sialomucin family, such as endomucin, endoglycan and M2000, and of the Ig superfamily, such as ICAM-1, ICAM-2, ICAM-3, LFA-1, LFA-3, Thy-1 and c-kit/CD117, were not included in this review. ND, not determined or not published.

Sialomucins
CD34L-selectinReviewed in Krause et al, 1996
CD43NDReviewed in Ostberg et al, 1998 & Rosenstein et al, 1999
CD45Heparan sulphateCoombe et al, 1994
PSGL-1 (CD162)P-, E- & L-selectinsBorges et al, 1997; Greenberg et al 2000; Zannettino et al, 1995; Levesque et al, 1999;  reviewed in Yang et al, 1999a & Mazo & von Andrian, 1999
CD164NDReviewed in Watt and Chan, 2000
PCLP-1L-selectinSassetti et al, 1998
Selectins
L-Selectin (CD62L)CD34, GlyCAM-1, MAdCAM-1, PSGL-1, Sgp-200, PCLP-1Reviewed in Gonzalez-Amaro & Sanchez-Madrid, 1999,Etzioni et al, 1999 & Simmons & Zannettino, 1997;  Robinson et al, 1999
E-Selectin (CD62E)PSGL-1, ESL-1, sialylated L-selectin 
P-Selectin (CD62P)PSGL-1, CD24 
Integrins
VLA-4 (α4β1) VLA-5 (α5β1)VCAM-1, VLA-4, Fibronectin FibronectinMiyake et al, 1991; Williams et al, 1991; Verfaillie et al,1991; Yanai et al, 1994; Hamamura et al, 1996; Frenetteet al, 1998;  Yokota et al, 1998; van der Loo et al 1998; Mazo et al, 1998; Papayannopoulou et al, 1995, 1998; Papayannopoulou & Craddock, 1997; Craddock et al, 1997; Yin et al, 1999; reviewed in Gonzalez-Amaro &  Sanchez-Madrid 1999, Levesque & Simmons, 1999, Whetton & Graham, 1999, Vermeulen et al, 1998, Papayannopoulou et al 1999, 2000,&  Mazo & von Andrain 1999
Miscellaneous
CD44Hyaluronic acid, Fibronectin, Collagen Types I and VI, Serglycin, OsteopontinReviewed in Ghaffari et al, 1999 & Borland et al, 1998
RHAMMHyaluronic acidPilarski et al, 1999
Immunoglobulin superfamily
PECAM-1 (CD31)PECAM-1, αvβ3,?Heparan sulphateReviewed in Newman, 1999 & Watt et al, 1995; Yong et al, 1998
VCAM-1(CD106)α4β1Reviewed in Gonzalez-Amaro & Sanchez-Madrid, 1999, Levesque & Simmons, 1999, Whetton & Graham, 1999,  Vermeulen et al, 1998, Papayannopoulou et al 1999, 2000, & Mazo & von Andrain, 1999

The sialomucins in haematopoiesis

  1. Top of page
  2. The importance of adhesion receptors in haematopoiesis
  3. The sialomucins in haematopoiesis
  4. The selectin family
  5. The integrin family
  6. CD44
  7. In vivo functions and summary
  8. Acknowledgment
  9. References

The sialomucins (reviewed in van Klinken et al, 1995) exhibit diverse, but important, roles in vivo, functioning as anti-adhesive agents and as adhesion receptors or co-receptors during haematopoietic cell homing and trafficking. Recently, there has been heightened interest in the sialomucins because of their proposed functions in regulating the proliferative status of haematopoietic precursor cells (reviewed in Simmons & Zannettino, 1997). The sialomucins have been defined by two criteria: (1) their high regional content of proline, threonine and/or serine residues (20% to 55% of their amino acid compositions), and (2) their dense local concentrations of O-linked carbohydrates that are attached to these serine and threonine residues, and constitute one or more mucin domains. The mucin domains are a primary feature of molecules such as PSGL-1 and CD43. However, others, such as CD164 (endolyn), CD34, CD45RA and PCLP1, are mosaic molecules with their mucin domains linked to non-mucin structural motifs, such as cysteine rich or immunoglobulin (Ig)-like domains. It is now known that several of the leucocyte/endothelial-associated mucins mediate cell–cell adhesion, involving interactions between their mucin-like domains and the N-terminal C-type lectin domains of their appropriate L-, E-, or P-selectin ligands (reviewed by Gonzalez-Amaro & Sanchez-Madrid, 1999). These interactions may be enhanced by co-operativity between mucin-like domains and non-mucin motifs on the same molecule, thereby forming part of an adhesion cascade. Although mucin receptors may be widely expressed, their function may differ on different cell types or on the same cell type under different states of activation and is dependent on the core peptide of the mucin and the cell-specific expression of glycosyl transferases (reviewed in Etzioni et al, 1999; Hartwell & Wagner, 1999). The genomic structure and chromosome localization of the mucin-like molecules diverge from each other to varying degrees. For example, the cDNA coding sequences for human CD43 and CD162 that contain mucin domains only are specified by single exons, while CD34 and CD45RA that contain multiple domains are composed of multiple exons. Ten sialomucins, CD34, PSGL-1, CD43, PCLP, CD164, GlyCAM-1, endomucin, endoglycan, M2000 and CD45RA, have been identified on haematopoietic progenitor cells and/or associated microenvironmental cells. On such cells, the sialomucins have a variety of functions that include mediating or regulating haematopoietic progenitor cell adhesion and/or the negative regulation of their growth and/or differentiation. A selection of these receptors will be reviewed below.

The CD34 sialomucin CD34 is a 90–110 kDa type 1 transmembrane cell surface glycoprotein that has universally been used as a marker for developmentally early haematopoietic ‘stem’/progenitor cells (reviewed in Krause et al, 1996; Goodell, 1999; Weissman, 2000a). It is not confined to these cells, but is also expressed on hepatocyte progenitor cells, bone marrow stromal cells, embryonic fibroblasts and on the vasculature in both the human and the mouse, being present on many large and small vessels, including post-capillary venules, capillaries in a wide variety of tissues and on high endothelial venules (HEVs) in peripheral lymph nodes (reviewed in Goodell, 1999). The highest concentration of CD34+ cells is found closely associated with the ventral floor of the dorsal aortic endothelium in the week 4–5 human embryo, at a site associated with the appearance of the definitive haematopoietic stem cell (Tavian et al, 1996). A similar population of CD34+ cells is found in the mouse (reviewed in Keller et al, 1999). Its pre-eminence as a stem cell marker has, however, been questioned (reviewed in Goodell, 1999), with several studies demonstrating low or absent expression of CD34 on putative quiescent adult stem or repopulating cells in the mouse and human. These studies have been interpreted as indicating that CD34 is an activation marker on adult haematopoietic stem cells (reviewed in Goodell, 1999). In the human, the mature CD34 polypeptide is composed of 354 amino acids and comprises a 259-amino-acid extracellular domain, a single 22-amino-acid transmembrane region and a cytoplasmic domain of 73 amino acids. The first 130 amino acids of the extracellular domain are heavily O-glycosylated and linked to a 100-amino-acid non-mucin cysteine-rich region. The genes for both murine and human CD34 are located on chromosome 1q32 (reviewed by Krause et al, 1996). The human CD34 coding sequence spans 26 kb of genomic DNA and is composed of eight exons that are separated by introns of 0·2 kb to 11 kb in length. The 5′ untranslated region (UTR) and signal sequence are encoded by exon 1, the mucin domain by exons 2 and 3, the cysteine-rich region by exons 4 and 5, the transmembrane region and part of the cytoplasmic tail by exon 7, and the remainder of the cytoplasmic region and 3′ UTR by exon 8. The cytoplasmic domains of human, murine and canine CD34 share more than 90% amino acid identity and contain two sites for protein kinase C (PKC) phosphorylation (R/KX1−3S/TX1−13R/K), suggesting conservation of function. PKC activation upregulates cell membrane CD34 expression. A splice variant of CD34 generated by the insertion of exon X and encoding a truncated protein lacking most of the cytoplasmic domain and potential target sites for PKC has been identified. Both isoforms are found in normal bone marrow cells, but their expression appears to vary with the relative differentiation state of the CD34+ haematopoietic progenitors. Two adhesion recognition motifs, VTXG and RNIAEIIKDI, occur in the CD34 extracellular domain (reviewed by Krause et al, 1996).

CD34 has two potential roles in vivo: (1) in leucocyte trafficking to both lymphoid and non-lymphoid sites, and (2) as a negative regulator of haematopoietic progenitor cell differentiation and proliferation (reviewed by Krause et al, 1996; Suzuki et al, 1996). In vitro studies have demonstrated that, on high endothelial venules (HEVs), CD34 is the major 90–110 kDa component of the peripheral node addressin (PNAd) complex of glycoproteins defined as MECA-79+ and that encompasses the L-selectin ligand activity for lymphocyte trafficking through peripheral lymphoid tissues (reviewed by Krause et al, 1996). However, CD34-null mice do not show major deficiencies in lymphoid trafficking to peripheral lymph nodes and the 90–110 kDa MECA-79 band associated with CD34 expression is not completely deleted from peripheral lymph nodes in these mice. CD34-deficient mice exhibit defective eosinophil migration to the lung in response to inflammation (Suzuki et al, 1996). This suggests that CD34 is not solely responsible for regulating lymphoid trafficking through lymph nodes. The importance of CD34 in directly mediating adhesion of haematopoietic progenitor cells to bone marrow stroma is unclear and it is possible that CD34 may function as an adhesion regulator. Thus, while murine CD34-deficient progenitors are able to adhere to a stromal cell line in vitro (Suzuki et al, 1996), mouse thymocytes overexpressing human CD34 adhere to human, but not murine, stromal cells (Healy et al, 1995).

CD34 may also function as a signalling receptor by negatively regulating haematopoietic progenitor cell differentiation and proliferation. In vitro transfection studies have revealed that the full-length, but not the truncated, CD34 isoform impedes terminal differentiation of M1 myeloid leukaemia cells to macrophages following interleukin 6 or leukaemia inhibitory factor stimulation (reviewed by Krause et al, 1996). Furthermore, a significant delay in both erythroid and myeloid differentiation has been observed during haematopoietic development in embryoid bodies derived from CD34-null embryonic stem cells. Although CD34-null mice have a normal adult peripheral blood profile, they exhibit decreased haematopoietic colony-forming progenitor cell numbers at haematopoietic sites including the yolk sac, fetal liver, bone marrow and spleen. Whether this is as a result of alterations in adhesion and migration of progenitors or to changes in their proliferative and differentiation responses is unclear. CD34-deficient progenitors do, however, have a reduced capacity to expand ex vivo in response to certain cytokines (Cheng et al, 1996), even though an additional study indicated that the cytoplasmic tail of CD34 does not transduce a proliferative signal (Hu & Chien, 1998). The kinetics of haematopoietic recovery/mobilization following 5-fluorouracil or granuloctye colony-stimulating factor (G-CSF) administration or sublethal irradiation are not significantly different in CD34-deficient and wild-type mice (Cheng et al, 1996; Suzuki et al, 1996).

Podocalyxin-like protein 1 (PCLP1) Human podocalyxin-like protein 1, a 160–165 kDa type I transmembrane glycoprotein of the glomerular podocyte glycocalyx, is present on vascular endothelial cell and high endothelial venules in peripheral lymph nodes (Kershaw et al, 1997a; Sassetti et al, 1998). In the mouse, PCLP1 is expressed on haematopoietic progenitor cells and on long-term repopulating cells, angioblasts and haemangioblasts in the AGM, with 12% of PCLP1+CD45 cells in the AGM also expressing flk1, a putative haemangioblast marker (Hara et al, 1999). The mature human PCLP1 polypeptide consists of 507 amino acids with the extracellular domain comprising 406 amino acids, 78 potential O-linked glycosylation sites and a potential glycosaminoglycan (GAG) attachment site (Kershaw et al, 1997a). The N-terminal mucin domain is linked to a cysteine-rich region lying adjacent to the cell membrane that is followed by a 26-amino-acid transmembrane region and a cytoplasmic domain of 75 amino acids. The latter contains one potential protein kinase C site and two potential casein kinase C sites. The protein structure of PCLP1 resembles CD34, with their cytoplasmic domains sharing 25% amino acid identity (Sassetti et al, 1998). The PCLP1 gene has been localized to human chromosome 7q32–33 (Kershaw et al, 1997b) and mouse chromosome 6 (Hara et al, 1999). PCLP1 on HEV is a ligand for L-selectin (Sassetti et al, 1998). It is tempting to speculate that PCLP1 may display overlapping or complementary functions with CD34, being involved in selectin–mediated adhesive interactions of haematopoietic progenitor cells to endothelium or leucocytes to HEV. Similar to CD34, PCLP1 may also play a role in negatively regulating haematopoietic differentiation, but this remains to be tested. Results for PCLP1-null mice have not been published.

Endolyn (CD164) Human CD164 is a newly identified 80–110 kDa sialomucin expressed by CD34+ haematopoietic progenitor cells, by a subset of repopulating CD34 precursors and by bone marrow stromal reticular cells (reviewed by Verfaillie, 1998; Watt et al, 1998, 2000; Zannettino et al, 1998; Almeida-Porada et al, 1999; Watt & Chan, 2000). It contains two mucin domains (I and II) linked by a non-mucin domain in its extracellular region, containing intramolecular disulphide bonds. Functional analyses in vitro have indicated that this receptor may play a key role in haematopoiesis by facilitating the adhesion of human CD34+ cells to bone marrow stroma and by negatively regulating CD34+CD38lo/– haematopoietic progenitor cell proliferation (Zannettino et al, 1998). These effects involve carbohydrate-dependent Class I and/or II epitopes recognized by the antibodies 105A5 and 103B2/9E10 on the first mucin domain (Doyonnas et al, 2000). The human CD164 molecule is widely expressed in all tissues, with differential expression of the 103B2/9E10 and 105A5 carbohydrate epitopes occurring on endothelial, epithelial and haematopoietic cells (Doyonnas et al, 2000; Watt et al, 2000; reviewed by Watt & Chan, 2000). The human and murine CD164 genes possess similar genomic structures (Kurosawa et al, 1999; Chan et al., 2000; reviewed by Watt & Chan, 2000; unpublished observations), with the longest human isoform CD164(E1–6) composed of six exons and interspersed with introns of 1·4–5·6 kb in length. This places CD164 within the sialomucin subgroup composed of multiple exons. Four human CD164 splice variants, CD164(E1–6), CD164(EΔ5) and CD164(EΔ4), and CD164(3′UTRΔ) have been described, with CD164(E1–6) appearing to be the dominant isoform. The human CD164(E1–6) mature polypeptide comprises 197 amino acids, with 32 potential O-linked glycosylation sites and a potential GAG attachment site in its extracellular domain. All three isoforms have the same transmembrane and cytoplasmic tail, with a potential tyrosine kinase phosphorylation site and a YHTL endosomal-trafficking motif at the carboxy terminal end that is not preceded by a glycine residue and that is known in other proteins to interact with the clathrin adaptor and adaptor-like complexes (Ihrke et al 2000; unpublished observations). Human, murine, rat and Drosophila CD164 share high levels of amino acid identity, particularly in their cytoplasmic and transmembrane sequences. CD164 is localized on both the cell surface and in the recycling endosomes and lysosomes (Ihrke et al, 2000; unpublished observations). While the human CD164 gene is located on chromosome 6q21, the mouse gene occurs in a syntenic region on chromosome 10B1-B2 (unpublished observations). Defining the nature and expression of the natural ligands for CD164 will provide insight into its normal function. Results for CD164 knock-out mice have not been published.

Leucocyte common antigen (CD45) Human CD45 is a 180–220 kDa type 1 transmembrane glycoprotein (reviewed in Trowbridge & Thomas, 1994). The gene for human CD45 is located on chromosome 1q31–32 and comprises 33 exons that extend over more than 120 kb of DNA. The first exon encodes the 5′ untranslated region, exon 2 the signal peptide, exons 3–15 the extracellular domain and exon 16 the transmembrane peptide. The cytoplasmic region is composed of two homologous phosphotyrosine phosphatase domains, the first encoded by exons 17–32 and the second by exons 25–32. Exon 33 encodes the carboxyl terminus and includes the entire 3′ untranslated region. The variable use of three exons, 4, 5 and 6, generates at least seven different isoforms of human CD45. However, in the mouse there are eight isoforms that differentially use exons 4, 5 and 6 and two for exons 7, 8 and 10. The full-length human CD45 isoform consists of a 1281-amino-acid mature polypeptide containing a 552-amino-acid extracellular domain, a 22-amino-acid hydrophobic transmembrane sequence and a 707-amino-acid cytoplasmic tail. The extracellular domain contains a mucin domain, a cysteine-rich region and three fibronectin type III repeats. CD45 is expressed by all cells of haematopoietic origin including CD34+CD164+PCLP1+ progenitors in the AGM, but not by erythrocytes and their immediate precursors, by the PCLP1+CD45 haemangioblasts (Hara et al, 1999) nor by muscle satellite stem cells with haematopoietic lineage potential (Jackson et al, 1999). Different cell types express different CD45 isoforms at precise stages of cell lineage differentiation and activation. These are identified by three sets of antibodies, CD45RA, CD45RB and CD45RC, that recognize mucin domain epitopes attached to peptides encoded by exons 4, 5 or 6, respectively, or by CD45RO antibodies that recognize the CD45 isoform lacking exons 4, 5 and 6. CD45RA is expressed at high levels on human CD34+ haematopoietic progenitor cells, with CD34+CD45RAhiCD71lo cells being mostly committed myeloid progenitors, CD34+CD45RAloCD71hi cells being enriched for erythroid progenitors and CD34+CD45RAloCD71lo cells being enriched for multipotent progenitors (Lansdorp et al, 1990). CD45 mediates the interaction of a murine multipotent haematopoietic cell line to stromal reticular cells via heparan sulphate (Coombe et al, 1994). In vivo, such interactions would bring haematopoietic progenitors into direct contact with stromal-derived cytokines presented by these heparan sulphate complexes (Gordon et al, 1987; Roberts et al, 1988). The effects of CD45 deficiency on T and B lymphoid functions, but not on haematopoietic stem cell development, have been reported.

Leukosialin (sialophorin; CD43) Human CD43 (reviewed in Ostberg et al, 1998; Rosenstein et al, 1999) is a major 95–135 kDa type 1 transmembrane sialoglycoprotein expressed widely on leucocytes. CD43 is expressed on almost all human haematopoietic progenitors, including the LinCD34hi cells (Bazil et al, 1996) and on week 4–5 embryonic CD34+ haematopoietic progenitors associated with the ventral floor of the dorsal aorta (Watt et al, 2000). The mature CD43 polypeptide is composed of 381 amino acids and contains a single extracellular mucin domain of 235 amino acids, a transmembrane region of 23 amino acids and a 123-amino-acid cytoplasmic tail. The human CD43 gene is located on chromosome 16p11.2 and comprises only two exons spanning 4·6 kb of genomic DNA. A single intron of 178 bp interrupts the sequence that specifies the 5′ UTR, with exon 2 containing 34 nucleotides of the 5′ UTR, the entire CD43 coding sequence and 3′ UTR.

CD43 is a functional paradox, appearing to have dual roles in leucocyte endothelial interactions, where it mediates both pro-adhesive and anti-adhesive events (reviewed in Ostberg et al, 1998; Rosenstein et al, 1999). The heavily sialylated structure of CD43 is consistent with a negatively charged molecule that may interfere with the ability of other adhesion molecules (e.g. β1 and β2 integrins) to interact with their ligands (Stockton et al, 1998; Woodman et al, 1998). However, the combined use of intravital microscopy and CD43-deficient mice/cells has demonstrated that CD43 negatively regulates T-lymphocyte homing, may be involved in cell polarization, may be pro-adhesive and may negatively regulate cell proliferation (reviewed in Ostberg et al, 1998; Woodman et al, 1998; He & Bevan, 1999; Rosenstein et al, 1999). CD43 may transduce signals involved in the regulation of haematopoiesis. Experimental evidence indicates that proliferating CD34hiLin cells, but not more primitive quiescent haematopoietic precursors, undergo apoptosis in response to the interaction of the CD43 antibody, MEM-59, with the CD43 receptor (Bazil et al, 1995, 1996). This suggests that engagement of CD43 may negatively regulate haematopoietic progenitor cell proliferation. The cognate ligand for such interactions has not been identified. Cross-linking of CD43 with antibodies also enhances VLA-4- and VLA-5-dependent adhesion of human CD34+CD38hi, but not CD34+CD38lo, cells to fibronectin via protein tyrosine kinase and PLC-γ activation pathways (Anzai et al, 1999).

P-selectin glycoprotein ligand-1 (PSGL-1; CD162) Human CD162 is a 210 kDa type I transmembrane glycoprotein (reviewed by Simmons & Zannettino, 1997; Yang et al, 1999a). Both human and murine CD162 have similar structures with highly conserved transmembrane and cytoplasmic domains. The human CD162 402-amino-acid polypeptide contains a signal sequence and pro-peptide with the extracellular domain of the mature polypeptide beginning at residue 42. The 269-amino-acid extracellular domain contains 15 decameric repeats, 74 potential O-linked glycosylation sites and, adjacent to the transmembrane region, a single cysteine that promotes dimerization. Its 23-amino-acid transmembrane sequence is followed by a cytoplasmic tail of 69 amino acids. The human CD162 gene is localized on chromosome 12q24 and comprises two exons extending over at least 12 kb of DNA and separated by a single 9 kb intron. The whole coding sequence and 3′ untranslated region are encoded by exon 2, while exon 1 encodes most of the 5′ untranslated region. CD162 is expressed on most leucocytes, where it has a role in tethering and rolling of leucocytes under conditions of hydrodynamic flow. CD162 is also expressed on some epithelia and on CD34+ haematopoietic progenitors [including granulocyte–macrophage colony-forming units (G/M-CFU), erythroid blast-forming units (BFU-E), pre-CFU, and CD38+ and CD38lo subsets] where it serves as a functional receptor for P-selectin, but not on CD34+CD19+ B-cell precursors, erythroid cells, megakaryocytes, platelets or normal vascular endothelia (Zannettino et al, 1995; Levesque et al, 1999; Yang et al, 1999a). It binds to E-selectin and L-selectin, with the former requiring the presence of the HECA 452 CLA epitope on CD162 for binding (reviewed in Yang et al, 1999a). CD162 must be α2,3-sialylated and α1,3-fucosylated, and contain a core-2 branched O-linked glycan at threonine 57 and sulphated tyrosines clustered at residues 46, 48 and 51 in its NH2 terminal region to bind P-selectin. L-selectin binding also requires this sulphotyrosine-containing domain. In contrast, CD162 requires sialylated and fucosylated core 2 branched O-glycans, but not tyrosine sulphation for binding to E-selectin.

Essentially, all myeloid progenitors bind to P-selectin via CD162 (Zannettino et al, 1995), as do mouse immature bone marrow-derived mast progenitors in vitro (reviewed in Yang et al, 1999a). Rolling of CD34+CD38lo cells is more efficient, slower and of longer duration than CD34+CD38+ cells or more mature CD34 cells and can be partially inhibited with the CD162 antibody PL-1, but not by the CD34 antibody Qbend/10 (Greenberg et al, 2000). Antibodies to an NH2-terminal epitope of murine CD162 have also been shown to block tethering and rolling of murine myeloid cells on P-selectin under flow (Borges et al, 1997). The binding of primitive haematopoietic/myeloid progenitors to P-selectin may mediate initial rapid shear-resistant adhesion and subsequent ‘rolling’ along endothelial cells as these progenitors enter the bone marrow or other haematopoietic microenvironments (Mazo & von Andrian, 1999; Greenberg et al, 2000). CD162, when it engages P-selectin, may also function as a signalling receptor, for example by inducing Mac-1 expression or enhancing β2 integrin adhesion to intercellular adhesion molecule (ICAM)-1 in neutrophils or inducing cytokines in monocytes and neutrophils (Evangelista et al, 1999; reviewed in Yang et al, 1999a). Recent studies suggest that CD162 is the only P-selectin receptor on human CD34+ cells and that engagement of CD162 on these cells by P-selectin negatively regulates haematopoietic cell growth (Levesque et al, 1999). The molecular mechanisms determining this inhibition differ at different stages of CD34+ cell maturity. P-selectin binding reduces the proliferative rate of CD34+CD38+ cells without affecting their viability, whereas 50% of the CD34+CD38lo cells undergo apoptosis in cultures containing either immobilized or soluble P-selectin and the interleukin (IL)3, IL6, G-CSF and stem cell factor (SCF) cytokines. The more primitive pre-CFU within the CD34+CD38lo/– subset remain viable. The apoptotic effects, but not the growth inhibition, can be overcome by adding Flt ligand (FL) and thrombopoietin (Tpo) to the cultures. Apoptosis (reviewed in Ruoslathi & Reed, 1994) is thought to be important for haematopoietic stem/progenitor cell homeostasis by preventing the overproduction of haematopoietic stem cells and controlling inaccurate cell positioning (annoikis or homelessness). The potential significance of these findings to haematopoietic development will be addressed in the section on selectins. CD162-null mice are fertile and develop normally, with erythromyeloid development appearing normal, but they show a modest neutrophilia and eosinophilia (Yang et al, 1999b). More detailed analyses of the effects of CD162 deficiency on haematopoietic development have not been reported.

The selectin family

  1. Top of page
  2. The importance of adhesion receptors in haematopoiesis
  3. The sialomucins in haematopoiesis
  4. The selectin family
  5. The integrin family
  6. CD44
  7. In vivo functions and summary
  8. Acknowledgment
  9. References

The selectins (reviewed in Gonzalez-Amaro & Sanchez-Madrid, 1999 and Vestweber & Blanks, 1999 and references therein) are a family of three proteins, E (endothelial, CD62E)-, P (platelet, CD62P)- and L (leucocyte, CD62L)-selectins, that mediate adhesive interactions between leucocytes and the endothelium and between leucocytes and platelets in the blood vascular compartment. Functionally, the selectins are involved in the earliest Ca2+-dependent phases of a cascade of events leading to leucocyte extravasation via the vascular endothelium to sites of inflammation. All three genes are located on human and mouse chromosome 1. Structurally, they are composed of a 120 amino terminal C-type Ca2+-dependent lectin domain or carbohydrate-recognition domain (CRD), followed by an epidermal growth factor (EGF)-like motif and a variable number (two to nine) of short consensus repeats (SCR) similar to those found in complement regulatory proteins. Both the EGF-like domains and the SCRs show a high degree of amino acid similarity between family members. The transmembrane domain is followed by a short cytoplasmic tail of 17–35 amino acids. E-selectin and P-selectin are expressed by endothelial cells following stimulation by inflammatory mediators, P-selectin is also on activated platelets and L-selectin is expressed constitutively on leucocytes, including CD34+ cells. In contrast to other tissues, resting endothelia in bone marrow and skin express E- and P-selectins constitutively (reviewed in Gonzalez-Amaro & Sanchez-Madrid, 1999; Mazo & von Andrian, 1999). Myeloid progenitors (CFU-GM) within the CD34+ population express L-selectin at high levels, while erythroid progenitors (BFU-E) exhibit low to undetectable levels. Hierarchically, more primitive progenitors (pre-CFU) identified as CD34+CD38lo/– cells express L-selectin at low to intermediate levels (reviewed in Simmons & Zannettino, 1997). Human L-selectin, the homologue of the 90 kDa MEL-14+ murine peripheral lymph node homing receptor, is a 75–110 kDa type I transmembrane glycoprotein. The L-selectin gene occupies at least 30 kb of genomic DNA and is composed of 10 exons that generate a 2·3-kb cDNA. This encodes a protein of 372 amino acids containing an amino terminal C-type lectin domain, a single EGF-like domain, two SCR domains, a single membrane spanning region and a cytoplasmic tail.

Selectins recognize the sialylated and fucosylated lactosamines, sialyl Lewis x (sLex) and sialyl Lewis a (sLea), when presented on the appropriate backbone. However, the most potent naturally occurring carbohydrate ligands for both L- and P-selectin are Lex and Lea derivatives, in which the hydroxyl group on carbon 3 of galactose is sulphated. Additional in vitro biological ligands for L-selectin include the sialomucins, GlyCAM-1, CD34, MAdCAM-1, PSGL-1/CD162, Sgp200 and PCLP1, provided that these possess the correct post-translational modifications (reviewed in Gonzalez-Amaro & Sanchez-Madrid, 1999). Sulphation of CD34 is required, for example, for HEV-derived CD34 to mediate L-selectin binding in vitro, although, as mentioned above, CD34-null mice do not appear to have defective lymphoid migration across HEV. L-selectin nevertheless appears important for the homing of naive lymphocytes via HEVs to peripheral lymph nodes and Peyer's patches, while E- and P-selectins, and possibly L-selectin on activated endothelial cells, contribute to the recruitment of leucocytes to sites of inflammation. P-selectin ligands include PSGL-1 and CD24, while E-selectin interacts with PSGL-1, ESL-1 and sialylated L-selectin (reviewed in Gonzalez-Amaro & Sanchez-Madrid, 1999).

Lymphocyte homing activity and leucocyte rolling and migration function to inflammatory sites are defective in L-selectin-null mice, especially in leucocyte rolling and adhesion to activated endothelia (reviewed in Hartwell & Wagner, 1999). In vitro, both primitive human adult bone marrow and fetal liver CD34+CD38lo/– or CD34+CD38+ progenitors and human and murine myeloid progenitors roll on E-, P- and L-selectins under conditions of shear (reviewed in Etzioni et al, 1999; Greenberg et al, 2000). Experiments on mice lacking one, two or three of the selectin genes (Robinson et al, 1999) indicate that P-selectin has a predominant role in leucocyte homeostasis and recruitment, but also co-operates with E- and L-selectins. Bone marrow from single selectin-deficient and E/L-null mice does not show significant abnormalities. E/L/P-null bone marrow resembles that found in P/E-deficient mice (Robinson et al, 1999). Double or triple P/E-, P/L- and P/L/E-selectin-deficient mice exhibit leucocytosis, increased cytokine [IL-3 and granulocyte–macrophage colony-stimulating factor (GM-CSF)] levels and haematopoietic changes, such as an increase in the myeloid to erythroid progenitor ratio in the bone marrow (Frenette et al, 1996; Robinson et al, 1999). This coincides with increased granulopoiesis in the bone marrow and spleen and increased erythropoiesis in the spleen (Frenette et al, 1996; Robinson et al, 1999). Furthermore, these knock-out mice also show defects in haematopoietic progenitor cell homing to the bone marrow, but not the spleen, after bone marrow transplantation into irradiated recipients (Frenette et al, 1998; reviewed in Hartwell & Wagner, 1999; Robinson et al, 1999). It is possible that E-selectin could regulate the rolling velocity of progenitors in vivo as has been demonstrated for neutrophils (reviewed in Etzioni et al, 1999). The function of L-selectin in haematopoiesis is less well defined. Blocking anti-L-selectin antibodies inhibits the generation of myeloid progenitors in stromal cell-dependent long-term bone marrow cultures and colony formation in semisolid clonogenic assays (reviewed in Simmons & Zannettino, 1997). However, intravital microscopy experiments (Mazo et al, 1998; reviewed by Mazo & von Andrian, 1999) indicate that in vivo L-selectin is not involved in murine haematopoietic progenitor cell rolling on bone marrow venules or sinusoidal endothelia, although a partial reduction in such rolling occurs in P/E−/– mice or in mice injected with blocking antibodies to P- and E-selectin.

Selectins also have a role in signal transduction with ligand interactions modifying the levels, activation states and/or function of molecules such as the β1 integrins, galectin-3 and Mac-1 (reviewed in Gonzalez-Amaro & Sanchez-Madrid, 1999). These results indicate that P- and E-selectins are important for normal leucocyte function and contribute to haematopoietic progenitor cell homing to the bone marrow.

The integrin family

  1. Top of page
  2. The importance of adhesion receptors in haematopoiesis
  3. The sialomucins in haematopoiesis
  4. The selectin family
  5. The integrin family
  6. CD44
  7. In vivo functions and summary
  8. Acknowledgment
  9. References

Structurally, the integrins are composed of two non-convalently associated α and β heterodimeric subunits. At least 16 α subunits (α1, α2, α3, α4, α5, α6, α7, α8, α9, αV, αL, αM, αX, αIIb, αIE and αD) and eight β subunits (β1, β2, β3, β4, β5, β6, β7 and β8) exist. While many α subunits associate with a single β subunit, several α subunits (e.g. α4, α6, αV) can associate with more than one type of β subunit, thereby generating at least 22 heterodimers (reviewed in Gonzalez-Amaro & Sanchez-Madrid, 1999). The integrin α subunits possess apparent molecular weights of 120–180 kDa with 20–60% amino acid sequence similarities. All are type 1 transmembrane proteins, containing seven tandem repeat domains of approximately 60 amino acids forming a β propeller cyclic structure in their extracellular domains, with three or four divalent cation binding sites homologous to the EF-hands of the calcium-binding proteins calmodulin and troponin. The transmembrane domain is followed by a 15- to 35-amino-acid cytoplasmic tail. Subunits α1, α2, αL, αMαD, αE and αX contain the I domain of 180 amino acids, similar to domains found in cartilage matrix protein, von Willebrand factor and complement factor, inserted between repeats 2 and 3. The divalent cation binding motif of the I domain is known as a MIDAS motif and is involved in ligand binding. The cytoplasmic domains of α subunits contain a conserved sequence (KXGFFKR) that binds calreticulin and modulates integrin avidity. The molecular weights of the integrin β subunits range from 90 kDa to 110 kDa except for β4 which is 210 kDa. They share 35–55% amino acid similarities, contain 56 conserved cysteines (except β4 which has 48) arranged in four repeating units, and I-like and MIDAS motifs. The N-terminal half of the protein is required for α/β heterodimerization. The cytoplasmic domains are 40–50 amino acids long, except for β4 with a 1018-amino-acid cytoplasmic tail containing four fibronectin type III repeats. Both α and β subunits interact with ligands that include extracellular matrix proteins (collagen, fibrinogen, fibronectin, lamin, thrombospondin, vitronectin, von Willebrand factor, tenascin and epiligrin) and/or immunoglobulin superfamily members, such as ICAM-1, ICAM-2, ICAM-3 and vascular cell adhesion molecule (VCAM)-1, with the α subunit providing specificity (reviewed in Gonzalez-Amaro & Sanchez-Madrid, 1999). These interactions allow intracellular signal transduction or cross-talk with other receptors such as cytokine receptors and they are mediated mostly via the β-chain (reviewed in Porter & Hogg, 1998). In this way, they play important roles in cell differentiation, leucocyte trafficking, platelet aggregation, cell activation, progenitor cell migration and homing, tissue organization, and initiating such cellular activities as division, secretion or gene expression (Gonzalez-Amaro & Sanchez-Madrid, 1999; reviewed in Levesque & Simmons, 1999).

The integrins are widely expressed, but only those known to be highly relevant to haematopoietic development will be described here. CD34+ haematopoietic progenitor cells, including CFU-GM and BFU-E, express most importantly the α4β1[very late antigen (VLA)-4, CD49d/CD29] and α5β1 (VLA-5, CD49e/CD29) integrins (reviewed in Levesque & Simmons, 1999). In addition, clonogenic myeloid and erythroid progenitors, but not pre-CFU, express the leucocyte integrin αLβ2 (CD11a/CD18, LFA-1), with Mac-1 (αMβ2, CD11b/CD18) being reported on murine haematopoietic progenitors that are more mature than haematopoietic stem cells, while a minor subpopulation of CD34+ cells, which include megakaryocyte progenitors, express the αIIbβ3 (CD41/CD61) integrin (reviewed in Whetton & Graham, 1999). The importance of the β1 integrins, VLA-4 and VLA-5 in haematopoiesis is particularly well documented and will be described below.

VLA-4 (α4β1) and VLA-5 (α5β1) structure and ligands The human α4 gene, which encodes a protein of 1038 amino acids with an apparent molecular weight of 130 kDa, is located on chromosome 2q31-q32. The four different isoforms (A, B, C and D) of the β1 subunit of VLA-4 each contain a 708-amino-acid extracellular domain and a 23-amino-acid transmembrane domain, but differ in that their cytoplasmic domains are composed of 47, 38, 73 and 50 amino acids respectively. The gene for the β1 subunit is located on chromosome 10p11.2. The human α5 gene, which encodes the α5 subunit of 1008 amino acids, is located on chromosome 12q-q13. VLA-4 is involved in both cell–cell and cell–matrix interactions and uses vascular cell adhesion molecule-1 (VCAM-1; CD106) and fibronectin (FN) as essential ligands. The first and fourth immunoglobulin domains of the seven-domain VCAM-1 are required for adhesion. The structure of FN is shown diagrammatically in Fig 3. VLA-5 binds to fibronectin via an RGD sequence in the cell binding domain (CBD) in the centre of the molecule and recognizes two other spatially distinct cell binding sites, with optimal interaction requiring co-operation of the RGD and a cell adhesion site in 3Fn9 (reviewed by Gonzalez-Amaro & Sanchez-Madrid, 1999; Yin et al, 1999). VLA-4 (α4β1) binds sites near the carboxyl terminus of FN (the IIICS domain), specifically and preferentially to the LDV tripeptide on the CS-1 region, that is distinct from the RGD sequence used by VLA-5. Additionally, the α4 subunit is a ligand for VLA-4. VLA-4 also binds to the 3Fn8 and 3Fn10 type III fibronectin motifs with the latter containing the RGD sequence (reviewed by Gonzalez-Amaro & Sanchez-Madrid, 1999; Yin et al, 1999). However, VLA-4/FN interactions are reduced by cyclic RGD peptides, but not by linear RGD-containing peptides that have been used to inhibit VLA-5/FN binding. FN is produced by bone marrow stromal cells in vitro and is highly expressed in the bone marrow microenvironment. VCAM-1 is expressed by activated endothelial cells stimulated by inflammatory cytokines such as IL-1, IL-4 and TNF-α. It is also present on stromal cells, macrophages and venous sinus endothelia in bone marrow (reviewed in Levesque & Simmons, 1999).

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Figure 3. The structure of fibronectin showing type I, II and III fibronectin repeats, the IIICS domain and the classical binding sites for VLA-5, proteoglycans and VLA-4, which are the cell binding (CBD), heparin binding (Hep) and CS-1 domains respectively. Regions required for VLA-4 mediated adhesion and migration and those that encompass the CH-296 and C-274 peptides are indicated by solid lines.

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The role of VLA-4 and VLA-5 in haematopoiesis Both VLA-4 and VLA-5 receptors play roles in haematopoietic development and migration, but like other adhesion receptors, the mechanisms by which they act have not been completely determined. Low affinity VLA-4 and VLA-5 receptors are expressed on 90% of human CD34+ bone marrow cells, including CFU-GM and BFU-E. They are also expressed by murine haematopoietic progenitors. High affinity/avidity states are induced in VLA-4 and VLA-5 rapidly, transiently and in successive waves by a variety of stimuli, including cytokines, chemokines and cell adhesion receptor interactions such as those involving PECAM-1 (reviewed by Levesque & Simmons, 1999). Cytokine-stimulated human CD34+ and CD34+CD38lo/– cells are reported to remain adherent to FN and VCAM-1 for at least 10 d in vitro. However, both cytokines and chemokines induce motility of murine and human haematopoietic progenitor cells on FN (reviewed in Levesque & Simmons, 1999). It has been suggested that VLA-5 is involved in the regulation of cell adhesion and matrix assembly, in cell migration beneath stromal cells and in maintaining the directional migration of cells, while VLA-4 is required for the speed of migration. Studies on human B-cell (α4β1hiα4β7hiα5β1 lo) lymphomas suggest that both the IIICS domain and central CBD promote cellular migration, although the latter domain is more important for chemotactic cell movement induced by cytokines (Yin et al, 1999). This has led Yin et al (1999) to propose that low affinity binding is preferred for cell motility and high affinity binding for stronger adhesion. In haematopoietic development, similar mechanisms might allow stronger adhesion to specific microenvironments via the IIICS domain, with low affinity binding sites on the central CBD of FN being more important for cell migration. Cytokines that induce adhesion of CD34+ cells to FN in vitro include IL-3, GM-CSF, SCF, G-CSF and Tpo, with maximal adhesion of clonogenic cells obtained with combinations of IL-1β, IL-3 and SCF or with IL-1β, IL-3, IL-6, GM-CSF, G-CSF and SCF (reviewed by Levesque & Simmons, 1999; Papayannopoulou, 2000). In contrast to this, the SDF-1 chemokine that functions as a chemoattractant for haematopoietic progenitors, downregulates cytokine-induced adhesion of CD34+ cells to FN (Gotoh et al, 1999). Tpo has been shown to stimulate adhesion of CD34+CD34lo/–, but not CD34+CD38+, human bone marrow cells to FN primarily, although not exclusively, via VLA-5 activation. SCF, IL-3, Tpo and SDF-1 transiently activate VLA-4, thereby increasing adhesion of haematopoietic progenitor cell lines and CD34+ bone marrow cells to VCAM-1 (reviewed in Levesque & Simmons, 1999; Peled et al, 1999). In this respect, it is of interest that SDF-1α- or CXCR4 (the SDF-1α receptor)-deficient mice have defective fetal liver and adult haematopoiesis (Nagasawa et al, 1996; Zou et al, 1998). Details of signalling mechanisms involving VLA-4 and VLA-5 integrins and mechanisms regulating integrin adhesiveness have recently been reviewed and will not be dealt with here (Gonzalez-Amaro & Sanchez-Madrid, 1999; Levesque & Simmons, 1999).

Antibodies to VLA-4 inhibit lymphopoiesis and erythropoiesis and retard myelopoiesis in long-term culture systems (Miyake et al, 1991; Williams et al, 1991). In the mouse, the development of erythroid cells on fetal liver and new-born spleen stroma in vitro requires SCF and is inhibited by anti-VLA-4 and anti-VCAM-1 antibodies, but not by anti-VLA-5 antibodies. VLA-5 mediates erythroid progenitor cell adhesion to FN, but under such conditions does not support erythropoiesis in vitro. Thus, in the stromal system, VLA-4/VCAM-1 interactions appear to be essential for the rapid expansion of erythroid progenitors, at least from fetal liver and spleen, perhaps by bringing them into close association with cytokines such as SCF (Yanai et al, 1994; Hamamura et al, 1996). FN binding to VLA-4 and VLA-5 can influence the proliferation and differentiation of progenitor cells. FN augments CFU-GM, BFU-E and mixed lineage CFU (CFU-GEMM) colony formation in the presence of specific cytokines, an effect abrogated almost completely by the addition of the CS1 fragment of FN, but not by the GRGDSP peptide, suggesting an important role for VLA-4 (Yokota et al, 1998). However, CS1-CBD fragments contain a growth supporting activity that resembles the whole FN molecule in vitro. In long-term bone marrow cultures, binding of human haematopoietic cells to a peptide containing the VLA-5 RGD binding site has been shown to increase with differentiation (Verfaillie et al, 1991).

Anti-VLA-4 antibodies, when injected in utero into mice, specifically induce anaemia, but have no effect on lymphoid or myeloid cell development, with erythroid progenitors, but no erythroblasts, being present in the fetal liver (Hamamura et al, 1996). The injection of anti-VCAM-1 antibodies does not result in anaemia, but causes peripheral blood leucocytosis, suggesting a possible role for VLA-4/FN interactions in murine fetal/neonatal erythropoiesis in vivo. VLA-4 and VLA-5 are also reported to be involved in regulating apoptosis, perhaps indicating an additional role in the negative regulation of haematopoiesis (reviewed in Gonzalez-Amaro & Sanchez-Madrid, 1999).

VLA-4 and VLA-5 are expressed on at least a proportion of primitive mouse and human haematopoietic cells, including repopulating cells (Craddock et al, 1997; van der Loo et al, 1998). VLA-4/VCAM−1 interactions have been shown to be key elements in homing to the bone marrow and mobilization of haematopoietic stem cells and their progeny in adult mice and primates (Papayannopoulou & Nakamoto, 1993; Papayannopoulou et al, 1995, 1998; Papayannopoulou & Craddock, 1997; Vermeulen et al, 1998 ; reviewed in Papayannopoulou, 1999, 2000). VLA-4 is required for the formation of day 12 spleen colony-forming units (CFU-S12) (Williams et al, 1991). Injection of the CS1-CBD fragment into mice has been reported to increase clonogenic progenitor numbers in the bone marrow and spleen, indicating a growth-supporting function (Yokota et al, 1998). However, preincubation of haematopoietic progenitor cells with the recombinant CH-296 peptide of FN containing VLA-4, VLA-5 and heparin binding domains (Fig 3) has been found by Van der Loo et al (1998) to reduce in vivo bone marrow engraftment and induce a change in distribution of HPP-CFC progenitors from bone marrow to spleen in the mouse. This is not observed with the C-274 peptide containing the central CBD. Both anti-VLA-4 and anti-VCAM-1 antibodies inhibit haematopoietic progenitor cell homing in mice 3 h after transplantation, and this is accompanied by increased peripheral blood progenitors and uptake of progenitors by the spleen (Papayannopoulou et al, 1995). Treatment of baboons with anti-VLA-4 antibodies mobilizes CD34+ haematopoietic progenitor cells from bone marrow to peripheral blood (Papayannopoulou & Nakamoto, 1993; reviewed in Papayannopoulou, 1999, 2000). These various effects have been explained by the ability of the blocking antibodies or FN fragments to inhibit interactions between existing or newly generated progenitors and stroma by alterations in the tissue distribution of circulating progenitors, stimulation of stem/progenitor cell migration or by interference with associated cytokine or other adhesion/ligand pathways. Elegant studies by Papayannopoulou et al (1998) have attempted to address some of these possibilities. Anti-VLA-4 treatment augments progenitor mobilization by G-CSF, SCF and FL (Craddock et al, 1997) in baboon and murine model systems, with mobilization of both CFU-S and long-term repopulating cells reported in mice. This mobilization is also induced with anti-VCAM-1 antibodies, but not with the CS-1 fragment of FN, suggesting a role for VLA-4/VCAM-1 or possibly VLA-4 with the central CBD of FN in these interactions. To determine whether these effects are due to VLA-4 downregulation or co-operation between cytokine receptors and VLA-4/VCAM-1 pathways, in vivo antibody blocking experiments were carried out on mice with cytokine-receptor deficiencies (Papayannopoulou et al, 1998). These studies demonstrated that anti-VLA-4-induced mobilization is dependent on a functional SCF receptor, but does not require G-CSF, IL-7 or IL-3α receptors. In contrast, anti-VCAM-1-induced mobilization required functional SCF and SCF receptors. These results were interpreted as evidence for a requirement of SCF/SCF receptor signalling in anti-VLA-4- and anti-VCAM-1-induced mobilization, with the possibility that SCF may induce stem/progenitor cell chemokinesis (reviewed in Whetton & Graham, 1999). In fact, anti-VLA-4 mobilization in mice is correlated with downmodulation of both VLA-4 and SCF receptor expression on murine bone marrow mobilized blast cells. These studies are supported by additional observations in moth-eaten mice that lack Shp-1 and show prolonged SCF receptor activation. They also exhibit a variety of haematological defects including splenomegaly with increased CFU-E, erythropoietin hypersensitivity and increased myelopoiesis, with elevated levels of clonogenic cells in the peripheral blood, but not the bone marrow. These effects are interpreted as evidence for an increased migration of cells from the bone marrow to the peripheral blood and spleen (reviewed in Papayannopoulou, 2000).

Further information on the relevance of β1 integrins in haematopoiesis has come from β1 integrin-deficient mice. In fact, β1-deficient chimaeric mice generated from implantation of β1 integrin-null ES cells into normal blastocysts generate β1-null haematopoietic stem cells that can produce different haematopoietic lineages, but cannot either colonize the fetal liver or be maintained in the fetal liver microenvironment (Hirsch et al, 1996). In vitro intravital microscopy experiments further demonstrate that, although murine haematopoietic progenitor cells roll on bone marrow microvessel endothelia via interactions with E- and P-selectins, the most significant interactions involve the VLA-4/VCAM-1 pathway (Mazo et al, 1998; reviewed in Mazo & von Andrian, 1999). Despite these observations, in P/E-deficient mice treated with anti-VCAM-1 or anti-α4 integrin antibodies, rolling is not completely abolished (reviewed in Mazo & von Andrian, 1999). This suggests that other adhesion receptor/ligand pairs such as CD44 or galactosyl/mannosyl-binding molecules contribute to haematopoietic progenitor cell homing to the bone marrow (Tavassoli & Hardy, 1990; reviewed in Vermeulen et al, 1998). Interestingly, homing of haematopoietic progenitor cells to the bone marrow, while reduced in P/E−/– mice, is further reduced by the administration of anti-VCAM-1 antibodies in vivo (Frenette et al, 1998). By 14 h after transplantation, anti-VCAM-1 treatment of P/E−/– mice also greatly augments the number of circulating haematopoietic progenitors, yet mice carrying a mutant VCAM-1 molecule can establish haematopoiesis (Friedrich et al, 1996). As with murine β1-null mutations, murine α4-null mutations are embryonic lethal. However, α4-null chimaeric mice exhibit apparently normal myelopoiesis in fetal and adult life, but exhibit a specific defect in T and B lymphoid development in bone marrow post-natally (Arroyo et al, 1996). Taken together, these results reveal some discrepancies in experimental data generated in vivo and in vitro, but suggest co-operative effects between VLA-4, VLA-5, proteoglycan/CD44 and selectins and their receptors in haematopoietic stem/progenitor cell homing and mobilization in vivo.

CD44

  1. Top of page
  2. The importance of adhesion receptors in haematopoiesis
  3. The sialomucins in haematopoiesis
  4. The selectin family
  5. The integrin family
  6. CD44
  7. In vivo functions and summary
  8. Acknowledgment
  9. References

The highly conserved human CD44 gene (reviewed by Borland et al, 1998; Ghaffari et al, 1999) is located on human chromosome 11pter–p13, spans approximately 50–60 kb of genomic DNA and contains at least 20 exons (Fig 4). There are 10 exons common to all human CD44 isoforms, while the other 10 (v1 to v10) that are differentially or variably spliced encompass exons 6a to 14. An in frame stop codon occurs in exon 6a. Exons 18 and 19 may be spliced out to produce short (exon 19 spliced out) and long (exon 18 spliced out) cytoplasmic tails. In total, these generate at least 18 different transcripts. CD44H or CD44s, which lacks the regions encoded by exons 6a to 14, is the predominant isoform. The mature CD44H polypeptide contains a 248-amino-acid extracellular domain, a 21-amino-acid transmembrane domain and a 72-amino-acid cytoplasmic tail, with the amino-terminus region containing five of the six N-linked glycosylation sites, exhibiting homology to the cartilage link proteins and interacting with glycosaminoglycans (GAGs), such as hyaluronic acid (HA), the major CD44 ligand. A number of membrane-proximal potential O-linked glycosylation sites and four serine–glycine GAG (chondroitin and heparan sulphate) attachment sites occur in CD44. Evidence of binding to fibronectin, the heparin binding domain of fibronectin, collagen types I and VI, serglycin and osteopontin also exists. These and the regulation of ligand binding by CD44 have been reviewed in detail by Ghaffari et al (1999). CD44 is widely expressed in haematopoietic and non-haematopoietic tissues (reviewed by Borland et al, 1998; Ghaffari et al, 1999), being present on murine day 10 CFU-S and clonogenic progenitors, as well as on human CD34+ cells including those with a primitive phenotype and those associated with the ventral floor of the dorsal aorta in the week 4–5 human embryo (Watt et al, 2000). Levels of CD44 are similar on bone marrow and GM-CSF mobilized peripheral blood CD34+ cells. Approximately 10% of normal human bone marrow CD34+ cells express CD44v10 or CD44v6, with the latter isoform being upregulated during monocytic differentiation (Rosel et al, 1999). CD44 on haematopoietic cells contains an attached chondroitin sulphate (Verfaillie et al, 1994) and may bind cytokines such as MIP-1β. The functional alterations in CD44 during leukaemic development have recently been reviewed by Ghaffari et al (1999).

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Figure 4. The human CD44 gene. (A) The genomic structure of human CD44. An additional v9a domain has been identified. (B) The exon usage in CD44H. Nomenclature for exons is based on that used by Borland et al, 1998.

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CD44 appears to have a role in the regulation of normal haematopoiesis, but through opposing mechanisms. For example, antibodies to the HA binding site of CD44 decrease the number of mature myeloid and lymphoid cells produced in murine long-term marrow cultures and the number of long-term culture initiating cells (LTC-IC) in stromal-dependent cultures initiated with human bone marrow CD34+CD38lo/– cells, a process that can be partially restored by the addition of MIP-1β (reviewed in Ghaffari et al, 1999). However, this antibody blocking effect is not observed in serum-free cultures containing cytokines. Other CD44 antibodies increase the production of progenitor cells in stroma-dependent long-term cultures. Although less than 20% of day 14 clonogenic cells encompassing those that express high levels of CD44 bind to HA in solid-phase assays, this binding can be increased by prestimulation with cytokines, SCF, IL-3 and GM-CSF, and with certain CD44 antibodies that bind to both the HA and to a non-HA binding domain (reviewed by Ghaffari et al, 1999). CD44 is also involved in CFU-S homing to murine bone marrow and more specifically to spleen (Vermeulen et al, 1998), while CD44 antibodies injected in vivo mobilize CFU-S and CFU-GM, but not BFU-E to peripheral blood. Injection of antibodies to CD44v10 or of soluble CD44v10 receptor into mice mobilizes committed progenitors, with increases in these progenitors in peripheral blood and spleen. The soluble CD44v10 molecule also binds to stromal cells and alters B-cell maturation (Rosel et al, 1999). CD44 thus plays a potential role in adhesion by haematopoietic progenitor cells to stromal cells, possibly through interactions with HA (Pilarski et al, 1999). Haematological impairment is seen in CD44 knock-out mice, although phenotypic changes appear relatively minor (Schmits et al, 1997). However, an altered tissue distribution of myeloid progenitors is seen. CFU-GM levels are reduced in bone marrow, but are increased in spleen and peripheral blood. While CD44 is not essential for engraftment or homing, G-CSF-treated CD44-null mice accumulate haematopoietic progenitor cells in the spleen, suggesting a defective release of progenitors from bone marrow or defective homing to the spleen (Schmits et al, 1997). A second HA binding receptor, RHAMM, has been identified on human CD34+ cells. It has been suggested that RHAMM mediates motility in co-operation with low avidity β1 integrin receptors, while CD44 participates in anchoring in co-operation with high avidity β1 integrin receptors (Pilarski et al, 1999).

The immunoglobulin superfamily Members of the immunoglobulin superfamily (IgSF) share common structural features, being composed of 70–110-amino-acid immunoglobulin variable (IgV) and/or constant (IgC) homology domains, containing two 5–10-amino-acid consensus sequences (L/I/VXL/IXC or F/YXCXV/AXH) (Williams & Barclay, 1988). Each Ig homology unit is generally encoded by a single exon. Recently, the IgSF has been divided into two subgroups, Ig-like cell adhesion molecules (Ig-CAMs) and molecules containing Ig-like immunoreceptor tyrosine-based inhibitory motifs (Ig-ITIMs) (reviewed in Newman, 1999), with the latter containing the consensus sequence, L/I/V/S-X-Y-X-X-L/V. Of all the IgSF members potentially involved in the regulation of haematopoiesis, only CD31 will be discussed in this review.

Platelet endothelial cell adhesion molecule-1 (PECAM-1/CD31) CD31 is a 115–140 kDa type I integral membrane protein comprising 711 amino acids, of which 574 form the extracellular region composed of six disulphide-linked IgC2 domains and 118 the cytoplasmic domain containing an ITIM motif (Watt et al, 1995; reviewed in Newman et al, 1999). It is expressed on human endothelial cells, macrophages and T cells in the haematopoietic microenvironment and on CD34+ haematopoietic progenitor cells at all stages of development (Watt et al, 1995; Tavian et al, 1996). The gene for CD31 is located on chromosome 17q23 and comprises 16 exons extending over more than 75 kb of DNA with the exons separated by introns of 86 bp to 12 kb in length. Several isoforms of human CD31 are generated by alternative splicing of exons 9, 13 or 14, with one encoding a soluble form lacking the transmembrane domain (Watt et al, 1995; reviewed in Newman et al, 1999). In vitro studies have implicated CD31 in vasculogenesis, angiogenesis and in the transendothelial migration of leucocytes including CD34+ cells. As a member of the Ig-ITIM family, CD31 functions as an inhibitory receptor (reviewed in Newman et al, 1999).

In haematopoietic tissues, CD31 may be involved in the formation of a stromal cell niche allowing interactions with haematopoietic progenitors. In vitro studies have shown that CD31 regulates progenitor cell adhesion in a subtle way through its ability to trigger adhesion mediated by β1 integrins (Leavesley et al, 1994). CD31 has been implicated in transendothelial migration of haematopoietic progenitor cells (Yong et al, 1998). For in vivo functional studies, CD31-deficient mice have been developed (Duncan et al, 1999). Defects in haematopoiesis have not been reported in CD31-null mice, suggesting that CD31 is not crucial for the maintenance of the haematopoietic microenvironment or for transendothelial migration. However, this does not rule out the possibility that compensatory mechanisms are induced that substitute for CD31 functions or that CD31 may mediate more subtle effects, such as regulating the rate of cell migration.

In vivo functions and summary

  1. Top of page
  2. The importance of adhesion receptors in haematopoiesis
  3. The sialomucins in haematopoiesis
  4. The selectin family
  5. The integrin family
  6. CD44
  7. In vivo functions and summary
  8. Acknowledgment
  9. References

We have attempted to review data on several classes of adhesion receptors that appear to be of some importance in haematopoietic stem/progenitor cell migration, homing and lodgement in microenvironmental niches, and in the subsequent determination of stem cell fate. The adhesion receptors described may also be involved in maintaining the architecture of the microenvironment in which the stem cells reside. The known in vivo functions of the adhesion receptors described are documented in Table II. It is evident that PSGL-1(CD162)/P-selectin and VLA-4/VCAM-1 are of major importance for the entry of haematopoietic progenitors into adult bone marrow via the sinusoidal endothelia and for their subsequent mobilization. The β1 integrins appear to be essential for colonization of fetal liver by haematopoietic stem cells, while CD44, VCAM-1, VLA-4, RHAMM and the P- and E-selectins regulate the distribution of stem/progenitor cells in different adult organs such as the bone marrow and spleen. However, both adhesion/receptor interactions analysed in vitro and those studied in vivo indicate that these receptor/ligand interactions are not sufficient to explain in detail the migration, homing and lodgement processes that occur during haematopoiesis. Other receptors, such as E-selectin, CD34, CD44, PCLP1 and CD164, extracellular matrix molecules such as FN, heparan sulphate proteoglycans and tenascin C, and those molecules that will be identified as part of the genomic project, are probably involved, although the exact molecular mechanisms controlling these events in fetal and adult life are far from resolved. Many of these processes will depend on the co-expression or activation of adhesion receptors/ligands through cytokine and chemokine receptors, such as the kit or SCF receptor and the CXCR4 receptor for SDF-1α. It is also probable that interactions between adhesion receptors and ligands may have a signalling role, regulating cytokine production and the proliferation, quiescence, differentiation and death of stem cells and their progeny. Indeed, the function of adhesion receptors may differ on different subsets of primitive and more mature haematopoietic progenitor cells. Such diverse effects are observed with engagement of the CD34, CD43, CD162 and CD164 sialomucins by their cognate or surrogate ligands on different haematopoietic progenitor cell subpopulations in vitro. These include inhibition of recruitment into the cell cycle, inhibition of differentiation and apoptotic effects on selected progenitor subsets. Because of major difficulties in isolating and assaying the very rare haematopoietic stem cell subset, many studies are based on analysis of haematopoietic progenitor cells as a whole and do not concentrate on the pluripotent stem cell, despite indications that immature haematopoietic progenitor subsets may home more efficiently to certain haematopoietic organs than more mature subsets. Furthermore, the in vitro and in vivo data do not necessarily correlate. Studies on cytokine-, cytokine receptor-, and adhesion receptor/ligand-null mice suggest that there is probably some overlap in function or redundancy in the use of receptors that will allow the recruitment of other receptors/ligands as functional substitutes for the missing molecules. Mice/cells lacking multiple receptors may assist in understanding these mechanisms more clearly. In antibody blockade experiments, it is not clear if antibodies mediate effects other than inhibiting adhesion or if there are multiple ligands for some adhesion receptors. It seems very probable, and there is some evidence, that haematopoietic development will rely on the co-operation of many cell surface receptors/ligands, with some being intimately involved in determining stem cell fate and others involved in fine tuning this process. For example, it has been suggested that the up- and downregulation/activation of different integrins and selectins are important processes for controlling the direction or speed of migration of mature leucocytes and perhaps their precursors. Such effects may not necessarily be detected in in vivo studies. Thus, despite many studies on the function of adhesion receptors on mature cells, there is still little detailed information available regarding haematopoietic stem cell trafficking and localization within microenvironmental niches in adult and fetal development. The identification of the full complement of receptor/ligand interactions and the molecular mechanisms that determine their involvement in regulating stem cell fate remains one of the major investigative areas in haematopoiesis.

Table II. In vivo functions of adhesion receptors during haematopoietic development in murine model systems.
Deficient or blocked cell surface receptor In vivo effect References
Sialomucins
CD34CD34−/–: normal adult peripheral blood profile; lower progenitor numbers in yolk sac, fetal liver, bone marrow and spleen; delayed embryonic haematopoiesis; & defective eosinophil migration to inflammed lungCheng et al, 1996; Suzuki et al, 1996
PSGL-1 (CD162)CD162−/–: Modest neutrophilia and eosinophiliaYang et al 1999b
Selectins
Double & triple knock-outs P/E-selectin−/– P/L-selectin−/– P/L/E-selectin−/–/–Leucocytosis, increased cytokine levels; increased myeloid to erythroid progenitor ratio in bone marrow; increased granulopoiesis in bone marrow & spleen; increased erythropoiesis in spleen; defective progenitor cell homing to bone marrow; defective rolling of progenitors on bone marrow venule & sinusoidal endothelia 1999; Mazo et al, 1998Frenette et al, 1996; Robinson et al, 1999; reviewed in Hartwell & Wagner, 1999, Etzioni et al, 1999 & Mazo & von Andrian,
Integrins
VLA-4 (α4β1) VLA-5 (α5β1) and their ligands VCAM-1 and FibronectinAnti-VLA-4 mAbs induce anaemia in utero, with absent fetal liver erythroblasts & mobilizes adult bone marrow progenitors; anti-VLA-4 and VCAM-1 mAbs inhibit homing of progenitors to bone marrow; anti-VCAM-1 mAbs cause peripheral blood leucocytosis; recombinant CH-296 fibronectin peptide blocks in vivo progenitor engraftment of bone marrow; α4−/– chimaeric mice show defective post-natal T and B lymphoid development; β1-/–stem cells fail to colonize fetal liver; VLA-4/VCAM-1 required in part for interaction with bone marrow endotheliaHamamura et al, 1996; Arroyo et al, 1996; van der Loo et al 1998; Hirsch et al, 1996; Mazo et al, 1998; reviewed in Papayannopoulou,1999, 2000, & Mazo & von Andrian, 1999
Miscellaneous
CD44Anti-CD44 mAbs inhibit CFU-S homing to bone marrow and spleen & mobilize CFU-S and CFU-GM; CD44−/–: reduced bone marrow and increased splenic and peripheral blood CFU-GM, with defective release of progenitors from bone marrow or defective homing to spleenVermeulen et al, 1998;  Schmits et al, 1997

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  2. The importance of adhesion receptors in haematopoiesis
  3. The sialomucins in haematopoiesis
  4. The selectin family
  5. The integrin family
  6. CD44
  7. In vivo functions and summary
  8. Acknowledgment
  9. References
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